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Biomedical subjects

O Simon

Publications and source records attributed to O Simon.

At least 55 records · Page 3Linked to original sources
PubMed1997

Non starch polysaccharide hydrolyzing enzymes as feed additives: detection of enzyme activities and problems encountered with quantitative determination in complex samples.

Chromogenic substrates, an agar diffusion assay and viscosity reduction were used to estimate beta-glucanase and xylanase activities in water soluble extracts of different feedstuffs and digesta supernatants. The dinitrosalicylic acid reducing sugar method was employed to calibrate results from different methods based on international units (IU, glucose equivalents). The detection of dye release from chromogenic substrates was a suitable method, allowing the detection of 0.05 IU of enzyme activity per ml of extract, although measurements in digesta supernatants were limited in linearity (0.1-0.5 IU/ml supernatant). With the agar diffusion assay the detection of enzyme activity was possible over a wider concentration range (extracts: 0.05-1 IU/ml, digesta supernatants: 0.1-1 IU/ml), but visual evaluation led to inaccurate measurement. Accuracy can be improved by computer based evaluation of digital images. The use of viscosity reduction produced linear standard curves from 0.01 to 0.5 IU/ml in feed extracts, but reliability of measurements depended on modification of substrates. Quantification of enzyme activities was influenced by matrix effects of complex samples. Cereal dependant differences were found in various extracts of feed mixtures and cereal extracts. Digesta supernatants partly inhibited enzyme activity, depending on the origin of the sample. Interaction of substrates with digesta components varied between methods. The sensitivity of the methods is comparable, however, all methods require specific calibrations to account for matrix- and enzyme specific effects.

Animal Feed↗
PubMed1997

[Post-lumbar puncture syndrome. Causes, prevention, treatments].

Spinal tape syndrome requires rapid diagnosis and adapted treatment. A relatively small diameter needle inserted parallel to the dura mata fibers is the best preventive measure. The ventral supine position for a few hours followed by a short walk is sufficient. Caffeine may also be prescribed and has been shown to be as effective orally or intravenously. For severe or persistent syndromes, the blood-patch is remarkably effective and simple to use.

Headache↗
PubMed1995

Genes encoding xylan and beta-glucan hydrolysing enzymes in Bacillus subtilis: characterization, mapping and construction of strains deficient in lichenase, cellulase and xylanase.

The gene encoding extracellular xylanase (xynA) was amplified as a 770 bp DNA fragment from Bacillus subtilis 168 chromosomal DNA by PCR. The genes encoding endo-beta-1,4-glucanase (eglS) and endo-beta-1,3-1,4-glucanase (bglS) were isolated from a genomic library of B. subtilis 168. The sequences of xynA and eglS were identical to those of the xylanase and cellulase genes from B. subtilis PAP115. Integrative plasmids containing DNA fragments with deletions in the coding region of the genes were constructed and used to replace the chromosomal eglS, bglS and xynA genes of B. subtilis 168. Strains without any detectable activity against xylan (Xyn-), carboxymethylcellulose (Egl-) or mixed linked beta-1,3-1,4-glucan (Egl- Bgl-) were obtained. The genes were mapped at 170 degrees (eglS), 175 degrees (xynA) and 340 degrees (bglS) on the B. subtilis chromosome.

Bacillus subtilis↗
PubMed1994

Microcalorimetric determination of the thermostability of three hybrid (1-3,1-4)-beta-glucanases.

Thermodynamic parameters of the three hybrid (1-3,1-4)-beta-glucanases H(A12-M), H(A12-M) delta Y13, and H(A16-M) composed of short N-terminal regions derived from the Bacillus amyloliquefaciens enzyme and a C-terminal region of the homologous Bacillus macerans enzyme were determined in 2 mM sodium cacodylate pH 6.0, 1.5M guanidine hydrochloride, containing 1 mM CaCl2 or 1 mM EDTA. Melting of H(A12-M) delta Y13 and H(A16-M) in the presence of calcium ions is characterized by two subtransitions; only one transition is observed in the case of H(A12-M). In calcium-free buffer each of the three hybrid enzymes melts in one two-state transition. Transition temperatures Tm and molar enthalpy changes delta H are reduced in the absence of calcium ions but the reduction is much more pronounced for H(A12-M) delta Y13 and H(A16-M) than for the less thermostable enzyme H(A12-M).

