Antiprotease activity of selected Slovak medicinal plants.

Fifty-six methanol extracts obtained from the barks, flowers, leaves and stems of 30 Slovak trees, bushes and herbs used in the traditional medicine of the Small Carpathians, Slovakia, have been screened for antiprotease (trypsin, thrombin and urokinase) activity using chromogenic bioassay. In this study, 14 extracts showed the strong inhibition activity to protease trypsin with IC50 values below 10 microg/mL. The highest inhibition activities were observed for methanol extracts of Acer platanoides IC50 = 1.8 microg/mL, Rhus typhina IC50 = 1.2 microg/mL and Tamarix gallica IC50 = 1.7 microg/mL. However, the results of extracts tested on thrombin were generally different from those observed for trypsin. The most marked inhibition activity to thrombin were estimated for extracts of Castanea sativa IC50 = 73.2 microg/mL, Larix decidua IC50 = 96.9 microg/mL and Rhus typhina IC50 = 20.5 microg/mL. In addition, Acer platanoides and Rhus typhina were the only extracts which showed inhibition activity to urokinase with IC50 = 171.1 microg/mL and IC50 = 38.3 microg/mL, respectively. In addition, Rhus typhina showed the broadest spectrum of inhibition activity to all tested serine proteases and seems to be a prospective new source of natural products as inhibitors of serine proteases.

The potential and practical applications of acylated flavonoids.

Flavonoids have many beneficial health effects. However, their practical applications are often strongly limited due to low solubility and stability in lipophilic media. In order to eliminate this drawback, enzymatic acylation with fatty acids has been introduced. Besides altering physico-chemical properties, bioavailability and biological properties of maternal compounds may also be improved. Although the research dealing with flavonoid ester synthesis is a matter of few years, and several aspects need to be clarified, there is a promising prospect of future application of these selectively acylated derivatives in therapy, cosmetics, and dietetic fields.

Viral proteinases--possible targets of antiviral drugs.

Viral infections represent various types of human, veterinary and plant diseases with a significant economic, ethic and demographic impact. Over the years a significant effort has been made to develop various means of prevention and therapy of viral diseases. Proteinases play an important role in the process of virus replication as well as in the pathophysiology of many viral diseases. The aim of this review is to assess the prospects of the application of proteinase inhibitors in antiviral therapy and to characterize viral proteinases of various classes. Six Human immunodeficiency virus (HIV) proteinase inhibitors have been approved for therapeutic use and can serve as examples of prospective application ofproteinase inhibitors to antiviral therapy.

Amylolytic enzymes: molecular aspects of their properties.

The present review describes the structural features of alpha-amylase, beta-amylase and glucoamylase that are the best known amylolytic enzymes. Although they show similar function, i.e. catalysis of hydrolysis of alpha-glucosidic bonds in starch and related saccharides, they are quite different. alpha-Amylase is the alpha --> alpha retaining glycosidase (it uses the retaining mechanism), and beta-amylase together with glucoamylase are the alpha --> beta inverting glycosidases (they use the inverting mechanism). While beta-amylase and glucoamylase form their own families 14 and 15, respectively, in the sequence-based classification of glycoside hydrolases, alpha-amylase belongs to a large clan of three families 13, 70 and 77 consisting of almost 30 different specificities. Structurally both alpha-amylase and beta-amylase rank among the parallel (beta/alpha)8-barrel enzymes, glucoamylase adopts the helical (alpha/alpha)6-barrel fold. The catalytic (beta/alpha)8-barrels of alpha-amylase and beta-amylase differ from each other. The only common sequence-structural feature is the presence of the starch-binding domain responsible for the binding and ability to digest raw starch. It is, however, present in about 10% of amylases and behaves as an independent evolutionary module. A brief discussion on structure-function and structure-stability relationships of alpha-amylases and related enzymes is also provided.

Monitoring of dihydroxyacetone production during oxidation of glycerol by immobilized Gluconobacter oxydans cells with an enzyme biosensor.

A bi-enzymatic biosensor for monitoring of dihydroxyacetone production during oxidation of glycerol by bacterial cells of Gluconobacter oxydans is presented. Galactose oxidase oxidizes dihydroxyacetone efficiently producing hydrogen peroxide, which reacts with co-immobilized peroxidase and ferrocene pre-adsorbed on graphite electrode. This mediator-based bi-enzymatic biosensor possesses very high sensitivity (4.7 µA/mM in phosphate buffer), low detection limit (0.8 µM, signal/noise = 3), short response time (22 s, 95% of steady-state) and broad linear range (0.002-0.55 mM in phosphate buffer). The effect of pH, temperature, type of buffer, as well as different stabilizers (combinations of a polyelectrolyte and a polyol) on the sensor performance were carefully optimized and discussed. Dihydroxyacetone produced during a batch conversion of glycerol by the pectate-immobilized bacteria in an air-lift reactor was determined by the biosensor and by reference spectrophotometric method. Both methods were compared and were in a very good correlation. The main advantage of the biosensor is a very short time needed for sample analysis (less than 1 min).

