An increase in the spread of antibiotic-resistant opportunistic microorganisms causes serious problems in the treatment of purulent infections, burns, and trophic ulcers. We tested the antimicrobial activity in vivo of three polyphenols, Resveratrol, Dihydroquercetin (Taxifolin), and Dihydromyricetin (Ampelopsin) from Norway spruce bark to promote the elimination of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans from wounds. Purulent infection was modelled on wounds in rats infected with suspensions containing 109 CFU (colony-forming unit)/mL of pathogens. The wound area was treated daily with solutions of the polyphenols or placebo for 14 days after the beginning of the treatment. The animals were examined daily, and each stage of the wound healing (inflammation, granulation, and maturation (marginal epithelialisation) was documented. The planimetric analysis of the wound recovery percentage was performed on the 3rd, 10th, and 14th day after the start of curing. Then, one echelon (three or four animals from each subgroup) was withdrawn from the experiment on days 3 (three animals), 10 (three animals), and 14 (four animals) for microscopy analysis of cytological composition of their wound defects by microscopy and microbiological analysis of their contamination with pathogens. Our results show that they are also able to suppress mast cell infiltration and stimulate lymphocyte and macrophage (monocyte) infiltration into the wound. Resveratrol stimulated the replacement of the scar with normal tissue (with a clear boundary between the dermis and epidermis) and the restoration of hair follicles. Resveratrol turned out to be significantly better than some commercial antimicrobial (Levomecol) and antifungal (Clotrimazole) ointments and can be proposed as a promising drug for topical use for the treatment of trophic ulcers and burns.

The gene phyA encoding phytase was isolated from Obesumbacterium proteus genomic library and sequenced. The cleavage site of the PhyA signal peptide was predicted and experimentally proved. The PhyA protein shows maximum identity of 53% and 47% to phosphoanhydride phosphorylase from Yersinia pestis and phytase AppA from Escherichia coli, respectively. Based on protein sequence similarity of PhyA and its homologs, the phytases form a novel subclass of the histidine acid phosphatase family. To characterize properties of the PhyA protein, we expressed the phyA gene in E. coli. The specific activity of the purified recombinant PhyA was 310 U mg(-1) of protein. Recombinant PhyA showed activity at pH values from 1.5 through 6.5 with the optimum at 4.9. The temperature optimum was 40-45 degrees C at pH 4.9. The Km value for sodium phytate was 0.34 mM with a Vmax of 435 U mg(-1).

Staphylococcus aureus is an extremely infectious and malignant pathogen among many bacteria species. The aim of this work is to provide a robust classification model that would be able to identify S. aureus independent of the culture growth stage and the variations in bacteria concentration in suspension and also one that would be able to identify the pathogen among both taxonomically close species of the same genus and taxonomically distant species of different genera, using Fourier transform infrared spectroscopy (FTIR). In total, the spectra of 141 isolates of 17 bacteria have been used. Based on a combination of principal component analysis (PCA) and linear discriminant analysis (LDA), an identification model providing 100% sensitivity and 98% specificity was built. Inherent reliability and flexibility of the model have been shown. The proposed method of analysis allows us to get closer to the diagnostic requirements in the field of clinical microbiology, and it can be utilized for typing of other pathogenic bacteria species.

A metalloprotease gene of Brevibacillus brevis (npr) was expressed in Escherichia coli in a soluble form as native Npr precursor. A significant fraction of the precursor was spontaneously processed, producing the N-terminal propeptide and the mature enzyme. A strong inhibition of the mature Npr by its own propeptide in the crude lysate was observed even in the absence of the covalent linkage between them. Pure precursor, propeptide and the mature Npr were isolated and kinetic parameters of the mature enzyme inhibition by the propeptide were determined. The inhibition is of the tight-binding competitive type with K i 0.17 nM. Inhibition of metalloproteases from Brevibacillus megaterium and thermolysine by the heterologous propeptide of the Npr from B. brevis was much weaker or none.z 1999 Federation of European Biochemical Societies.Key words: Proenzyme; Tight-binding inhibition ; Brevibacillus brevis IntroductionMany proteases in various organisms are synthesized as inactive precursors, preproproteins or zymogens. Generally it is almost impossible to isolate a bacillar protease precursor from a natural strain, since it appears in the cultural medium as a processed mature form. When expressed in E. coli or other Gram-negative bacteria, these proteins are accumulated in insoluble inclusion bodies formed by non-active precursor, as found for subtilisin E [1], K-lytic protease from Lysobacter enzymogenes [2] and yeast carboxypeptidases Y [3], or they get rapidly processed producing mature form, as in the case of elastase from P. aeruginosa [4] and K-lytic protease from L. enzymogenes [2]. Therefore protease expression in a form of the non-active precursor is a challenging task. For that reason protease precursors and mechanisms of there activation were not thoroughly studied.The presequences usually serve as signal peptides for a transport through the plasma membrane. A functional role of the propeptides is less clear. Several functions were proposed for the propeptides. The propeptide may be required for productive folding and/or maintaining the protease in an inactive state inside the cell, as well as for interaction with the transport machinery of the cell which is important for e¡ec-tive secretion of these proteins [5].The role of protease prosequences is most extensively studied in the serine proteases: subtilisin E from B. subtilis [6,7], K-lytic protease from L. enzymogenes [8] and the vacuolar carboxypeptidase Y from yeast S. cerevisiae [3]. In all mentioned examples the propeptide assists the folding of the respective enzymes in vivo and in vitro, and blocks the enzymatic activity when covalently attached to the`mature' moiety of the enzyme. In subtilisin E and K-lytic protease their propeptides appear as tight-binding inhibitors of mature enzymes in trans. A propeptide of carboxypeptidase Y has a low a¤n-ity for its mature enzyme in vitro. The propeptides of cystein proteases papain and cathepsin B are found to inhibit their respective mature enzymes [9,10]. In a cystein vacuolar protease cathepsi...