Biomedical subjects

Vladimír Kren

Publications and source records attributed to Vladimír Kren.

At least 19 recordsLinked to original sources

PubMed2007

Sucrose feeding during pregnancy and lactation elicits distinct metabolic response in offspring of an inbred genetic model of metabolic syndrome.

The importance of early environment, including maternal diet during pregnancy, is suspected to play a major role in pathogenesis of metabolic syndrome and related conditions. One of the proposed mechanisms is a mismatch between the prenatal and postnatal environments, leading to misprogramming of the metabolic and signaling pathways of the developing fetus. We assessed whether the exposure to high-sucrose diet (HSD) alleviates the detrimental effects of sucrose feeding in later life (predictive adaptive hypothesis) in a highly inbred model of metabolic syndrome, the PD/Cub rat. Rat dams were continuously fed either standard or HSD (70% calories as sucrose) starting 1 wk before breeding, throughout pregnancy, at birth, and until weaning of the offspring. After weaning, all male offspring were fed HSD until the age of 20 wk, when detailed metabolic and morphometric profiles were ascertained. The early life exposure to a sucrose-rich diet resulted in distinct responses to longtime postnatal HSD feeding. Offspring of the sucrose-fed mothers displayed higher adiposity and substantial increases in triglyceride liver content together with unfavorable distribution of cholesterol into lipoprotein subfractions. On the other hand, their adiponectin concentrations were significantly higher, and insulin sensitivity of skeletal muscle was enhanced compared with the offspring of standard diet-fed mothers. Triglycerides, free fatty acids, overall glucose tolerance, and the insulin sensitivity of adipose tissue were comparable in both groups. In the genetically identical animals, maternal HSD feeding elicited a variety of subtle effects but did not lead to predictive adaptive protection from most HSD-induced metabolic derangements.

Animals↗

PubMed2006

Purification and characterization of a nitrilase from Aspergillus niger K10.

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg(-1)) at 45 degrees C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of D: -sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.

Amino Acid Sequence↗

PubMed2006

Induction and characterization of an unusual alpha-D-galactosidase from Talaromyces flavus.

An extracellular alpha-d-galactosidase from Talaromyces flavus CCF 2686 with extremely broad and unusual acceptor specificity is produced exclusively in the presence of the specific inducer--6-deoxy-D-glucose (quinovose). The procedure for the preparation of this very expensive substance has been modified and optimized. Surprisingly, any of other common alpha-D-galactosidase inducers or substrates, e.g., D-galactose, melibiose and raffinose, did not stimulate its production. The crude alpha-D-galactosidase preparation was purified by anion-exchange chromatography and three isoenzymes with different substrate specificities were identified. The main isoenzyme (alphaGal1) was further purified by cation-exchange chromatography and fully characterized. When compared with other alpha-galactosidases and also with other isoenzymes produced by T. flavus, it showed a markedly different regioselectivity and also negligible hydrolytic activity towards melibiose. Moreover, it was active on polymeric substrates (locust bean gum, guar gum) and significantly inhibited by alpha-D-galactopyranosyl azide, D-galactose, D-xylose, melibiose, methyl alpha- and beta-D-galactopyranoside and lactose.

Deoxyglucose↗

PubMed2006

Novel double-congenic strain reveals effects of spontaneously hypertensive rat chromosome 2 on specific lipoprotein subfractions and adiposity.

We have developed a new, double-congenic rat strain BN-Lx.SHR2, which carries two distinct segments of chromosome 2 introgressed from the spontaneously hypertensive rat strain (SHR) into the genetic background of congenic strain BN-Lx, which was previously shown to express variety of metabolic syndrome features. In 16-wk-old male rats of BN-Lx and BN-Lx.SHR2 strains, we compared their glucose tolerance and triacylglycerol and cholesterol concentrations in 20 lipoprotein subfractions and the lipoprotein particle sizes under conditions of feeding standard and high-sucrose diets. Introgression of two distinct SHR-derived chromosome 2 segments resulted in decreased adiposity together with aggravation of glucose intolerance in the double-congenic strain. The BN-Lx.SHR2 rats were more sensitive to sucrose-induced rise in triacylglycerolemia. Although the total cholesterol concentrations of the two strains were comparable after the standard diet and even lower in BN-Lx.SHR2 after sucrose feeding, detailed analysis revealed that under both dietary conditions, the double-congenic strain had significantly higher cholesterol concentrations in low-density lipoprotein fractions and lower high-density lipoprotein fractions. We established a new inbred model showing dyslipidemia and mild glucose intolerance without obesity, attributable to specific genomic regions. For the first time, the chromosome 2 segments of SHR origin are shown to influence other than blood pressure-related features of metabolic syndrome or to be involved in relevant nutrigenomic interactions.