Novel glucose non-interference biosensor for lactose detection based on galactose oxidase-peroxidase with and without co-immobilised beta-galactosidase.

Two types of amperometric biosensors for lactose detection based either on co-immobilisation of two enzymes (galactose oxidase with peroxidase) or co-immobilisation of three enzymes (beta-galactosidase, galactose oxidase and peroxidase) were constructed. A graphite rod with pre-adsorbed ferrocene was used as a working electrode. The use of galactose oxidase instead of the frequently used glucose oxidase resulted in the construction of a glucose-non-interfering lactose sensor. Co-immobilisation of peroxidase with galactose oxidase allowed the effect of borate on the extension of the linear range and the effect of the working potential on galactose oxidase activation to be studied. The presence of beta-galactosidase greatly enhances the sensor's sensitivity, but its linear range is narrower than that of the sensor without beta-galactosidase. Addition of DEAE-dextran and inositol to the enzyme layer improved the half-life more than 16-fold compared with the sensor without stabilisers. A response time between 60 and 75 s (90% of the steady-state value) and a detection limit for lactose determination from 44 to 339 microM (signal-to-noise ratio = 3) were observed depending on the conditions. The precision of measurements of standard lactose solution for the trienzymatic and bienzymatic sensors was 2.19 and 2.02%, respectively. The precision of analysis of dairy products varied from 0.24 to 5.24%. Analyses of real samples showed good correlation with HPLC analysis; eight samples and 10 standard lactose solutions without pre-treatment were analysed in 1 h.

Use of bioluminometry for determination of active yeast biomass immobilized in ionotropic hydrogels.

The technique of bioluminometry was used to determine the biomass concentration of yeast cells immobilized in ionotropic hydrogel beads, including alginate, pectate, and kappa-carrageenan. The method uses determination of ATP extracted from viable cells, the concentration of which is then expressed as the active biomass concentration. Seven yeast strains divided into three categories (brewing, wine-making, and ethanol-producing yeasts) were tested, and different biomass concentrations were determined in all three immobilization materials. The described method is characterized by a good correlation (up to 99%) to classical dry biomass determination. The method is quicker, easier, and not so laborious, providing sufficient determination accuracy, and can be used for a rapid estimation of viable biomass in most biotechnological processes using immobilized living cells.

Receptor-ligand interaction and molecular modelling.

A number of computational methods for the description of the ligand-receptor interaction that were developed in the past decade are reviewed in this paper. The central two sections introduce the methods that are already established as useful tools for the qualitative and quantitative description of ligand-receptor complexes, either when the detailed atomic structure of the receptor is known or not. The following section gives two examples of the application of the described methodology on two limiting cases.

Effect of growth supplements and whey pretreatment on butyric acid production by Clostridium butyricum.

Improved production of butyrate (up to 19 g/l) from whey by Clostridium butyricum was achieved by adding either yeast extract (5 g/l) or biotin (50 µg/l). Hydrolysed lactose and proteolysed whey were less effective even with added biotin.

Influence of carbohydrates and polyols on L-lactic acid production and fatty acid formation by Rhizopus arrhizus.

Growth and L-lactic acid production on 24 different carbohydrates and polyols (glycerol, mannitol and sorbitol) by Rhizopus arrhizus CCM 8109 were determined. The D- but not the L-forms of xylose, fructose, galactose, mannose, glucose, cellobiose, maltose and sucrose and partially hydrolysed starch were converted to L-lactic acid. Changes in lipid formation and fatty acid composition were detected in biomass grown on the different sugars. In the presence of polyols, growth and considerable production of lipids were observed with little or no lactate production. Invertase was mainly associated with the mycelium during growth on sucrose, whereas glucoamylase and α-amylase were produced extracellularly during growth on starch.

Study of the complex formation between amine local anesthetics and uncouplers of oxidative phosphorylation carbonyl cyanide phenylhydrazones.

Spectroscopic evidence is presented which indicates that the anionic uncoupler carbonyl cyanide-4-nitro-2-chloro-phenylhydrazone and the amine local anesthetics form a complex in aqueous solution. The complex formation studies were carried out for several pharmacologically important tertiary amines and some primary amines. Their relative potencies to form a complex with uncoupler have followed the order: procaine < trimecaine < tetracaine < dibucaine < dodecylamine < dicyclohexylamine < hexadecylamine. As to the more lipophilic nature of the complex the emphasized penetration into octanol and reinforced retention into mitochondria was observed. The higher ability of the complex to colapse the mitochondrial membrane potential confirms this fact. The effective concentration of amine local anesthetics to form a complex was correlated with their physicochemical properties namely lipophilicity and acidobasicity. The highest effectivities for complex formation is shown by the most lipophilic and the most ionized molecules of amines. Present results point to the importance of considering the role of amine anesthetic-uncoupler complex in interpreting physiological or ion transport data in which these substances have been used together.

