Design, synthesis and molecular modelling of 1-amidinopiperidine thrombin inhibitors.

Design, synthesis and biochemical evaluation of a series of novel non-covalent thrombin inhibitors with a 1-amidinopiperidine moiety are presented. Replacement of the planar benzamidine group in azaphenylalanine derivatives with 1-amidinopiperidine resulted in lower activity but higher selectivity for this type of compounds. The binding conformation of inhibitors in the active site of thrombin was revealed by molecular modelling studies.

Isolation and characterization of Mn(III) tartrate from Phanerochaete chrysosporium culture broth.

High initial Mn(II) concentration results in accumulation of a Mn(III) tartrate complex in the growth medium of Phanerochaete chrysosporium. Since Mn(III) is the major oxidant in ligninolysis by manganese peroxidase, the role of accumulated complex should not be neglected when degradation experiments by a crude culture filtrate are performed. To study the Mn(III) complex oxidative potential it was isolated by absorption to polyamide followed by desorption with an alkaline methanol solution. High performance liquid chromatography analysis and atomic absorption spectroscopy confirmed that the isolate was Mn(III) tartrate. Oxidation of 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonate) was used for testing the temperature and pH stability of the isolate that also intensively oxidized 2,6-dimethoxyphenol. In comparison with the non-isolated complex in the culture filtrate, the isolate showed increased temperature and pH stability. The oxidative potential of the isolated Mn(III) tartrate was additionally tested by decolorization of the synthetic dye Indigo carmine.

The effect of agitation and nitrogen concentration on lignin peroxidase (LiP) isoform composition during fermentation of Phanerochaete chrysosporium.

Convective Interaction Media (CIM) monolithic columns were applied for the HPLC monitoring of Phanerochaete chrysosporium lignin peroxidase (LiP) isoforms during cultivation. The influence of the agitation mode (circular, elliptic) and rate (130 and 200 rpm), as well as the initial nitrogen concentration (1.6-6 mM) in the growth medium was investigated. Identical rotation rate but different agitation modes resulted in different LiP activities and isoenzyme compositions. On the other hand, at different agitation types and rates, similar LiP activities were obtained at different isoenzyme compositions. Although LiP H2 and LiP H6/H7 were predominant isoenzymes obtained at various cultivation conditions, relative isoenzyme amounts differ considerably when initial nitrogen concentration was changed between 1.6 and 5 mM.

Passing of fluorescein derivatives into the hyphae of Phanerochaete chrysosporium.

Fluorescein derivatives added into the growth medium were decolorized during submerged cultivation of Phanerochaete chrysosporium. The highest decrease of absorbance A450 was observed in the growth phase regardless of the presence of inducers Tween 80 or poly(ethylene glycol) (PEG). Fluorescein linked to PEG was prepared and, after addition to cultures, shown to stimulate the production of lignin peroxidase. Passing of fluorescing substances into hyphae (observed by confocal microscopy) showed that they were concentrated on some structures inside hyphae.

Recent advances in the development of catalytic inhibitors of human DNA topoisomerase IIα as novel anticancer agents.

DNA topoisomerases comprise an important family of enzymes that catalyse the induction of topological changes (e.g. relaxation/ supercoiling, catenation/decatenation and knotting/unknotting) in the DNA molecule. These enzymes perform their functions by creating transient either single-stranded or double-stranded breaks in the DNA molecule. Due to their ability to modulate the topology of the DNA molecule, DNA topoisomerases play vital roles in replication, transcription, chromosome separation and segregation, and thus represent an important collection of design targets for novel anticancer drugs. The aim of this review is to provide an overview of the development of catalytic inhibitors of the human topoisomerase IIα enzyme--an important member of the DNA topoisomerase family--as potential novel anticancer agents. The group of catalytic topoII inhibitors is classified into four types according to their molecular mechanism of action: inhibitors that bind to the ATP binding site, inhibitors that prevent the ATP hydrolysis step and trap the enzyme in a closed clamp, inhibitors that block the DNA cleavage and inhibitors that prevent the enzyme binding to the DNA. One of the important considerations highlighted throughout this review is the structure-based perspective of inhibitor design, giving the reader a medicinal chemist's perspective on this vibrant and active field of drug design research.

Small molecule antagonists of integrin receptors.

