Biomedical subjects

O Olsen

Publications and source records attributed to O Olsen.

At least 73 records · Page 4Linked to original sources

PubMed1995

Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-beta-glucanase.

In beta-glucans those beta-1,4 glycosidic bonds which are adjacent to beta-1,3 bonds are cleaved by endo-1,3-1,4-beta-glucanases (beta-glucanases). Here, the relationship between structure and activity of the beta-glucanase of Bacillus macerans is studied by x-ray crystallography and site-directed mutagenesis of active site residues. Crystal structure analysis at 2.3-A resolution reveals a jelly-roll protein structure with a deep active site channel harboring the amino acid residues Trp101, Glu103, Asp105, and Glu107 as in the hybrid Bacillus beta-glucanase H(A16-M) (Keitel, T., Simon, O., Borriss, R., and Heinemann, U. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5287-5291). Different mutant proteins with substitutions in these residues are generated by site-directed mutagenesis, isolated, and characterized. Compared with the wild-type enzyme their activity is reduced to less than 1%. Several mutants with isosteric substitutions in Glu103 and Glu107 are completely inactive, suggesting a direct role of these residues in glycosyl bond hydrolysis. The kinetic properties of mutant beta-glucanases and the crystal structure of the wild-type enzyme are consistent with a mechanism where Glu103 and Glu107 are the catalytic amino acid residues responsible for cleavage of the beta-1,4 glycosidic bond within the substrate molecule.

Amino Acid Sequence↗

PubMed1995

Unbiased vs. conservative estimators of etiological fractions: examples of misclassification from studies of occupational lung cancer.

Theoretical studies emphasize the importance of making unbiased etiological fraction estimates. In empirical works, however, the published estimates are usually conservative. The purpose of the present report is to study, empirically, the numerical magnitude of such conservative biases. Examples from the literature on occupational exposure and lung cancer are reported. It is demonstrated that conservative bias may decrease a numerical estimate by more than a factor 10 and that decreases by a factor 2 or 3 are not unusual. It is concluded that it is important, in future review studies, to pay attention to the magnitude of the conservative biases in the published empirical estimates and to put most emphasis on the least biased estimates in the review process.

Asbestos↗

PubMed1995

Glycan modification of a thermostable recombinant (1-3, 1-4)-beta-glucanase secreted from Saccharomyces cerevisiae is determined by strain and culture conditions.

High level biosynthesis and secretion of the thermostable hybrid (1-3, 1-4)- beta-glucanase H(A16-M) has been achieved in Saccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1P) and the Bacillus macerans (1-3, 1-4)-beta-glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man8-13GlcNAc2, although only traces of Man9GlcNAc2 were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gln in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steric hindrance to glycan modification by the glycans themselves. H(A16-M)-Y produced under control of either the yeast protease B or the yeast 3'-phosphoglycerate kinase promoter, each in two different Saccharomyces strains revealed a dependence of N-glycan profile on both strain and culture conditions. The extent of O-glycosylation was found to be nine mannose units per H(A16-M)-Y molecule. An attempt to identify the linkage-sites for the O-glycans by amino acid sequencing failed, suggesting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to structural information available for H(A16-M)-Y.

Bacillus↗

PubMed1995

Synergism between Erwinia pectate lyase isoenzymes that depolymerize both pectate and pectin.

Phytopathogenic Erwinia bacteria cause tissue maceration by secretion of pectinolytic enzymes such as pectate lyase (PL). Sequencing of overlapping genomic fragments from Erwinia carotovora subsp. atroseptica established the organization of a 7.5 kbp region encoding PL isoenzymes. Two intergenic regions of 656 and 645 bp separate three enzyme coding regions of 1125 bp exhibiting approximately 80% positional identity. The promoters of each of the three genes contain a segment with high homology to the binding sequence of the E. chrysanthemi KdgR transcription repressor, implying similar mechanisms of gene regulation in the two bacterial species. Separate expression of the pel genes in the Escherichia coli-pT7-7 system and purification of their products yielded PLs at 7-33 mg (I culture)-1 with greater than 95% purity. Availability of the recombinant enzymes allowed determination of the kinetic differences amongst the PL isoforms, PL1, PL2 and PL3. The results show that PL is not strictly confined to depolymerization of pectate since each isoenzyme more readily degrades 31% esterified pectin. Addition of isoenzyme combinations revealed no synergism with respect to degradation of pectate or 31% esterified pectin. However, addition of enzyme combinations containing PL3 enhanced the activity towards 68% esterified pectin, against which individual PL activities were low, by up to 64%. These data suggest that the combination of PL isoenzymes extends the range of pectic substrates which the bacterium can degrade.

Amino Acid Sequence↗

PubMed1995

Low doses of pentagastrin stimulate gastric lipase secretion in man.