Adipose Tissue↗

PubMed2006

Preparative production and separation of 2-acetamido-2-deoxymannopyranoside-containing saccharides using borate-saturated polyolic exclusion gels.

A new separation method based on the combination of exclusion and ion exchange chromatography in borate buffer was developed. It allows semi-preparatory and preparatory separation of isobaric N-acylhexosamines (C-2 epimers) and corresponding methyl glycosides (anomers and tautomers). Three types of polyolic gels were tested for these separations. Ion-exchange HPLC was used as a rapid and reliable method for the quantification of the respective analytes. NMR studies of the interactions of N-acetylhexosamines with borate confirmed the importance of a proper stereochemical arrangement of acetamido sugars for their interactions with borate anions.

Borates↗

PubMed2006

Extension of the Nef reaction to C-glycosylnitromethanes.

Acid-catalysed methanolysis of 3,4,5,6-tetra-O-acetyl-1,2-dideoxy-l-arabino-hex-1-enitol proceeds via a cascade set of consecutive reactions resulting in its regiospecific conversion to a mixture of alpha- and beta-C-L-arabinofuranosylmethanal dimethyl acetals and a mixed internal methyl acetal. Structures of the final products of the overall process provide unique evidence that a kinetically controlled, five-membered-ring closure precedes a six-membered-ring closure in reversible systems capable of giving both five-membered and six-membered all-sp3-atom rings. Determination of the reaction intermediate enabled extension of the Nef reaction to C-glycosylnitromethanes. Protonated aci-nitro forms of C-glycosylnitromethanes that are resistant to the Nef reaction in aqueous acidic media undergo a modified Nef reaction in acidified methanol, and the corresponding C-glycosylmethanal dimethyl acetals with alpha-L-arabinopyranosyl, beta-D-glucopyranosyl, beta-D-galactopyranosyl, beta-D-mannopyranosyl and beta-L-rhamnopyranosyl configurations were obtained in moderate yields.

Kinetics↗

PubMed2006

New derivatives of silybin and 2,3-dehydrosilybin and their cytotoxic and P-glycoprotein modulatory activity.

Large series of O-alkyl derivatives (methyl and benzyl) of silybin and 2,3-dehydrosilybin was prepared. Selective alkylation of the silybin molecule was systematically investigated. For the first time we present here, for example, preparation of 19-nor-2,3-dehydrosilybin. All prepared silybin/2,3-dehydrosilybin derivatives were tested for cytotoxicity on a panel of drugs sensitive against multidrug resistant cell lines and the ability to inhibit P-glycoprotein mediated efflux activity. We have identified effective and relatively non-cytotoxic inhibitors of P-gp derived from 2,3-dehydrosilybin. Some of them were more effective inhibitors at concentrations lower than a standard P-gp efflux inhibitor cyclosporin A. Another group of 2,3-dehydrosilybin derivatives also had better inhibitory effects on P-gp efflux but a cytotoxicity comparable with that of parent 2,3-dehydrosilybin. Structural requirements for improving inhibitory activity and reducing toxicity of 2,3-dehydrosilybin were established. Effect of E-ring substitution as well as an influence of the substituent size at the C-7-OH position of A-ring on P-gp-inhibitory activity was evaluated for the first time in this study.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Effects of silymarin flavonolignans and synthetic silybin derivatives on estrogen and aryl hydrocarbon receptor activation.

