Biomedical subjects

Daniel Horák

Publications and source records attributed to Daniel Horák.

13 recordsLinked to original sources

PubMed2006

Isolation of polymerase chain reaction-ready bacterial DNA from Lake Baikal sediments by carboxyl-functionalised magnetic polymer microspheres.

Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (P(HEMA-co-EDMA)) microspheres were used for the isolation of polymerase chain reaction (PCR)-ready DNA from samples of Baikal sediments. DNA was isolated using the phenol extraction method or the Soil DNA Isolation Kit. The occurrence of false-negative results in PCR caused by the presence of extracellular inhibitors in DNA samples was solved using solid phase reversible DNA immobilisation. PCR-ready DNA was reversibly adsorbed to the microspheres in the presence of 8.0% (w/v) poly(ethylene glycol) (PEG 6000) and 2.0M sodium chloride concentrations. The adsorbed DNA was released from the microspheres in a low ionic strength TE buffer. The quality of isolated DNA was checked by PCR amplification.

Bacillus↗

PubMed2006

Functionalized magnetic micro- and nanoparticles: optimization and application to micro-chip tryptic digestion.

The preparation of an easily replaceable protease microreactor for micro-chip application is described. Magnetic particles coated with poly(N-isopropylacrylamide), polystyrene, poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(glycidyl methacrylate), [(2-amino-ethyl)hydroxymethylen]biphosphonic acid, or alginic acid with immobilized trypsin were utilized for heterogeneous digestion. The properties were optimized, with the constraint of allowing immobilization in a microchannel by a magnetic field gradient. To obtain the highest digestion efficiency, sub-micrometer spheres were organized by an inhomogeneous external magnetic field perpendicularly to the direction of the channel. Kinetic parameters of the enzyme reactor immobilized in micro-chip capillary (micro-chip immobilized magnetic enzyme reactor (IMER)) were determined. The capability of the proteolytic reactor was demonstrated by five model (glyco)proteins ranging in molecular mass from 4.3 to 150 kDa. Digestion efficiency of proteins in various conformations was investigated using SDS-PAGE, HPCE, RP-HPLC, and MS. The compatibility of the micro-chip IMER system with total and limited proteolysis of high-molecular-weight (glyco)proteins was confirmed. It opens the route to automated, high-throughput proteomic micro-chip devices.

Amyloid beta-Peptides↗

PubMed2006

Functional polymer hydrogels for embryonic stem cell support.

Embryonic stem (ES) cells are pluripotent cells with the ability to differentiate among all embryonic and adult cell lineages. Derivation of human ES cells opened up the way for treatment of many serious disorders by stem cell-based transplantation therapy. One of the most exciting challenges in development of transplantation therapies is to repair the damaged part of the organ or tissue by transplantation of undifferentiated ES cells or their differentiated derivatives within three-dimensional polymer scaffold. This method allows both renewal of structure and restoration of function of the organ. To address this issue, new polymer hydrogels were synthesized and tested. Cationic hydrogel slabs were synthesized by bulk radical copolymerization of 2-hydroxyethyl methacrylate (HEMA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) with ethylene dimethacrylate (EDMA) or 1-vinyl-2-pyrrolidone (VP) with N,N'-divinylethyleneurea (DVEU) or EDMA in the presence of saccharose or NaCl as a porogen. Swelling studies of the synthesized copolymers showed a high water content in the swollen state. Biocompatibility was studied with the use of feeder-independent mouse ES cell line D3. Cells grown either on the surface or inside synthesized polymer slabs suggest that the tested slabs are not toxic. The ability of ES cells to proliferate was only partially limited in PHEMA slabs crosslinked with EDMA compared with standard culture conditions. When cultured for a limited period of time, ES cells retained their undifferentiated state independently of properties of the hydrogel slabs, presence or absence of surface charges, type of crosslinking agent and matrix (PHEMA or PVP). Notably, prolonged culture in superporous hydrogel slabs initiated ES cell differentiation. Compared with unmodified PHEMA, the number of proliferating ES cells was still lower in the presence of cationic polymers.

Animals↗

PubMed2006

Magnetic IDA-modified hydrophilic methacrylate-based polymer microspheres for IMAC protein separation.

Preparation of a new type of magnetic non-porous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres with hydrophilic properties containing coupled iminodiacetic acid (IDA) is described. The prepared microspheres were used for the immobilization of Ni(II) or Fe(III) ions to show their application in protein binding studies. Human IgG was bound to magnetic Ni(II)-IDA-modified microspheres and conditions of its adsorption and elution were optimized. Non-specific binding of the protein to magnetic microspheres in the absence of Ni(II) ions was low. Fe(III) ions immobilized on magnetic IDA-modified microspheres were used for the specific binding of porcine pepsin, as a model phosphoprotein. The ability of phosphate buffer to release the adsorbed enzyme from the microspheres and a low adsorption of the dephosphorylated protein indicate the participation of phosphate groups in the pepsin interaction. The elaborated method represents a rapid technique that can be used not only for the separation of proteins but also for analytical purposes.