The complex and widespread family of integrin receptors is involved in numerous physiological processes, such as tissue remodeling, angiogenesis, development of the immune response and homeostasis. In addition, their key role has been elucidated in important pathological disorders such as cancer, cardiovascular diseases, osteoporosis, autoimmune and inflammatory diseases and in the pathogenesis of infectious diseases, making them highly important targets for modern drug design campaigns. In this review we seek to present a concise overview of the small molecule antagonists of this diverse and highly complex receptor family. Integrin antagonists are classified according to the targeted integrin receptor and are discussed in four sections. First we present the fibrinogen alpha(IIb)beta3 and the vitronectin alpha (V)beta(3) receptor antagonists. The remaining selective integrin antagonists are examined in the third section. The final section is dedicated to molecules with dual or multiple integrin activity. In addition, the use of antibodies and peptidomimetic approaches to modulate the integrin receptors are discussed, as well providing the reader with an overall appreciation of the field.

Distinct labelling of fusion events in rat lactotrophs by FM 1-43 and FM 4-64 is associated with conformational differences.

AIM: Conformational analysis of fluorescent styryl dyes FM 1-43 and FM 4-64 was undertaken to clarify if distinct activity-dependent labelling of single lactotrophs vesicles and plasma membrane by two dyes is associated with their structural differences. METHODS: The activity-dependent labelling of single vesicles and plasma membrane by FM 1-43 and FM 4-64 was studied using confocal microscopy. The fluorescence intensity of vesicles fused with the plasma membrane, and the plasma membrane alone was measured; the ratio of their respective peak amplitudes was calculated. The conformational analysis of FM 1-43 and FM 4-64 was further undertaken by employing the Monte Carlo approach to search the conformational space of these molecules. RESULTS: In FM 1-43 staining of vesicles and plasma membrane, the ratio of the fluorescence peak amplitudes (vesicle vs. plasma membrane) was 2.6 times higher in comparison with FM 4-64 staining. In FM 4-64 molecule the low-energy conformations are distributed in three conformational states (consisting of 3, 4 and 2 conformers respectively) in which the proportion of the molecules residing in a given state is 62%, 28% and 9% respectively. In FM 1-43 the conformation distribution is limited to just one conformational state with three approximately equally populated conformers what can be explained by greater intrinsic rigidity of the molecule. CONCLUSIONS: The observed structural characteristics of FM 1-43 molecules may account for a higher increase in quantum yield and/or binding affinity upon incorporation of the dye into the vesicle matrix and therefore stronger fluorescence emission in comparison with FM 4-64.

Detection and identification of natural water-soluble colouring matters in foods.

The natural colouring matters of foods can be isolated and identified simultaneously with the synthetic dyes by the benzalkonium method. In the course of isolation anthocyanins, carmine, betanin, caramel and riboflavin are separated from synthetic dyes, as well as from one another, with the exception of first two, which are separated from one another by chromatography or distinguished by oxidation.

Stimulation of ligninolytic enzyme production in Phanerochaete chrysosporium by polyoxyalkanes.

AIMS: Poly(ethylene glycol) (PEG) and some substances similar to PEG in chemical structure were tested as stimulators of ligninolytic enzyme production in shaken culture of Phanerochaete chrysosporium. METHODS AND RESULTS: The substances that caused high enzymatic activity were linear polymers [poly(ethylene glycol), poly(propylene glycol), poly(butylene glycol) and poly(vinyl alcohol)] and cyclic polymers (crown ether). They can have terminal groups other than -OH [PEG (di)methyl ether, PEG sulphate, PEG derivative with the amino group and xanthate]. The maximum lignin peroxidase activities were compared with the surface pressure caused by the stimulator. Addition of polymers composed of charged monomer units did not increase the enzymatic activity and the fungi did not grow at all on addition of polymers having a fixed positive charge. CONCLUSIONS: Lignin peroxidase activity was increased after the addition of polymers with uncharged monomer units. It was higher and its maximum was reached in a shorter time on addition of polymers with higher molecular weights. SIGNIFICANCE AND IMPACT OF STUDY: Beside Tweens there are several polymers that stimulate ligninolytic enzyme production in shaken culture of P. chrysosporium. Their characteristics are: similarity to PEG in chemical structure, having uncharged monomer units and high molecular weight.

Analysis of Fungal Pellets by UV-Visible Spectrum Diffuse Reflectance Spectroscopy.