Bacillus↗
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

Molecular and active-site structure of a Bacillus 1,3-1,4-beta-glucanase.

The three-dimensional structure of the hybrid Bacillus 1,3-1,4-beta-glucanase (beta-glucanase; 1,3-1,4-beta-D-glucan 4-glucanohydrolase, lichenase, EC 3.2.1.73) designated H(A16-M) was determined by x-ray crystallography at a resolution of 2.0 A and refined to an R value of 16.4% using stereochemical restraints. The protein molecule consists mainly of two seven-stranded antiparallel beta-pleated sheets arranged atop each other to form a compact, sandwich-like structure. A channel crossing one side of the protein molecule accommodates an inhibitor, 3,4-epoxybutyl beta-D-cellobioside, which binds covalently to the side chain of Glu-105, as seen in a crystal structure analysis at 2.8-A resolution of the protein-inhibitor complex (R = 16.8%). That Glu-105 may be indispensible for enzyme catalysis by H(A16-M) is suggested by site-directed mutagenesis of this residue, which inevitably leads to an inactive enzyme.

Amino Acid Sequence↗
PubMed1991

Crystallization of the hybrid Bacillus (1-3,1-4)-beta-glucanase H(A16-M).

The extremely thermostable hybrid Bacillus (1-3,1-4)-beta-glucanase H(A16-M) has been crystallized with polyethylene glycol by vapour diffusion. The single crystals diffract to a resolution of 2.2 A. The protein crystallizes in the orthorhombic space group P2(1)2(1)2(1) with a = 70.22(7) A, b = 72.56(1) A, c = 49.97(1) A, and has one molecule per asymmetric unit.

Bacillus↗
PubMed1991

Hybrid Bacillus (1-3,1-4)-beta-glucanases: engineering thermostable enzymes by construction of hybrid genes.

Hybrid (1-3,1-4)-beta-glucanase genes were constructed by extension of overlapping segments of the (1-3,1-4)-beta-glucanase genes from Bacillus amyloliquefaciens and B. macerans generated by the polymerase chain reaction (PCR). Four hybrid genes were expressed in Escherichia coli cells. The mature hybrid enzymes contain a 16, 36, 78, or 152 amino acid N-terminal sequence derived from B. amyloliquefaciens (1-3,1-4)-beta-glucanase followed by a C-terminal segment derived from B. macerans (1-3,1-4)-beta-glucanase. Biochemical characterization of parental and hybrid enzymes shows a significant increase in thermostability of three of the hybrid enzymes when exposed to an acidic environment thus combining two important enzyme characteristics within the same molecule. At pH 4.1, 85%-95% of the initial activity was retained after 1 h at 65 degrees C in contrast to 5% and 0% for the parental enzymes from B. amyloliquefaciens and B. macerans. After 60 min incubation at 70 degrees C, pH 6.0, the parental enzymes retained 5% or less of the initial activity whilst one of the hybrids still exhibited 90% of the initial activity. Of the parental enzymes B. macerans (1-3,1-4)-beta-glucanase had the lower specific activity while the hybrid enzymes exhibited specific activities that were 1.5- to 3-fold higher. These experimental results demonstrate that exchange of homologous gene segments from different species may be a useful technique for obtaining new and improved versions of biologically active proteins.

Amino Acid Sequence↗
PubMed1991

Effect of sulfur mustard on murine lymphocytes.

The effect on spleen cells of a single in vivo treatment with sulfur mustard was analyzed in mice 1 week after intoxication. A marked decrease in the number of total spleen cells was observed in mice receiving high doses of sulfur mustard. Flow cytometric analysis indicated that B-lymphocytes were relatively more affected than T-lymphocytes by this toxic compound. However, the function of remaining B-cells, measured by thymidine incorporation and immunoglobulin secretion in the presence of lipopolysaccharide, was not significantly impaired. In addition, sulfur mustard did not depress T-lymphocyte function since their proliferation in response to concanavalin A or to an anti-CD3 antibody was not affected by the treatment. These results suggest that whereas some observations reported in patients can be found in a murine model, additional in vitro studies with human lymphocytes could more adequately provide further information on sulfur-mustard-induced alterations of the immune system.