BACKGROUND: Gastric lipase is an important enzyme for dietary triglyceride digestion in normal subjects. Its regulation is unknown, as is the relation between the quantity and activity of the enzyme. METHODS: In a dose-response study we investigated the effect of low doses of pentagastrin (less than 1000 ng/kg/h) on the output of gastric lipase measured by a kinetic assay and an enzyme-linked immunosorbent assay (ELISA). RESULTS: In five healthy volunteers stepwise intravenous pentagastrin infusions of 0, 50, 100, 500, and 1000 ng/kg/h resulted in a stepwise increase in the lipase output, as measured with ELISA. However, the lipolytic activity, measured with a kinetic assay, decreased as the pH of the gastric contents decreased. CONCLUSION: We conclude that secretion of the gastric lipase is stimulated by pentagastrin, but the simultaneous secretion of acid counteracts the lipolytic activity of the enzyme when food is not present.

Adult↗

PubMed1994

[Home delivery and scientific reasoning].

Doctors commonly assume that it is safer for all women to give birth in hospital rather than at home. Nevertheless, all statistical comparisons relevant to Nordic women today show that for healthy pregnant women it is at least as safe to give birth at home--and perhaps even safer. Furthermore, many randomised clinical trials consistently show that several of the elements which characterize home births make the births proceed much easier. The question is raised, in what ways it is possible to convince obstetricians that they should base their judgements and advice regarding place of birth on empirical evidence rather than on "well established" but pre-scientific dog-mas.

Attitude of Health Personnel↗

PubMed1994

The effect of gastrin-releasing peptide on porcine pancreaticobiliary bicarbonate secretion is mediated by secretin.

The effect of gastrin-releasing peptide (GRP) (250, 500, 1000 pmol/kg.h) on the pancreaticobiliary bicarbonate secretion, the pancreatic protein secretion, and the plasma concentrations of secretin and cholecystokinin (CCK) was studied in the anaesthetized pig. Infusion of GRP (1000 pmol/kg.h) increased the portal plasma concentrations of secretin from 0.9 to 13.6 pmol/l and CCK from 1.2 to 38.4 pmol/l, the pancreatic bicarbonate secretion from 0.01 to 5.6 mmol/h, the hepatic bicarbonate secretion from 0.5 to 4.1 mmol/h, and the pancreatic protein secretion from 3 to 680 mg/h. Blocking of CCK-A receptors by MK-329 did not significantly change the effect of GRP, whereas prevention of secretin release by removal of the small intestine caused a 13-fold reduction in the GRP-induced pancreatic bicarbonate secretion and completely abolished the effect on hepatic bicarbonate secretion but did not change the effect on pancreatic protein secretion. We conclude that the effect of GRP on pancreaticobiliary bicarbonate secretion is not mediated through the release of CCK but more likely through the release of secretin and that the effect on pancreatic protein secretion is possibly a direct effect of GRP.

Animals↗

PubMed1994

Duodenal mucosal bicarbonate secretion in pigs is accompanied by compensatory changes in pancreatic and biliary HCO3- secretion.

BACKGROUND: The purpose of the study was to examine the effect of stimulation and inhibition of duodenal mucosal bicarbonate secretion on pancreatic and hepatic bicarbonate secretion in response to acid. METHODS: The effect of inhibition (indomethacin) or stimulation (misoprostol) of duodenal mucosal bicarbonate secretion on pancreatic and biliary bicarbonate secretion in response to intraduodenal infusion of HCl or intravenous infusion of secretin was studied in anaesthetized pigs. RESULTS: The hepatic and pancreatic response to exogenous secretin was not significantly altered by stimulation/inhibition of duodenal bicarbonate secretion. However, pancreatic and biliary bicarbonate secretion in response to duodenal acidification was significantly augmented by inhibition of duodenal mucosal bicarbonate secretion; conversely, it was reduced by stimulation of duodenal bicarbonate secretion. The increase in plasma secretin levels in response to duodenal acidification was reduced by stimulation and augmented by inhibition of duodenal mucosal bicarbonate secretion. CONCLUSIONS: Duodenal mucosal bicarbonate secretion can serve as a modulator of both pancreatic and biliary bicarbonate secretion in response to luminal acidification, possibly through regulation of the release of secretin.

Acid-Base Equilibrium↗

PubMed1994

Etiologic fractions for physical work load, sports and overweight in the occurrence of coxarthrosis.

