Biomedical subjects

I Miksík

Publications and source records attributed to I Miksík.

At least 19 recordsLinked to original sources

PubMed2006

Corticosterone metabolism in chicken tissues: evidence for tissue-specific distribution of steroid dehydrogenases.

Glucocorticoids influence the function of numerous tissues. Although there are a very large number of studies that have investigated the local metabolism of glucocorticoids in mammals, the knowledge of this metabolism in birds is limited. The local concentration of corticosterone is critical for both glucocorticoid- and mineralocorticoid-dependent activity, and we have therefore carried out studies of corticosterone metabolism in various chicken organs. It was found that corticosterone was metabolized to 20-dihydrocorticosterone, and in some tissues also to 11-dehydrocorticosterone and 11-dehydro-20-dihydrocorticosterone. The activity of 20-hydroxysteroid dehydrogenase (20HSD), responsible for the transformation of corticosterone to 20-hydroxy derivatives, was abundant in the kidney and intestine, with lower levels in the liver and testis. Low levels of 20HSD were detected in the brain and ovaries. In contrast, 11-hydroxysteroid dehydrogenase (11HSD) activity was only found in the kidney and intestine. No activity was observed in the brain, testis, or ovaries. The treatment of chickens with estrogens stimulated 20HSD activity in the kidney, intestine, and oviduct and 11HSD activity in the liver and oviduct. Kinetic studies for corticosterone yielded an apparent Km for 11HSD in the nanomolar (Km = 21 +/- 5 nmol.l(-1)) and for 20HSD in the micromolar range (Km = 3.7 +/- 0.3 micromol.l(-1)). When progesterone or 5alpha-dihydrotestosterone were used instead of corticosterone, the tissues reduced the former to 20beta-dihydroprogesterone and the latter to both 5alpha,3alpha- and 5alpha,3beta-dihydrotestosterone. The data presents the first evidence for corticosterone metabolism via 11beta-, 3alpha/3beta-, and 20beta-hydroxysteroid dehydrogenases in various chicken organs and provide support for the theory of prereceptor modulation of glucocorticoid signals in avian tissues.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed2006

Separation of poly(amidoamine) (PAMAM) dendrimer generations by dynamic coating capillary electrophoresis.

The separation of compounds possessing amino groups (peptides, proteins, polyamino compounds) by capillary zone electrophoresis suffers from the interaction (sticking) of these solutes with the capillary wall. This sticking can result in the absence or incomplete separation of compounds or even in their retention in the capillary. Polyamidoamine (PAMAM) dendrimers are a class of spherical polymers with primary amino groups at the surface. These compounds can be separated reasonably well at acidic pH but not at neutral pH. A new method based on the dynamic coating of the capillary was developed for the separation of these compounds at pH 7.4. The method comprises separation in a fused-silica capillary (57 cm total length, 50 cm to the detector, ID 75 microm) and a background electrolyte consisting of a Tris-phosphate buffer (50 mmol/L, pH 7.4) and 0.05% (w/v) polyethyleneimine. This system is suitable for the separation of 7 generations of dendrimers (generations 0-6). The dynamic coating agent (polyethyleneimine) also improves the separation at acid pH.

Dendrimers↗

PubMed2006

Cloning and expression of chicken 20-hydroxysteroid dehydrogenase.

The ligand specificity and activation of steroid receptors depend considerably on the enzymatic activities involved in local pre-receptor synthesis and the metabolism of the steroids. Several enzymes in particular, steroid dehydrogenases have been shown to participate in this process. Here we report the isolation of 20-hydroxysteroid dehydrogenase (ch20HSD) cDNA from chicken intestine and the distribution of ch20HSD mRNA and 20-reductase activity in various avian tissues. Using a reverse transcription PCR and comparison with the known sequences of mammalian 20betaHSDs, we have isolated a new ch20HSD cDNA. This cDNA predicted 276 amino acid residues that shared about 75% homology with mammalian 20betaHSD. Sequences specific to the short-chain dehydrogenase/reductase superfamily (SDR) were found, the Gly-X-X-X-Gly-X-Gly cofactor-binding motif (residues 11-17) and the catalytic activity motif Tyr-X-X-X-Lys (residues 193-197). The cDNA coding for ch20HSD was expressed in Escherichia coli by placing it under isopropylthiogalactoside (IPTG) inducible control. Both the IPTG cells of E. coli and the isolated recombinant protein reduced progesterone to 20-dihydroprogesterone, corticosterone to 20-dihydrocorticosterone and 5alpha-dihydrotestosterone to its 3-ol derivative. The 20-reductase and 3-reductase activities of ch20HSD catalyzed both 3alpha/beta- and 20alpha/20beta-epimers. The mRNA transcripts of ch20HSD were found in the kidney, colon, and testes; weaker expression was also found in the heart, ovaries, oviduct, brain, liver, and ileum. 20-Reductase activity has been proven in tissue slices of kidney, colon, ileum, liver, oviduct, testis, and ovary; whereas the activity was nearly absent in the heart and brain. A similar distribution of 20-reductase activity was found in tissue homogenates measured under V(max) conditions. These results suggest that chicken 20HSD is the latest member of the SDR superfamily to be found, is expressed in many avian tissues and whose precise role remains to be determined.