Animals↗
PubMed1990

The influence of two behavioral regimens on the distribution of sleep and wakefulness in narcoleptic patients.

Thirty-two hours (night-day-night) of polygraphic recordings were performed on 14 patients with a diagnosis of narcolepsy-cataplexy. Half of the patients stayed in bed during the day, whereas the other half were seated at a table. Patients were free to nap whenever they wanted to. Patients under continuous bedrest slept 2-3 times more during the day than patients who were sitting at the table. Rapid-eye-movement (REM) sleep and slow-wave sleep (SWS, stages 3 and 4) were nearly absent during daytime sleep in the table group, but not in the bed group. The differential behavioral regimes during the day resulted in different amounts of SWS in the consecutive night sleep. Although SWS increased from the first to the second night in the table group, it decreased in the bed group. This result suggests that the presumably homeostatic regulation of SWS is intact in narcoleptic patients.

Adult↗
PubMed1989

Utilisation of free and protein dietary lysine in chicks estimated with isotopes.

1. Utilisation of supplementary free L-lysine hydrochloride was estimated in growing chicks and compared to that of protein-bound lysine. The technique used is based on the oxidative catabolism of either free (U-14C)-L-lysine or the labelled lysine incorporated into yeast proteins. 2. The animals received a wheat-wheat gluten diet which was L-lysine-supplemented with either unlabelled yeast proteins or a mixture of synthetic amino acids simulating the yeast proteins or L-lysine hydrochloride alone. 3. At 13 and 15 days after hatching, expiry of (14C)--carbon dioxide was followed 8 h after dosing with the appropriate radiolabelled diet. After 4 h, 0.66% of the protein-bound and 5.3 to 5.7% of the free lysine radioactivity appeared as 14C--carbon dioxide. 4. It is concluded that under these conditions lysine from both sources was utilised more efficiently than had been assumed hitherto, protein-bound lysine being slightly better utilised than free lysine.

Animal Feed↗
PubMed1987

Estimation of the endogenous N proportions in ileal digesta and faeces in 15N-labelled pigs.

Four 40 kg castrated male pigs fitted with simple 'T' cannulas in the terminal ileum were given 15N-labelled ammonium salts, added to a low protein diet, for 6 days. Excretion of 15N in urine and faeces was monitored daily throughout the labelling and subsequent experimental periods. During the experimental period the pigs were given a diet based on wheat and fish meal, supplemented with varying levels of partially hydrolysed straw meal to give crude fibre contents ranging from 40 to 132 g/kg. After adaptation to the particular levels of straw meal, faeces and ileal digesta were collected during successive 24 h periods. N digestibility values were determined by the chromic oxide ratio method. The retention of 15N labelled non-specific N was 0.46 of the dose given. The validity of using urine values as a measure of 15N abundance in endogenous N was demonstrated by the similarity of 15N abundance in urine immediately before slaughter at the end of the experiment and in the digestive secretory organs thereafter. The average amount of endogenous N passing the terminal ileum was 3.4 g/day or 0.30-0.50 of total ileal N flow. This was not affected by dietary fibre level. The proportion of faecal N which was of endogenous origin was similar to that in ileal digesta, suggesting similar utilization of endogenous and residual dietary N by hindgut bacteria. Half the endogenous N entering the large intestine was reabsorbed there. Increasing dietary crude fibre from 40 to 132 g/kg increased faecal endogenous N excretion from 1.3 to 2.0 g/animal and day.

Animals↗
PubMed1986

Studies on the secretion of amino acids and of urea into the gastrointestinal tract of pigs. 3. Secretion of urea determined by continuous intravenous infusion of 15N-urea.