Silymarin, a standardized mixture of flavonolignans, or its major constituents could be effective for prevention and treatment of hepatic damage or skin cancer. However, their potential side effects, such as modulation of endocrine functions via the disruption of estrogen receptor (ER) and/or aryl hydrocarbon receptor (AhR) activation, are largely unknown. In the present study, we investigated impact of silymarin, its constituents and a series of their synthetic derivatives on ER- and AhR-mediated activities using in vitro reporter gene assays. We found that none of the compounds under study affected the AhR-mediated activity in rat hepatoma cells. Contrary to that, several compounds behaved as either partial or full ER agonists. Silymarin elicited partial ER activation, with silybin B being probably responsible for a majority of the weak ER-mediated activity of silymarin; silybin A and other flavonolignans were found to be inactive and potent ER agonist taxifolin is only a minor constituent of silymarin. To our knowledge, this is probably the first time, when receptor-specific in vitro effects of separated diastereomers have been demonstrated. In contrast to silymarin constituents, the synthetic silybin derivatives, potentially useful as chemoprotective agents, did not modulate the ER-mediated activity, with exception of 23-O-pivaloylsilybin. Interestingly, 7-O-benzylsilybin potentiated ER-mediated activity of 17beta-estradiol despite possessing no estrogenic activity. In conclusion, our data suggest that estrogenicity of some silymarin constituents should be taken in account as their potential side effect when considered as chemopreventive compounds. These results also stress the need to study biological activities of purified or synthesized diastereomers of silybin derivatives.

Animals↗

PubMed2005

Genetic analysis of complex cardiovascular traits in the spontaneously hypertensive rat.

Identification of the genetic determinants of common diseases is a major challenge for current biomedical research. Combining linkage analyses of essentially monogenic cis-regulated expression phenotypes with oligogenic intermediate physiological phenotypes represents a promising approach for identification of quantitative trait loci at the molecular level. In the present review, a genetic analysis of cardiovascular phenotypes studied at several levels of complexity in rat recombinant inbred strains is described.

Animals↗

PubMed2005

A chemoenzymatic route to mannosamine derivatives bearing different N-acyl groups.

The chemoenzymatic route to 2-deoxy-2-propionamido-D-mannose (1b), 2-butyramido-2-deoxy-D-mannose (2b) and 2-deoxy-2-phenylacetamido-D-mannose (3b) involved N-acylation of 2-amino-2-deoxy-D-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido-D-glucose (1a), 2-butyramido-2-deoxy-D-glucose (2a) and 2-deoxy-2-phenylacetamido-D-glucose (3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy-D-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 degrees C and 8.0-9.0, respectively. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose and 2-acetamido-2-deoxy-6-O-sulfo-D-glucose but not 2-acetamido-2-deoxy-1-O-phospho-D-glucose or 2-acetamido-2-deoxy-D-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al3+, Ca2+, Co2+, Cu2+, Mn2+ or Zn2+ and activated by 1 mM Mg2+. The concentrated enzyme is highly stable at 4 degrees C in the presence of 0.1 M ammonium sulfate.

Acylation↗

PubMed2005

Silybin and silymarin--new effects and applications.

This article aims to review critically literature published mainly within this millennium on the new and emerging applications of silymarin, the polyphenolic fraction from the seeds of Silybum marianum and its main component silybin. Silymarin and silybin used so far mostly as hepatoprotectants were shown to have other interesting activities as e.g., anticancer and canceroprotective. These activities were demonstrated in a large variety of illnesses of different organs as e.g., prostate, lungs, CNS, kidneys, pancreas and others. Besides the cytoprotective activity of silybin mediated by its antioxidative and radical-scavenging properties also new activities based on the specific receptor interaction were discovered--e.g., inhibition and modulation of drug transporters, P-glycoproteins, estrogenic receptors, nuclear receptors and some others. New derivatives of silybin open new ways to its therapeutic applications. Pharmacology dealing with optically pure silybin diastereomers may suggest new mechanisms of its action.

Humans↗

PubMed2005

The efficacy of glucosamine and chondroitin sulfate in the treatment of osteoarthritis: are these saccharides drugs or nutraceuticals?

This review summarizes recent knowledge on the efficacy of glucosamine (GS) and/or chondroitin sulfate (CS) in the therapy of mild to moderate osteoarthritis (OA). OA, the most common joint disease is a significant source of disability, quality of life impairment and a considerable burden to any health care system. In the Czech Republic, glucosamine sulfate (GS) and chondroitin sulfate (CS) are available both as prescription drugs and as food supplements. Based on available data both are useful in the earlier stages of OA when combined with other modalities such as weight loss and exercises. They appear to relieve pain and improve range of the joint motion. In addition, they also display mild anti-inflammatory effects. However, controversy still exists over their ability to change significantly the natural history of the osteoarthritic joint. This effect is not easy to demonstrate for any other treatment modalities apart from joint replacement. Monitoring the cure efficacy by X-ray has been recently criticised and hence future techniques are anticipated for this reason. Further, long-term oral administration is required to obtain slightly increased levels of GS and/or CS in human blood. Both reviewed saccharides are well tolerated with negligible adverse reactions. In conclusion, the authors suggest that GS and CS should be classified as food supplements only.