20-Hydroxysteroid Dehydrogenases↗

PubMed2006

Intestinal inflammation modulates expression of 11beta-hydroxysteroid dehydrogenase in murine gut.

The effect of glucocorticoids is controlled at the pre-receptor level by the activity of 11beta-hydroxysteroid dehydrogenase (11HSD). The isoform 11HSD1 is an NADP+ -dependent oxidoreductase, usually reductase, that amplifies the action of glucocorticoids due to reduction of the biologically inactive 11-oxo derivatives cortisone and 11-dehydrocorticosterone to cortisol and corticosterone. The NAD+ -dependent isoform (11HSD2) is an oxidase that restrains the effect of hormones due to 11beta-oxidation of cortisol and corticosterone to their 11-oxo derivatives. Although the immunosuppressive and anti-inflammatory effects of glucocorticoids are well known, the relationship between inflammation and local metabolism of glucocorticoids is not well understood. In this study, we demonstrated that colitis induced by dextran sulfate sodium modulates colonic 11HSD1. Experimentally induced intestinal inflammation stimulated colonic NADP+ -dependent but not NAD+ -dependent 11HSD activity. Colonic 11HSD1 mRNA was increased, whereas 11HSD2 mRNA was not changed. Additional parallel studies revealed a similar pattern of 11HSD1 mRNA induction in mesenteric lymph nodes and intestinal intraepithelial lymphocytes, but not in spleen and peritoneal macrophages. These data suggest that inflammation modulates local metabolism of glucocorticoid and support the notion that pre-receptor regulation of endogenous corticosteroids might play a role in inflammatory processes.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed2004

Capillary electrophoretic separation of proteins and peptides by ion-pairing with heptanesulfonic acid.

Heptanesulfonic acid as ion-pairing agent was used for the separation of mixtures of low and high molecular mass peptides/proteins by capillary electrophoresis. The separation conditions used were: capillary 37 cm (30 cm to the detector) x 75 microm i.d., voltage 10 kV, phosphate buffer 50 mmol/l, ion-pairing agent heptanesulfonic acid at three different concentrations, namely, 0, 20 or 100 mmol/l, pH 2.5. The separation reflected the ion-pairing equilibria between the ion-pairing agent and the peptide/protein analytes. The influence of ion-pairing on sample mobility (running time) was more pronounced in case of the higher-molecular peptides as compared to the low molecular ones. This difference offers the possibility to separate low and high molecular peptides/proteins that under the absence of the ion-pairing agent would co-migrate. The principle of this approach was demonstrated on a randomly selected set of peptides/proteins; the practical applicability was demonstrated on a set of CNBr peptides arising from a naturally occurring mixture of collagen types I and III.

Animals↗

PubMed2004

Corticosteroid regulation of colonic ion transport during postnatal development: methods for corticosteroid analysis.

Many mammalian species including human are immature at birth and undergo major developmental changes during suckling and weaning period. This problem is also conspicuous for the gastrointestinal tract that undergoes abrupt transitions coinciding with birth and weaning. This review deals with the maturation of ion transport functions in colon, the intestinal segment that plays an important role in sodium and potassium absorption and secretion. The purpose of the present review is to summarize the mechanism of sodium and potassium transport pathways and show how these transport processes change postnatally and how hormones, particularly corticosteroids, modify the pattern of development. Finally we describe some of the ways, how to analyze corticosteroid metabolism in target tissue.

Adrenal Cortex Hormones↗

PubMed2003

Preparative procedures and purity assessment of collagen proteins.

