Biomedical subjects

Jirí Janata

Publications and source records attributed to Jirí Janata.

8 recordsLinked to original sources

PubMed2006

High-throughput quantification of lincomycin traces in fermentation broth of genetically modified Streptomyces spp. Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography with UV detection.

A new separation and quantification method using ultra-performance liquid chromatography (UPLC) with UV detection was developed for detection of lincomycin traces in fermentation broth of different Streptomyces spp. A similar high-performance liquid chromatography (HPLC) protocol was simultaneously developed for comparison purposes. Both methods were validated and showed a linear range of detector response for quantification of lincomycin in concentration from 3.125 to 1000.0 microgml(-1) with correlation coefficient 0.999 and recoveries ranging from 81.5 to 89.85% with precision < or =5%. Compared with the HPLC, the UPLC method offered high sample throughput and about 10 times lower consumption of solvents. The developed assays were used for determination of lincomycin production in genetically manipulated production strain Streptomyces lincolnensis and for determination of lincomycin production after heterologous expression of lincomycin biosynthetic gene cluster in non-producing strain Streptomyces coelicolor.

Chromatography, High Pressure Liquid↗

PubMed2006

Enhanced performance of a filter-sensor system.

In this paper are addressed two important, but seemingly unrelated issues: long term performance of a gas sensing array and performance of an air purification unit. It is shown that when considered together, the system can be regarded as a "smart filter". The enhancement is achieved by periodic differential sampling and measurement of the "upstream" and "downstream" gases of a filter. The correctly functioning filter supplies the "zero gas" from the downstream for the continuous sensor baseline correction. A key element in this scheme is the synthetic jet that delivers well-defined pulses of the two gases. The deterioration of the performance of the "smart filter" can be diagnosed from the response pattern of the sensor. The approach has been demonstrated on removal/sensing of ammonia gas from air.

Air Conditioning↗

PubMed2005

Gold-polyaniline composite. Part I. Moving electrochemical interface.

The controlled growth of gold nanoclusters in polyaniline is predicated by the ability of PANI to switch from conducting to insulating form and by the fact that imine nitrogens form a strong complex with chloroaurate anions. The principal technique used in this study has been chronopotentiometric stripping. The chronopotentiograms indicate that the electrochemical interface for the Pt/Au/polyaniline/electrolyte shifts back and forth from the solution/PANI interface to Au/PANI interface. By adjusting experimental conditions the size and the size distribution of Au clusters can be controlled.

Aniline Compounds↗

PubMed2005

Gold-polyaniline composites. Part II. Effects of nanometer sized particles.

The amount of electronic charge transferred between gold particles and polyaniline depends not only on the electron affinity of the two materials but also on the size of the gold particles. As measured by X-ray photoelectron spectroscopy, for particles < approximately 5 nm the binding energy of the electrons is size dependent. This "nano-effect" has its origin in the electrostatics of particles. It is demonstrated as a measurable shift of the binding energy of the Au(4f7/2) photoelectrons emitted from Au particles embedded in a polyaniline matrix. Gold nanoparticle size was evaluated by high resolution transmission electron microscopy.

Aniline Compounds↗

PubMed2005

Cleavage site selection within a folded substrate by the ATP-dependent lon protease.

Mechanistic studies of ATP-dependent proteolysis demonstrate that substrate unfolding is a prerequisite for processive peptide bond hydrolysis. We show that mitochondrial Lon also degrades folded proteins and initiates substrate cleavage non-processively. Two mitochondrial substrates with known or homology-derived three-dimensional structures were used: the mitochondrial processing peptidase alpha-subunit (MPPalpha) and the steroidogenic acute regulatory protein (StAR). Peptides generated during a time course of Lon-mediated proteolysis were identified and mapped within the primary, secondary, and tertiary structure of the substrate. Initiating cleavages occurred preferentially between hydrophobic amino acids located within highly charged environments at the surface of the folded protein. Subsequent cleavages proceeded sequentially along the primary polypeptide sequence. We propose that Lon recognizes specific surface determinants or folds, initiates proteolysis at solvent-accessible sites, and generates unfolded polypeptides that are then processively degraded.

Adenosine Triphosphate↗

PubMed2004

Substrate evokes translocation of both domains in the mitochondrial processing peptidase alpha-subunit during which the C-terminus acts as a stabilizing element.

All three tryptophan residues in alpha-subunit of mitochondrial processing peptidase (MPP) were subsequently substituted. While substitutions of Trp223 led to misfolded non-functional protein, mutations of Trp147 and/or Trp481 did not affect the enzyme processing activity. Thus, fluorescence properties of the mutants with fewer tryptophans were used for observation of both alpha-MPP domain translocation and visualization of conformational changes in the interdomain linker evoked by substrate. We found that in the presence of substrate the C-terminal penultimate Trp481 was approaching Trp223, which is localized at the border of N-terminal domain and interdomain linker. Also, excision of the alpha-MPP C-terminal 30 amino acid residues (DeltaC30) led to a complete loss of protein function. Even shorter deletions of the alpha-MPP C-terminus destabilized the protein slightly (DeltaC2) or dramatically (DeltaC17). It suggests that the extreme C-terminus of alpha-MPP provides mechanical support to the C-terminal domain during its extensive conformational change accompanying the substrate recognition process.

Amino Acid Substitution↗

PubMed2004

l-3,4-Dihydroxyphenyl alanine-extradiol cleavage is followed by intramolecular cyclization in lincomycin biosynthesis.

The LmbB1 protein, participating in the biosynthesis of lincomycin, was heterologously expressed in Escherichia coli, purified in its active form, and characterized as a dimer of identical subunits. Methods for purification and analysis of the LmbB1 reaction product were developed. Molecular mass and fragmentation pattern of the product revealed by capillary electrophoresis-mass spectrometry were in agreement with its proposed structure, 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydro-1H-pyrrole-2-carboxylic acid. The LmbB1 is therefore a dioxygenase catalysing the 2,3-extradiol cleavage of the l-3,4-dihydroxyphenyl alanine aromatic ring. The final LmbB1 reaction product, a unique compound found in biosynthesis of lincomycin and expected in anthramycins, arises through subsequent cyclization of the primary cleavage product, 2,3-secodopa. A possible role of LmbB1 in 2,3-secodopa cyclization and alternative ways of the cyclization in the formation of biosynthetically related compounds, muscaflavin and stizolobinic acid, are discussed.

Chromatography, Ion Exchange↗

PubMed2002

Chemical plume tracking. 3. Ascorbic acid: a biologically relevant marker.

Dynamic characteristics of the amperometric sensing system for tracking of turbulent chemical plumes have been studied. The correlation analysis requires that such sensors respond rapidly and have long-term stability and minimal or low-flow sensitivity. Moreover, for practical purposes, such sensors must respond to an electrochemical marker that would be found in or at least be compatible with the diet of marine animals. Ascorbic acid is such a compound and can be electrochemically oxidized. Its long-term dynamic behavior on several types of electrodes has been studied. It has been found that a Pt electrode coated with polyaniline satisfies all the above requirements. However, this system has a peculiar dynamic behavior that affects the results of the correlation analysis, namely, at the higher frequencies. The behavior of such system in the virtual plume setup has been characterized and its usability for detection of fluctuating concentration of ascorbic acid in saline solution has been confirmed.

Animals↗