Chondroitin Sulfates↗

PubMed2004

Oxidised derivatives of silybin and their antiradical and antioxidant activity.

Carboxylic acids derived from silybin (1) and 2,3-dehydrosilybin (2) with improved water solubility were prepared by selective oxidation of parent compounds and a new inexpensive method for preparation of 2,3-dehydrosilybin from silybin was developed and optimised. The antioxidative properties of the above-mentioned compounds and of side product 3a from oxidation of compound 1 were determined by cyclic voltammetry, free radical scavenging (DPPH, superoxide) assays, and by inhibition of in vitro generated liver microsomal lipid peroxidation. Dehydrogenation at C((2))-C((3)) in flavonolignans (silybin vs 2,3-dehydrosilybin; silybinic acid vs 2,3-dehydrosilybinic acid) strongly improved antioxidative properties (analogously as in flavonoids taxifolin vs quercetin). Thus, in antioxidative properties, dehydrosilybin was superior to silybin by one order, but its water solubility is too low for application in aqueous milieu. On the other hand, 2,3-dehydrosilybinic acid is a fairly soluble derivative with antilipoperoxidation and antiradical activities better than that of silybin.

Animals↗

PubMed2004

Generation of an alpha-L-rhamnosidase library and its application for the selective derhamnosylation of natural products.

A screening of 16 different fungal strains was performed under different cultivation conditions, using L-rhamnose or L-rhamnose-containing flavonoid glycosides (rutin, hesperidin, and naringin) as specific inducers. No significant constitutive production of alpha-L-rhamnosidases was detected in noninduced cultures, while high levels of these glycosidase activities were obtained using different inducers. New species, so far unknown for the production of alpha-L-rhamnosidases, were identified. More than 30 different alpha-L-rhamnosidase samples were prepared by ammonium sulfate precipitation. Substrate specificity of this alpha-L-rhamnosidase library was tested with various L-rhamnose-containing natural compounds (flavonoids, terpenoids, and saponins). Most of the enzymatic preparations showed broad substrate specificity, and some of them were also acting on sterically hindered substrates (e.g., quercitrin). The screening of the library under different reaction conditions showed the coexistence, in the same preparation, of more than one alpha-L-rhamnosidase activities with different substrate specificity and different stability towards organic cosolvents. To exploit this enzymatic library for synthetic applications, the presence of contaminating alpha-L-arabinosidases and beta-D-glucosidases was investigated. The latter enzymes were observed in several preparations, while alpha-L-arabinosidase content was generally quite low. The selective derhamnosylation of the saponin desglucoruscin was performed on a preparative scale. The enzyme obtained by rhamnose induction of the Aspergillus niger K2 CCIM strain showed high activity towards this substrate and negligible alpha-L-arabinosidase contamination. Therefore, it was chosen as a catalyst for the selective derhamnosylation reaction, which provided the desired product in 70% yield.

Enzyme Activation↗

PubMed2004

Enzymatic synthesis of complex glycosaminotrioses and study of their molecular recognition by hevein domains.