Collagens represent a large family (25 members identified so far) of closely related proteins. While the preparative procedures for the members that are ubiquitous and present in tissues in large quantities (typically fibre and network forming collagens types I, II, III, IV and V) are well established, the procedures for more recently discovered minor collagen types, namely those possessing large non-collagenous domain(s) in their molecule, are mostly micropreparative and for some collagenous proteins even do not exist. The reason is that the proof of their existence is based on immunochemical staining of tissue slices and nucleic database searching. Methods of preparation and identification of constituting alpha-polypeptide chains as well as collagenous and non-collagenous domains are also reviewed. Methods for revealing non-enzymatic posttranslational modifications (particularly of the fibre forming collagen types) are briefly described as well.

Chromatography, Liquid↗

PubMed2001

Evaluation of peptide electropherograms by multivariate mathematical-statistical methods. I. Principal component analysis.

Depository effects in slowly metabolised proteins, typically glycation or the estimation of products arising from the reaction of unsaturated long-chain-fatty acid metabolites (possessing aldehydic groups) are very difficult to assess owing to their extremely low concentration in the protein matrix. In order to reveal such alterations we applied deep enzymatic fragmentation resulting in a set of small peptides, which, if modified, are likely to change their electrophoretic properties and can be visualised on the resulting profile. Peptide maps of collagen (a mixture of collagen types I and III digested by bacterial collagenase) were applied as the model protein structure for detecting the nonenzymatic posttranslational changes originating during various physiological conditions like high fructose diet and hypertriglyceridemic state. Capillary electrophoresis in acidic media (sodium phosphate buffer, pH 2.5) was used as the separation method capable of (partial) separation of over 60 peptide peaks. Two to 13 changes were revealed in the profiles obtained reflecting the physiological conditions of the animals tested. Combination of peptide profiling with subsequent t-test evaluation of individual peak areas and principal component analysis based on cumulative peak areas of individual sections of the electropherograms allowed to determine in which section (part) of the electropherogram the physiological state indicating changes occurred. Simultaneously it was possible to reveal the qualitative differences between the four physiological regimes investigated (i.e., which regime affects the collagen molecules most and which affects them least). The approach can be used as guidance for targeted preseparation of the very complex peptide mixture.

Animals↗

PubMed2001

Binding of substituted phenol and aniline derivatives to the corn protein zein studied by high-performance liquid chromatography.

The interaction of 12 substituted phenol, three aminophenol and four substituted aniline derivatives with the corn protein zein was studied on zein-coated silica and alumina stationary phases by high-performance liquid chromatography using bidistilled water as mobile phase. Solutes were eluted from the zein-coated supports with different retention times indicating that they bind to the protein with different forces. They were more strongly retained on silica-based than on alumina-based support proving that the original adsorptive character of the support remains even after impregnation. The retention of solutes on both zein-coated stationary phases significantly depended on the steric and electronic parameters of solutes and was independent of the calculated and measured lipophilicity parameters, indicating that hydrophobic forces are not included in the interaction of zein with these class of solutes. It has been concluded that the interaction is governed by steric and electrostatic forces.

Aniline Compounds↗

PubMed2001

Effect of molecular parameters on the binding of phenoxyacetic acid derivatives to albumins.

The interaction of 12 phenoxyacetic acid derivatives with human and serum albumin as well as with egg albumin was studied by charge-transfer reversed-phase (RP) thin-layer chromatography (TLC) and the relative strength of interaction was calculated. Each phenoxyacetic acid derivative interacted with human and bovine serum albumins whereas no interaction was observed with egg albumin. Stepwise regression analysis proved that the lipophilicity of the derivatives exert a significant impact on their capacity to bind to serum albumins. This result supports the hypothesis that the binding of phenoxyacetic acid derivatives to albumins may involve hydrophobic forces occurring between the corresponding apolar substructures of these derivatives and the amino acid side chains.

Acetates↗

PubMed2000

Comparison of different electrokinetic separation modes applicable to a model peptide mixture (collagen type I and III CNBr fragments).

A number of electromigration separation modes were applied to the separation of CNBr-released peptides from rat tail tendon collagen (microemulsion electrokinetic chromatography, methanol- or ethanol-modified background electrolytes and the separation in the presence of molecular sieving effect exerting polymer, both in the presence and absence of SDS). Electrodriven separations with a Hypersil C8 packed capillary were investigated as well. The best separations were obtained with either the molecular sieving effect exerting polymer (polyethylene oxide) in the background electrolyte (whether SDS was present or absent) or with the electrodriven chromatography using the C8 reversed-phase packed capillary. In the latter separation system, it was possible to separate 25-27 peaks of the theoretically expected 24 peptides in the analyzed mixture of which 17 were at least tentatively identified. The additional peaks apparently stem from the incomplete cleavage of the parent collagen alpha chains. Successful separations can be done either with predominating molecular sieving or hydrophobic partitioning mechanism.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2000

Capillary electrophoretic separation of proteins and peptides using Pluronic liquid crystals and surface-modified capillaries.