Adsorption↗

PubMed2005

Ferrite supports for isolation of DNA from complex samples and polymerase chain reaction amplification.

The influence of cobalt ferrite particles, with non-modified or modified surface, on the course of polymerase chain reaction (PCR) was investigated. DNA isolated from bacterial cells of Bifidobacterium bifidum was used in PCR evaluation of magnetic microspheres. The presence of cobalt ferrite particles inhibits PCR amplification. The effect is not dependent on the functional groups of the modifying reagents used (none, amino, carboxyl). Amplification was improved after the magnetic separation of magnetic particles. Proposed indirect method enabled verification of the suitability of designed particles for their application in PCR assays. Magnetic particles coated with alginic acid under high PEG and sodium chloride concentration were used for the isolation of PCR-ready bacterial DNA from various dairy products. DNA was isolated from crude bacterial cell lysates without phenol extraction of samples. Bifidobacterium and Lactobacillus DNAs were identified in dairy products using PCR.

Bifidobacterium↗

PubMed2005

Magnetic hydrophilic methacrylate-based polymer microspheres for genomic DNA isolation.

Carboxyl groups containing magnetic and non-magnetic microspheres were used in solid-phase reversible immobilization (SPRI) of genomic DNA. Magnetic non-porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate)--P(HEMA-co-EDMA), poly(glycidyl methacrylate)--PGMA and P(HEMA-co-GMA) microspheres with hydrophilic properties were prepared by dispersion copolymerization of the respective monomers in the presence of colloidal iron oxides. DNA from chicken erythrocytes and DNA isolated from bacterial cells of Bifidobacterium longum was used for testing of adsorption/desorption properties of magnetic microspheres. The occurrence of false negative results in polymerase chain reaction (PCR) caused by the presence of extracellular inhibitors in DNA samples has been solved using SPRI. The P(HEMA-co-EDMA) and P(HEMA-co-GMA) microspheres were used for isolation of DNA from different dairy products followed by PCR identification of Bifidobacterium strains.

DNA↗

PubMed2005

Methacrylate-based chromatographic media.

This review summarizes the preparation and application of chromatographic separation media based on methacrylate monomers with a major focus on highly crosslinked macroporous beads prepared from 2-hydroxyethyl methacrylate and glycidyl methacrylate, respectively. The effects of process variables such as composition of the polymerization mixture that includes monomers, porogenic solvents, and free radical initiator, suspension stabilizer, reaction temperature, and stirring are detailed for both classical and templated suspension polymerization. In addition, specific features of the preparation of monodisperse beads are also discussed. The performance of methacrylate-based separation media is demonstrated on numerous separations in a variety of chromatographic modes.

Chromatography↗

PubMed2004

Magnetic hydrophilic methacrylate-based polymer microspheres designed for polymerase chain reactions applications.

Magnetic hydrophilic non-porous P(HEMA-co-EDMA), P(HEMA-co-GMA) and PGMA microspheres were prepared by dispersion (co)polymerization of 2-hydroxyethyl methacrylate (HEMA) and ethylene dimethacrylate (EDMA) or glycidyl methacrylate (GMA) in the presence of several kinds of magnetite. It was found that some components used in the preparation of magnetic carriers interfered with polymerase chain reaction (PCR). Influence of non-magnetic and magnetic microspheres, including magnetite nanoparticles and various components used in their synthesis, on the PCR course was thus investigated. DNA isolated from bacterial cells of Bifidobacterium longum was used in PCR evaluation of non-interfering magnetic microspheres. The method enabled verification of the incorporation of magnetite nanoparticles in the particular methacrylate-based polymer microspheres and evaluation of suitability of their application in PCR. Preferably, electrostatically stabilized colloidal magnetite (ferrofluid) should be used in the design of new magnetic methacrylate-based microspheres by dispersion polymerization.

Bifidobacterium↗

PubMed2004

Poly(2-hydroxyethyl methacrylate)-based slabs as a mouse embryonic stem cell support.