Hevein, a protein found in Hevea brasiliensis, has a CRD domain, which is known to bind chitin and GlcNAc-containing oligosaccharides. By using NMR and molecular modeling as major tools we have demonstrated that trisaccharides containing GalNAc and ManNAc residues are also recognized by hevein domains. Thus far unknown trisaccharides GlcNAcbeta(1-->4)GlcNAcbeta(1-->4)ManNAc (1) and GalNAcbeta(1-->4)GlcNAcbeta(1-->4)ManNAc (2) were synthesized with the use of beta-N-acetylhexosaminidase from Aspergillus oryzae. This method is based on the rather unique phenomenon that some fungal beta-N-acetylhexosaminidases cannot hydrolyze disaccharide GlcNAcbeta(1-->4)ManNAc (5) contrary to chitobiose GlcNAcbeta(1-->4)GlcNAc (4) that is cleaved and, therefore, cannot be used as an acceptor for further transglycosylation. Both trisaccharides 1 and 2 were prepared by transglycosylation from disaccharidic acceptor in good yields ranging from 35% to 40%. Our observations strongly indicate that the present nature of the modifications of chitotriose (GlcNAcbeta(1-->lcNAcbeta(1-->4)GlcNAc, 3) at either the non-reducing end (GalNAc instead of GlcNAc) or at the reducing end (ManNAc instead of GlcNAc) do not modify the mode of binding of the trisaccharide to hevein. The association constant values indicate that chitotriose (3) binding is better than that of 1 and 2, and that the binding of (with ManNAc at the reducing end) is favored with respect to that of 2 (with ManNAc at the reducing end with a non-reducing GalNAc moiety).

Acetylglucosamine↗

PubMed2004

Fluorescent labelled thiourea-bridged glycodendrons.

GlcNAc-coated glycodendrimers, which are polyvalent glycomimetics, display strong in vitro affinity for the rat natural killer cell protein-1A (NKR-P1A), a C-type lectin-like receptor of natural killer (NK) cells in rats, humans and some strains of mice. Administration of these compounds in vivo results in a substantial increase in the antitumour activity with involvement of the natural cell immunity. To clarify the in vitro and in vivo fate of these molecules, we synthesized labelled glycodendron analogues of the previously studied glycodendrimers. Labelling with fluorescent tags enabled the localization of the glycodendrons in white blood cells, tumours and other tissues by using different imaging techniques such as fluorescence and confocal microscopy. These studies are useful for probing the mechanism of action and fate of artificial ligands and the cell receptors involved.

Animals↗

PubMed2004

In vitro antiplasmodial activities of semisynthetic N,N'-spacer-linked oligomeric ergolines.

Starting from three monomeric ergolines (terguride 1, festuclavine 2, pergolide 3) N,N'-spacer-linked oligomeric derivatives were prepared using different aliphatic or arylalkyl spacers. The compounds have been evaluated for their in vitro antiplasmodial activity against the chloroquine-sensitive strain poW and the chloroquine-resistant clone Dd2 of Plasmodium falciparum. Additionally, the cytotoxic effects against mouse fibroblasts (NIH 3T3) in vitro, and human hepatocytes were evaluated. All monomers displayed only a weak antiplasmodial effect, but N-1,N-1'-spacer-linked dimerization substantially enhanced their antiplasmodial activity. The best activities were observed for compounds showing a distance of six carbon atoms between two monomers, which can be obtained by aliphatic or p-xylene linkers. The N-6,N-6'-spacer-linked depropylpergolide dimer 3i exhibited the highest antiplasmodial activity of all compounds tested (IC(50) values: 0.14 and 0.13 microM against poW and Dd2, respectively). Unfortunately, it displayed toxic effects against the mouse fibroblast cell line NIH 3T3 (IC(50): 0.1+/-0.09 microM) and also against human hepatocytes at 100 microM (LDH-leakage: 15.58+/-0.87 microkat/L; GSH-level: 8.15+/-0.78 nmol/10(6) cells). However, the N-1,N-1'-spacer-linked trimer of festuclavine (2f), and also the N-1,N-1'-spacer-linked tetramer of terguride (1g) possessed remarkable antiplasmodial activities (IC(50): 0.54 and 1.53 microM, respectively, against Dd2) lacking cytotoxicity.

3T3 Cells↗

PubMed2004

Altered spermatogenesis in the hypodactylous rats.

Germinal epithelium of seminiferous tubules in adult, infertile hypodactylous males displays significant reduction in the number of germ-line cells. Detection of apoptosis in the germ-line cells during postnatal differentiation was performed to elucidate the mechanism of the decreased number of germ cells in the testes of adult rats. Evaluation of DNA fragmentation and expression of activated caspase-3 in germ cells did not confirm marked germ cell death during the onset of spermatogenesis as a main cause of significant reduction of germ cells in Hd/Hd testes of adult males. The primary cause of spermatogenesis defect seems to be rather associated with a disorder in the cell cycle regulation and interrelation of germ-line cells with Sertoli cells.

Animals↗