Three pigs, of 34 kg live weight, were each fitted with re-entrant cannulas both in the duodenum and terminal ileum and catheters in the jugular vein and in the carotid artery. Pigs received a diet based on wheat and dried skimmed milk in equal amounts at 12 h intervals. During the preliminary period the digesta flowing from both duodenal and ileal cannulas were collected over 12 h after feeding on two consecutive days and half of them were reintroduced into the gut and half were stored at -20 degrees C. During the experimental period 15N-urea was infused into the jugular vein for 12 hours starting with the morning meal. Total amount of urea infused was 5 g containing 1.22 g 15N-excess. The digesta from both proximal duodenal and ileal cannulas were collected and stored, while the digesta from the preliminary period were reintroduced into the respective distal cannulas. Blood samples were taken at different time of infusion. At the end of infusion period the animals were sacrificed and samples of the contents of the digestive tract and tissues were taken. Urea flux calculated according to atom-% 15N-excess of urea N in plasma was 1.23 to 2.37 g/kg body weight/day. In the duodenal digesta 94.5 +/- 0.2 and in ileal digesta 57.1 +/- 7.39 per cent of 15N were in the TCA soluble fraction. The total amount of 15N in the duodenal digesta was 1.7 to 6.3 times greater than in the ileal digesta. Only small amount of 15N was found in the caecum and almost none in the contents of colon and rectum. It is concluded that urea is secreted into all parts of the digestive tract, the main sites of urea secretion being pancreatic juice and/or bile as well as the small intestine. The total amount of urea secreted is assumed to be similar to the daily urea excretion.

Amino Acids↗
PubMed1986

Studies on the secretion of amino acids and of urea into the gastro intestinal tract of pigs. 1. Secretion of leucine into the stomach and upper part of the duodenum during continuous intravenous infusion of 14C-leucine.

Three pigs of 34 kg live weight were fitted with a re-entrant cannula in the duodenum, and with two catheters placed in the jugular vein and carotid artery. They were fed 1.2 kg/d of wheat-dried-skimmed milk diet. Digesta from the proximal duodenal cannula were collected for 12 h on 2 consecutive days; 50% were reintroduced into the respective distal cannula and 50% were stored at -20 degrees C. Three days later 14C-leucine was infused into the jugular vein for 12 h, starting with the morning meal. During this period the digesta from the proximal cannula were collected and stored for analysis while the digesta collected previously were reintroduced into the distal cannula. Blood samples were taken from the carotid artery. The total flow of duodenal digesta in 12 h was 5760 +/- 530 g. On average 70 percent of the radioactivity in digesta was associated with the TCA-precipitable fraction. During hours 0-3 and 11-12 of infusion 60-70% and 96-98% of the radioactivity in the TCA precipitable fractions was in leucine. In the TCA soluble fraction only 30-40 per cent of the radioactivity was associated with leucine. At the plateau 2.1 and 3.3% of infused 14C-leucine and of radioactivity were recovered in the duodenal digesta. The calculated amount of endogenous protein passing the duodenum was 20.4 g/d/pig. The results are discussed in relation to previous studies on protein synthesis and secretion in which 14C- and 15N-amino acids were used.

Amino Acids↗
PubMed1986

Studies on the secretion of amino acids and of urea into the gastro intestinal tract of pigs. 2. Net secretion of leucine into the small and large intestines.

The experiment was carried out on 3 pigs of 34 kg body weight equipped with re-entrant cannulas both in the duodenum and terminal ileum. Furthermore, catheters were inserted into the external jugular vein and into the carotid artery. During a preliminary period digesta from both cannulas were collected and stored. This digesta was than reintroduced during a 12 hours period of intravenous infusion of 14C-leucine while outflowing digesta from the proximal parts of the cannulas were collected, stored and analyzed for leucine content, 14C-radioactivity and specific radioactivity of leucine. This paper reports the net secretion of 14C-activity and of 14C-leucine by the small intestine and the recovery of both in the content of the digestive tract at the end of the infusion. It was concluded that endogenous leucine enters the lumen of the small intestine through the intestinal wall mainly in a protein bound form. Free leucine is secreted, however, only in small amounts. Metabolites of leucine were also secreted into or formed in the lumen of the small intestine. A proportion of endogenous leucine was reabsorbed during the passage of digesta along the small intestine.

Amino Acids↗