Poly(2-hydroxyethyl methacrylate) (PHEMA) crosslinked with ethylene dimethacrylate (EDMA) or N,O-dimethacryloylhydroxylamine (DMHA) was obtained in the form of slabs by bulk radical polymerization. Two porosity-inducing methods were investigated, phase separation using a low-molecular-weight porogen and a salt-leaching technique using NaCl and saccharose. Compared with the phase separation, the salt-leaching created open porous structures with voids of the size and shape of crystallites. To address its potentials in the context of stem cell therapies, undifferentiated mouse embryonic stem cells D3 (ES D3 cells) were seeded on the slabs and analyzed for the ability to grow on different types of non-degradable and/or degradable porous PHEMA hydrogels. The cells were able to proliferate only on PHEMA crosslinked with EDMA or 2 wt% DMHA. In order to assess the effect of gelatin, which is routinely used for ES cell cultures, PHEMA slabs were soaked in gelatin solutions and compared the number of cells on gelatin-treated and untreated slabs 4 days after cell seeding. Surprisingly, the number of cells was only slightly higher on gelatin-treated slabs.

Animals↗

PubMed2003

Immunomagnetic separation and detection of Salmonella cells using newly designed carriers.

Magnetic nonporous poly(HEMA-co-EDMA) and poly(HEMA-co-GMA) microspheres were prepared by dispersion copolymerisation of 2-hydroxyethyl methacrylate (HEMA) and ethylene dimethacrylate (EDMA) or glycidyl methacrylate (GMA) in the presence of magnetite. They were functionalized by polyclonal Salmonella antibodies via the trichlorotriazine method. Salmonella cells were then successfully identified using cultural and polymerase chain reaction (PCR) methods after their immunomagnetic separation. The PCR sensitivity of target cell detection was negatively influenced by the presence of some compounds used in the process of particle preparation. In some cases, magnetic poly(HEMA-co-EDMA) microspheres with immobilized proteinase K were used for degradation of intracellular inhibitors present in Salmonella cells.

Electrophoresis, Agar Gel↗

PubMed2002

Characterization of deoxyribonuclease I immobilized on magnetic hydrophilic polymer particles.

Magnetic bead cellulose particles and magnetic poly(HEMA-co-EDMA) microspheres with immobilized DNase I were used for degradation of chromosomal and plasmid DNAs. Magnetic bead particles were prepared from viscose and magnetite powder. Magnetic poly(HEMA-co-EDMA) microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite. Divalent cations (Mg(2+), Ca(2+), Mn(2+) and Co(2+)) were used for the activation of DNase I. A comparison of free and immobilized enzyme (magnetic bead particles) activities was carried out in dependence on pH and activating cation. The maximum of the activity of immobilized DNase I was shifted to lower pH compared with free DNase I. DNase I immobilized on magnetic bead cellulose was used 20 times in the degradation of chromosomal DNA. Its residual activity was influenced by the nature of activating divalent cation. The immobilized enzyme with decreased activity was reactivated by Co(2+) ions.

Cations, Divalent↗

PubMed2002

Oriented immobilization of galactose oxidase to bead and magnetic bead cellulose and poly(HEMA-co-EDMA) and magnetic poly(HEMA-co-EDMA) microspheres.

In order to obtain an active and stable oxidation reactor for daily use in biochemical laboratory we decided to immobilize galactose oxidase orientedly through a carbohydrate chain to the magnetic carriers. We used hydrazide derivatives of non-magnetic and magnetic bead cellulose and of magnetic and non-magnetic poly(HEMA-co-EDMA) microspheres. Activation of the enzyme molecules was done by sodium periodate in the presence of supplements (fucose, CuSO4, catalase). Orientedly immobilized galactose oxidase presents high storage stability and lower susceptibility to inappropriate microenvironmental conditions. Reactor reactivated by three pulses of D-galactose retained practically 100% of its native activity after 6 months. The positive properties of both magnetic carriers were entirely confirmed.

Animals↗

PubMed2002

Poly(HEMA)--based embolic material in endovascular surgery of liver.

Physico-chemical and medico-biological methods, including hematology and cytology, were used in an evaluation of properties of poly(2-hydroxyethyl methacrylate) (poly(HEMA))-based embolic material indicated in the occlusion of branches of hepatic artery of patients with focal alterations of the liver. The elastographic method helped in predicting mechanical properties of the hydrogel material. Poly(HEMA) was mechanically stable for endovascular applications, exhibited no significant loss in elasticity and possessed consistency resembling the soft tissue of the organism. Analysis of blood in contact with poly(HEMA) hydrogel provided a control of its biological inertness. Recently, 315 patients with focal alterations of the liver, including hemobilic hemorrhages and both primary and metastatic tumors, underwent endovascular occlusion with poly(HEMA) emboli as a pre-surgical step or as a simple occlusive measure. Compared with a direct operation on hemangiomatous injuries in the liver without endovascular occlusion, embolization with poly(HEMA) allowed to reduce 2.5-3 times bleeding in the operational zone. At the same time, the poly(HEMA) embolic material induced activation of general hemostatic reaction in the postembolization period.

Adolescent↗