Separation of model mixtures of peptides/proteins carried out in a hydrophilically coated capillary in 10 mmol/l Tris and 75 mmol/l phosphate buffer containing 7.5% (w/w) Pluronic F127 copolymer (apparent pH 2.9) revealed that the separation is predominantly driven by the charge/mass ratio with little or no sieving effect. Using a coated capillary helped to remove current fluctuations that are observed in the fused-silica capillaries in the presence of the Pluronic copolymer. With peptides bearing distinct positive charge (polylysine of Mr around 3300) molecular sieving helps more detailed separation of individual species. Polyamino acids carrying negative charge can be brought to the detector window in the reversed polarity mode, however, no detailed separation of the individual species involved was observed under the conditions used. With a naturally occurring mixture of collagen fragments released by CNBr treatment of the protein the sequence of emerging peptides (positive polarity mode) with no relation to the rel. mol. mass could be revealed. It is concluded that separation of proteins/peptides in the presence of Pluronic in the background electrolyte occur on the charge/mass ratio basis with molecular sieving effects acting as a secondary partition mechanism.

Electrophoresis, Capillary↗

PubMed2000

External electric field control of electroosmotic flow in non-coated and coated fused-silica capillaries and its application for capillary electrophoretic separations of peptides.

The influence of an external electric field on the electroosmotic flow in the noncoated (bare) fused-silica capillaries and in the fused-silica capillaries with covalent coating of the inner surface by the polymer of a new acrylamido derivative, N-(acryloylaminoethoxy)ethyl-beta-D-glucopyranose, has been tested in the capillary electrophoretic separations of peptide analytes. The effect of magnitude and polarity of the external electric field on the flow-rate of the electroosmotic flow, the migration times of charged analytes and the separation efficiency and resolution of separations of synthetic oligopeptides, diglycine, triglycine, glycyl-proline and prolyl-glycine, by capillary zone electrophoresis has been evaluated. The effect of the external electric field on the velocity of the electroosmotic flow was much higher in the bare fused-silica capillaries than in the coated capillaries. Better separation of the analyzed peptides was achieved in the coated fused-silica capillaries. An external electric field proved to be an effective tool for control of the electroosmotic flow and for optimization of the speed and resolution of capillary electrophoretic separations of synthetic peptides.

Electrophoresis, Capillary↗

PubMed2000

Capillary electrophoretic separation of vitamins in sodium dodecyl sulfate containing buffers with lower aliphatic alcohols and n-hexane as organic modifiers.

The effect of lower organic alcohols as co-surfactants (methanol, ethanol, n-propanol, isopropanol, propanediol, n-butanol and isoamylalcohol) and n-hexane as an organic modifier in 12.5 mol/l phosphate buffer with varying SDS concentration was investigated using a set of vitamins and p-hydroxybenzoic acid as the test mixture. It was demonstrated that optimum separations can be achieved particularly at high concentrations of the surfactant; the selectivity can be changed by adding a co-surfactant; while propanol and isopropanol show the same properties as co-surfactants, the most efficient alcohols were isoamylalcohol and propanediol. n-Butanol was capable of selective separation of p-hydroxybenzoic acid in the test mixture. Addition of ethanol appears most effective at higher concentrations (while all the other alcohols are effective already at 5% concentration, the best results with ethanol were obtained when it constituted 20% of the background electrolyte). 5% Concentration of methanol resulted in poor separation of the test mixture, however if 300 microl/10 ml of hexane were added to 20 mmol/l SDS containing phosphate buffer, the resulting separation was practically the same as with 50 mmol/l SDS.

Alcohols↗

PubMed2000

Advanced separation methods for collagen parent alpha-chains, their polymers and fragments.

Current techniques used for collagen alpha-chains and their CNBr fragments are reviewed. Ion exchange, gel permeation, reversed-phase and affinity chromatography are discussed mainly from the preparative aspects as these are both the techniques of choice to remove biological matrix contaminants always present in collagen preparations and techniques routinely used for preparative purposes. Among electromigration procedures gel electrophoresis is widely used both for intact collagen alpha-chains and their fragments. Recently this technique was applied also for miniaturised preparations. Immunoblotting techniques serve more specific detection of otherwise hard to distinguish different collagen polypeptide chains. Capillary electromigration techniques brought recently new aspects of understanding the behaviour of collagen proteins upon different separation modes and seem to represent a smart perspective for better quantitation of individual collagen species.