The application of the UV-visible spectrum diffuse reflectance spectroscopy for the determination of intracellular pH in vivo, for determination of cytochrome content, and for the noninvasive in vivo detection of the redox state of fungal mitochondrial cytochromes in filamentous fungi is introduced. The time course of the intracellular pH values, mitochondrial cytochromes, and CO-binding pigments content and the correlations between the actual redox state of cytochrome aa(3) and saturation of growth medium with oxygen in pellets of the basidiomycete Phanerochaete chrysosporium were determined. As the test microorganism, the yeast Saccharomyces cerevisiae was used. UV-visible spectrum diffuse reflectance spectroscopy proved to be a promising method for the quick and simple analysis of light-impermeable biological structures for which the classical transmittance spectrophotometric methods are difficult to implement.

Physiological Aspects of Biosynthesis of Lignin Peroxidases by Phanerochaete chrysosporium.

Methods based on UV-visible diffuse reflectance spectroscopy were used to study the physiological aspects of lignin-peroxidase biosynthesis by Phanerochaete chrysosporium. Here we introduce the use of cytochrome aa(3) as an indicator of active fungal biomass and of its redox state to calculate the oxygen mass transport coefficient between the growth medium and the fungal cell interior. When lignin peroxidase biosynthesis was enhanced by the addition of Tween 80 or Tween 20 to the growth medium, a higher proportion of reduced cytochrome aa(3) and a higher oxygen diffusion barrier were observed compared with control cultures. In cultures supplemented with Tween 80 or Tween 20, a higher oxygen mass transport coefficient between the growth medium and the interior of the fungal cell was also found. The beginning of the lignin peroxidase activity in these cultures was found to coincide with a temporary cessation in the dry biomass increase and a reduction in the relative active-biomass concentration. During the lignin peroxidase activity, a decrease in the intracellular pH and an increase in the growth medium pH were determined in cultures supplemented with Tween 80.

A method of fast separation of lignin peroxidases using convective interaction media disks.

The HPLC separation of lignin peroxidase isoenzymes using Convective Interaction Media disks containing quaternary amine and diethylaminoethyl ion-exchange active groups is proposed. In contrast to standard HPLC procedures the separation can be performed within a few minutes without considerably affecting the separation resolution. The method is reproducible and gives a linear response of integrated peak area to protein concentration for all measured isoenzymes. The separation resolution is retained unchanged by applying crude culture filtrate instead of a sample previously frozen and dialyzed. The optimized method might therefore be used for on-line monitoring of lignin peroxidase isoenzyme composition during fermentation. On the other hand, the proposed method is comparable in time to the original method of lignin peroxidase activity measurement (proposed by Tien and Kirk), providing additionally the isoenzyme composition.

Amino acids in chitin - glucan complex ofAspergillus niger.

DeproteinatedA. niger biomass contains several covalently bound amino acids. The most abundant are arginine, serine, and proline in molar ratio of 3:2:2. One order of magnitude less is the amount of valine, phenylalanine, leucine and glycine. On deacetylation and separation of chitosan from glucan, the main three amino acids remain bound predominantly to chitosan, whereas the hydrophobic amino acids accompany mainly glucan. The presence of arginine could be the cause of stronger basicity of fungal chitosan compared to polyglucosamine.

Laccase production by Phanerochaete chrysosporium--an artefact caused by Mn(III)?

AIMS: The possibility of laccase production by Phanerochaete chrysosporium was studied. METHODS AND RESULTS: A relatively high initial Mn(II) concentration (1-4 mM) in the growth medium leads to the development of reddish-brown coloration and intensive oxidation of 2.2'-azino-bis(3-etilbenz-tiazolin-6-sulfonate) (ABTS). The peak of ABTS oxidation was obtained approximately 1 day after the peak of MnP activity. CONCLUSION: ABTS oxidation was not caused by manganese peroxidase (MnP) nor by laccase but was the consequence of the action of Mn(III) which was stabilised in the growth medium. Decomposition of the complex took place after the biomass was removed from the growth medium and especially after the aeration of the culture was interrupted. SIGNIFICANCE AND IMPACT OF THE STUDY: Mn(III) seems to be the cause of false positive laccase reactions. More reliable data on MnP activity can be obtained if the complex is decomposed by the fungus before MnP activity is measured in the medium.