OBJECTIVES: The aim of this study was to estimate the impact of physical work load, sports, and overweight on the incidence of coxarthrosis. METHODS: A case-referent study was made of 239 male recipients of a hip prosthesis and 302 men randomly selected from the general population. Information was obtained by means of an interview and questionnaire. Exposures to physical work load and sports were measured as the cumulative number of hours of exposure up to 49 years of age. Overweight was measured as the estimated body mass index at 30 or 40 years of age. RESULTS: The etiologic fraction related to the three risk factors was 40% for physical work load, 55% for sports, and 15% for overweight. Various measures of physical work load were considered, but they all proved to be correlated. CONCLUSIONS: Approximately 80% of the idiopathic coxarthrosis was explained by the presence of the three risk factors.

Age Factors↗

PubMed1993

Determinants for the enhanced thermostability of hybrid (1-3,1-4)-beta-glucanases.

Hybrid (1-3,1-4)-beta-glucanases which contain an N-terminal region derived from the Bacillus amyloliquefaciens enzyme and a C-terminal region of the closely related B. macerans enzyme may exhibit a thermostability superior to both parental enzymes. A systematic series of hybrid enzymes were constructed in order to delineate the amino acid residues that affect protein stability. Hybrid enzymes with between one and four of the N-terminal residues for the mature B. amyloliquefaciens (1-3,1-4)-beta-glucanase exhibit no significant changes in biochemical characteristics as compared with the parental B. macerans enzyme. However, significantly enhanced thermostability was observed in the hybrid enzyme containing an N-terminal segment of eight amino acid residues derived from the B. amyloliquefaciens enzyme. Site-directed mutagenesis revealed that the combined effect of Gln1, Thr2, Ser5 and Phe7 confer enhanced stability on hybrid enzymes, probably by improving the hydrogen bonding that stabilizes the interactions between the N-terminal and the centre of the folded molecule, as well as between the two termini of the polypeptide chain. Furthermore, deletion of Tyr13 in the hybrid enzyme containing the 12 N-terminal amino acids from the B. amyloliquefaciens (1-3,1-4)-beta-glucanase results in a dramatic increase in stability at 70 degrees C with the half-life of 6 min increased to around 4 h. This is twofold higher than the hitherto most stable hybrid enzyme in which the N-terminal domain consisted of 16 residues of the B. amyloliquefaciens enzyme.

Amino Acid Sequence↗

PubMed1993

Sodium azide mutagenesis: preferential generation of A.T-->G.C transitions in the barley Ant18 gene.

The molecular basis for the absence of anthocyanins and proanthocyanidins in four independent sodium azide-induced ant18 mutants of barley was examined by sequencing the gene encoding dihydroflavonol 4-reductase in these mutants. Sodium azide generated 21 base substitutions, which corresponds to 0.17% of the 12,704 nucleotides sequenced. Of the substitutions, 86% were nucleotide transitions, and 14% were transversions. A.T-->G.C base pair transitions were about 3 times more frequent than G.C-->A.T transitions. No deletions or mutation hot spots were found. The absence of dihydroflavonol 4-reductase activity in ant18-159, ant18-162, and ant18-164 plants is caused by missense mutations in the respective genes. By using microprojectile bombardment, a plasmid harboring the wild-type Ant18 gene was introduced into ant18-161 mutant cells and resulted in the development of anthocyanin pigmentation, which demonstrates that the mutation is corrected by expression of the introduced gene. On the other hand, a plasmid derivative with the two ant18-161-specific base transitions at the 5' splice site of intron 3 prevented complementation. It is concluded that the absence of detectable mRNA for dihydroflavonol 4-reductase in ant18-161 cells is due to the mutations in the pre-mRNA splice donor site.

Alcohol Oxidoreductases↗

PubMed1993

[Work load and cardiovascular risk factors. A cross-sectional study of employed Danish men and women].

As part of World Health Organisation initiated MONICA project, 2000 men and women aged 30, 40, 50 and 60 from the general population were invited to undergo a medical examination with special emphasis on cardiovascular disease. A total of 1504 (75%) participated, 1209 of whom were employed. The participants answered a questionnaire on working, social, and health conditions and underwent clinical examinations that included the measurement of blood pressure and serum cholesterol triglycerides, high density lipoprotein, fibrinogen and glycosylated haemoglobin (HbA1C) concentrations. Using the demand control model for measuring job strain suggested by Karasek, the employed people were classified according to those who had suffered job strain and those who had not in two different ways. The subjective classification was based on the participants' statements regarding demand and control in their jobs, whereas the objective classification was based on job title and mode of payment. More women than men were classified as having high strain jobs. After adjusting for age and sex no significant association was found between coronary risk factors and subjective job strain. A tendency for an association between fibrinogen and job strain was found. Body mass index and HbA1C concentration were significantly associated with objective job strain independent of confounders.

Adult↗

PubMed1993

Theoretical studies of Rhizomucor miehei lipase activation.