Animals↗

PubMed2000

11Beta-hydroxysteroid dehydrogenase activity in spontaneously hypertensive and Dahl rats.

The role of the enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD) in hypertension remains unknown even if it appears that the inappropriately decreased 11betaHSD activity might be involved in a process that leads to high blood pressure. The possible changes of 11betaHSD were therefore investigated in rats with spontaneous or salt-induced hypertension. The adult male rats of the following genotypes were used: spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto rats (WKY), Dahl salt-sensitive rats fed either a high-salt diet containing 8% NaCl (DS-HS) or low-salt diet containing 0.2% NaCl (DS-LS), and Dahl salt-resistant rats fed the same diets (DR-HS, DR-LS). 11betaHSD was investigated in colon, aorta, renal cortex, and renal medulla and was assessed as percentage conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone in the presence of NAD or NADP. The results demonstrated that genotype exerts a significant effect on 11betaHSD. 11betaHSD activity was significantly increased in colon and renal medulla of SHR compared with WKY rats. No significant differences were observed in renal cortex and aorta. In Dahl rats kept on a low-salt diet, 11betaHSD activity was significantly higher in colon, renal medulla, and cortex of DS-LS than in DR-LS rats but no difference was observed in aorta. The differences disappeared in age-matched DS and DR rats fed the high-salt diet. Increased dietary sodium intake stimulated the activity of 11betaHSD in renal cortex and medulla of DR rats and decreased the activity in colon of DS rats. We conclude that the development of spontaneous and salt-induced hypertension is not associated with decreased activity of 11betaHSD. However, the results showed that salt intake is able to modulate the activity of 11betaHSD and that 11betaHSD in DS and DR rats responds to high dietary salt intake in a different manner.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1999

Binding of lead to collagen type I and V and alpha2(I) CNBr (3,5) fragment by a modified Hummel-Dreyer method.

Binding of lead (as lead acetate) to collagen type I alpha, and alpha2 chains, collagen type V and a large cyanogen bromide fragment of type I collagen [alpha2(I)CB(3,5)] was investigated by the large-zone Hummel-Dreyer method. It was demonstrated that two categories of binding sites exist in the collagen molecule, the number of which correlates rather well with the available aspartic and glutamic acid residues. Similar results were obtained for all collagen chains (fragments) used. The number of sites thus obtained was compared with the cross-striation pattern (reflecting areas where lead is bound) of the SLS form of collagen type I (alpha1 chain); it is suggested that the number of bands seen in the SLS form reflects primarily the number of available aspartic acid residues in the molecule. The association constants obtained are comparable with the low affinity interactions seen e.g., between Cu and bovine serum albumin.

Collagen↗

PubMed1999

Separation of proteins and peptides by capillary electrophoresis in acid buffers containing high concentrations of surfactants.

Separations of proteins at acid pH in the presence of a high concentration of surfactant [sodium laurylsulfate (SDS), 50 mmol/l] was investigated. The purpose of using high concentrations of SDS as background electrolyte modifier was threefold: First, the surfactant exerts a washing effect upon the capillary wall thus preventing binding of analytes and possible clogging of the capillary. Second, it was revealed that even under very acid conditions (below pH 3) the surfactant is capable of forming associates with protein analytes which still bear considerable negative charge and can be separated on this basis. Third, the system can be applied not only for protein mixtures sufficiently soluble in neutral to alkaline media (leukocyte lysates, standard proteins), but it can be used also with proteins, that are under such conditions virtually insoluble and their solubilization is possible in acid buffers only (eggshell proteins or collagen CNBr fragments). The result was that adsorption to the capillary wall was minimized and the analytes were separated as negatively charged associates with high efficiency. With collagen fragments partition was possible on the affinity differences of the peptides to the surfactant micelles and inner wall of the capillary. Theoretical plate counts approaching 100,000 were easily achieved even with proteins which under the more conventional operation conditions exhibit considerable sticking to the capillary wall. The other feature of this system is that the associates move very rapidly to the anode. Owing to the low pH, endoosmotic flow is negligible, and therefore the system has to be operated at reversed polarity.

Buffers↗