Simultaneous production and purification of Bacillus subtilis alpha-amylase.

Production of alpha-amylase with B. subtilis CCM 2722 in an aqueous two-phase polyethylene glycol/dextran system integrated with product purification by affinity chromatography on crosslinked starch during cultivation was studied. The medium was drawn from the bioreactor to the external settler during fermentation. After phase separation in the settler the dextran-rich bottom phase with cells was returned to the bioreactor. The PEG-rich top phase was pumped to the column with crosslinked starch and returned to the bioreactor after alpha-amylase adsorption. The same volumetric productivities, 0.53 U/mL/h, were reached in both batch and described process, but total productivity of the latter method was much higher owing to shortening upstream and downstream processing time. The enzyme of 98% homogenity in 95% yield was obtained after its elution from the column.

Biochemical, morphological and cytochemical studies of enhanced autolysis of Saccharomyces cerevisiae. 2. Morphological and cytochemical studies.

Morphological and cytochemical observation of Saccharomyces cerevisiae undergoing of induced autolysis were done in response to various chemical inducers of autolysis (NaCl, ethanol, fresh autolyzate). Changes in the inner structure of yeast cells were monitored by transmission electron microscopy and the surface of the cell wall was observed by scanning electron microscopy during autolysis. Cytochemical characterization of autolyzed cells was performed using four synthetic substrates for determination of proteinase activities but only carboxypeptidase Y could be detected in the vacuolar membranes. The morphological studies supported the data obtained from biochemical studies and confirmed that optimized conditions of autolysis have a significant effect on the structural changes of autolyzed yeast.

Biochemical, morphological and cytochemical studies of enhanced autolysis of Saccharomyces cerevisiae. 1. Biochemical studies.

Biochemical aspects of induced autolysis of Saccharomyces cerevisiae were observed in the presence of various physical and chemical enhancers of autolysis (increased temperature, changes of pH, NaCl, ethanol, fresh autolyzate). Direct assays of proteinases, nucleases, glucanases and acid phosphatases in homogenized autolyzed cells were performed. In addition, the degradation products of proteins, nucleic acids, polysaccharides and phosphate from phosphorylated compounds were determined in the supernatant of autolyzate after centrifugation. These results suggested that the inducers affected transport processes and that they had mostly negative effects on the activities of the above-mentioned enzymes.

Degradation of Delor 103, a technical mixture of polychlorinated biphenyls, by selected bacteria.

Ability to utilize a technical mixture of polychlorinated biphenyls (PCB), Delor 103, as the sole carbon source, has been tested in 14 bacterial strains. For the five best growing strains (Alcaligenes latus, Alcalgenes eutrophus, Comamonas testosteroni, Micrococcus varians and Pseudomonas putida), the dependence of the degradation of individual PCB congeners on the number of chlorine substituents is discussed.

Uncoupling effect of protonophoric and nonprotonophoric analogs of carbonyl cyanide phenylhydrazone on mitochondrial oxidative phosphorylation.

Analogs of carbonyl cyanide phenylhydrazone providing no reaction with nucleophilic groups and lacking acidobasic properties, respectively, were synthesized for study of mechanism of uncoupling effect on oxidative phosphorylation. Their retention, influence on proton transport, abilities to SH--groups modify and to stimulate respiration in rat liver mitochondria, together with their physico-chemical properties, namely lipophilicity, acidobasicity and reactivity were characterized. The substitution of acidic hydrogen of the imino group resulted in the loss of both acidobasicity and uncoupling effect on oxidative phosphorylation. A decreased reactivity resulted from the substitutions of nitrile groups with the uncoupling activity remaining preserved.

Kinetics of drug activities as influenced by their physico-chemical properties: antibacterial effects of alkylating 2-furylethylenes.

A method is presented allowing for direct incorporation of the time of exposure into the relationship between biological and physico-chemical properties of drugs. The approach employs kinetics of the drug-receptor interaction based on mass action law, whereby biological response is considered as proportional to the receptor modification, and the time-dependent drug concentration in the vicinity of receptors is expressed by a disposition function. The function with variable physico-chemical properties and time relates the intracellular drug concentration to the dose. General description of individual steps in the development of a quantitative structure-time-activity relationship (QSTAR) is illustrated in detail using the data on antibacterial effects of alkylating 2-furylethylenes. It is shown that common approaches to description of quantitative structure-activity relationships (QSAR), working with a prefixed time of exposure, represent special cases of the method presented and can even be improved using its conclusions.