Computational methods have been used to study the extensive conformational change of Rhizomucor miehei lipase upon activation. The present study considers the possible activation route, the energies involved and molecular interactions during the conformational change of the lipase in a hydrophobic environment. The conformational change was studied by conventional molecular dynamics methods and with a combined molecular dynamics and mechanics protocol, in which the conformational change was simulated by restraining C alpha pseudotorsional angles in small steps between the two crystallographically observed positions of the lid. In the closed conformer of the enzyme the active site is completely buried under a short helical loop, 'the lid'. The activation of the lipase consists of a movement of the lid, which results in an open conformer with an exposed active site. From the results of the simulations in the present work we suggest that the lipase in a hydrophobic environment is stabilized in the open form by electrostatic interactions.

Computer Simulation↗

PubMed1993

Expression of the dihydroflavonol reductase gene in an anthocyanin-free barley mutant.

The barley gene encoding dihydroflavonol-4-reductase (DFR) was delivered by micoprojectile bombardment into leaf sheath tissue of the anthocyanin-free barley mutant ant 18-162, a mutant which lacks DFR activity-probably because of a missense mutation in the structural gene for DFR. The delivered gene complemented the mutation, as evidenced by the synthesis of anthocyanin in individual leaf sheath cells of the bombarded tissues. Pigment synthesis appeared two days after gene delivery and both the number of pigmented cells and the intensity of pigmentation increased over the following days. Depending on the physiological condition of the host plants, up to 15 pigmented cells per 10 tissue segments were detected. These results demonstrate that the Ant 18 gene of barley encodes dihydroflavonol-4-reductase. A series of gene constructs encoding DFR were expressed in the anthocyanin-free mutant tissue. The genomic clone complemented the mutation whereas an equivalent plasmid with all introns deleted did not. The highest number of pigmented cells was obtained using plasmids containing the DFR-coding sequence interrupted by intron 1 of the genomic clone, indicating that the presence of an intron stabilizes the DFR message.

Alcohol Oxidoreductases↗

PubMed1993

Impact of social network on cardiovascular mortality in middle aged Danish men.

STUDY OBJECTIVE: To estimate quantitatively (the aetiological fraction) the impact of poor social network on premature death from cardiovascular disease in middle aged, white men. DESIGN: The causality of the relationship has already been discussed in a large review, and it is assumed to be well documented. The numerical estimation of the impact was based on a review of all published cohort studies on the relationship between social network and mortality in white, middle aged men. RESULTS: The studies reviewed are all of high epidemiological quality and present a consistent and stable dose-response pattern. The aetiological fraction was estimated to be 30%, with a plausible range of 20-40%. CONCLUSIONS: Social network was an important, independent, risk factor for cardiovascular disease in white, middle aged men. It had a strong impact on mortality, comparable to that of traditional risk factors. Social network should have a more central role in future epidemiological research into cardiovascular disease. The factors that result in a strong social network should be identified and strategies applicable in preventive work should be developed.

Cardiovascular Diseases↗

PubMed1993

Secretin and portal blood flow.

Using a duplex Doppler technique, we investigated the effect of low doses of secretin on the portal blood flow. In eight healthy volunteers successive intravenous secretin infusions of 0, 8, and 32 pmol x kg-1 x h-1 resulted in proportional increases in plasma secretin levels. The portal venous flow, however, was unaffected. A bolus injection of 930 pmol of secretin caused plasma secretin levels to increase 100-fold, whereas blood flow in the portal vein increased only by a factor three. This suggests that secretin in the present dose range is of no quantitative importance as a regulator of portal venous flow under physiologic conditions.

Adult↗

PubMed1992

Effects of oleic acid and endogenous bile on duodenal secretion of somatostatin in man.

We studied the effects of intraduodenal oleic acid on the release of somatostatin to plasma and the correlation between endogenous bile output and plasma somatostatin. In five normal persons infusion of 0, 5, 10, 20, and 40 mM oleic acid dose-dependently increased the levels of somatostatin during as well as after gallbladder emptying. The difference between somatostatin concentration during and after gallbladder emptying was not significant. The amylase secretion also was significantly correlated to the dose of fat, whereas the output of bile salts was the same for all fat doses used. Our observations indicate that intraduodenal oleic acid--and not bile salts--releases somatostatin from the gut.

Adult↗

PubMed1992

Fat and gastric acid secretion.

To evaluate the importance of the terminal carboxyl group of the oleic acid molecule in the inhibition of gastric acid secretion, 6 normal persons were stimulated twice with duodenal perfusates containing either 20 mM oleic acid or 20 mM oleyl alcohol. Oleic acid significantly inhibited the gastric acid secretion stimulated by pentagastrin (100 ng/kg/h) and increased the levels of secretin in plasma. The effect of oleyl alcohol was insignificant. It is concluded that the carboxyl group of the fat molecule has an important role in the inhibition of gastric acid secretion, and the effect could in part be attributable to the release of secretin into plasma.

Adult↗