Selective antibiofilm properties and biocompatibility of nano-ZnO and nano-ZnO/Ag coated surfaces.

Spread of pathogenic microbes and antibiotic-resistant bacteria in health-care settings and public spaces is a serious public health challenge. Materials that prevent solid surface colonization or impede touch-transfer of viable microbes could provide means to decrease pathogen transfer from high-touch surfaces in critical applications. ZnO and Ag nanoparticles have shown great potential in antimicrobial applications. Less is known about nano-enabled surfaces. Here we demonstrate that surfaces coated with nano-ZnO or nano-ZnO/Ag composites are not cytotoxic to human keratinocytes and possess species-selective medium-dependent antibiofilm activity against Escherichia coli, Staphylococcus aureus and Candida albicans. Colonization of nano-ZnO and nano-ZnO/Ag surfaces by E. coli and S. aureus was decreased in static oligotrophic conditions (no planktonic growth). Moderate to no effect was observed for bacterial biofilms in growth medium (supporting exponential growth). Inversely, nano-ZnO surfaces enhanced biofilm formation by C. albicans in oligotrophic conditions. However, enhanced C. albicans biofilm formation on nano-ZnO surfaces was effectively counteracted by the addition of Ag. Possible selective enhancement of biofilm formation by the yeast C. albicans on Zn-enabled surfaces should be taken into account in antimicrobial surface development. Our results also indicated the importance of the use of application-appropriate test conditions and exposure medium in antimicrobial surface testing.

Evaluation of the potential hazard of lanthanides to freshwater microcrustaceans.

The use of lanthanides in different sectors of industry has significantly increased during the last decades. Although the "anthropogenic" anomalies of lanthanides in the soils, surface and ground waters have already been registered, the ecotoxicological effects of these elements and their fate in the environment are still insufficiently investigated. In this study acute and long-term toxicity of selected lanthanides (La, Ce, Pr, Nd and Gd) nitrates to freshwater crustaceans Daphnia magna, Thamnocephalus platyurus and Heterocypris incongruens were studied and critically evaluated. The data obtained show that (i) due to the methodical nuances the acute toxicity data of lanthanides are not reliable and have doubtful scientific value even for preliminary toxicity screening and thus should not be used for risk assessment; (ii) toxicity of lanthanides in the 21-day D. magna reproduction test was high whereas the mortality of parent daphnids was more sensitive endpoint than reproduction; (iii) the long-term LC50 values for lanthanides varied from 0.3 to 0.5 mg Ln/L and the differences between individual Ln were not statistically significant. All in all, the results of this study allow us to conclude that the environmental risk assessment of lanthanides should be performed only using long-term toxicity tests. In the environmental risk assessment, lanthanides may be considered as a uniform group of elements with additive mode of action until future investigations will not reveal differences in the ecotoxicity mechanisms of these elements.

Vipera lebetina venom contains all types of snake venom metalloproteases.

Snake venoms contain four classes of metalloproteases that all have a typical zinc-chelating sequence (HEXXHGXXH). N-terminal sequences and internal sequences of different purified metalloproteases were determined using Edman sequencing and LC MS/MS technique. Oligonucleotides were designed and used as primers for cDNA cloning from Vipera lebetina venom gland cDNA library. We found that isoforms of fibrinolytic enzyme lebetase Le-4 and Le-3 are synthesized in different way: Le-4 is synthesized as P-I type metalloprotease, Le-3 is synthesized with disintegrin-like domain as P-II type protease and processed post-translationally. An endothelial cell apoptosis-inducing heterodimeric glycosylated metalloprotease, V. lebetina apoptosis-inducing protease (VLAIP), belongs to P-III type containing metalloprotease, disintegrin-like and cysteine-rich domains. All these enzymes hydrolyze the Aalpha-chain and more slowly the Bbeta-chain of fibrinogen. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes apoptosis. V. lebetina venom contains also P-IV type-specific coagulant factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein composed of a heavy chain and two C-type lectin-like light chains linked by disulfide bonds. The heavy and light chains of VLFXA are synthesized from different genes.

Calbindin D(9k): a protein optimized for calcium binding at neutral pH.

The binding of calcium ions by EF-hand proteins depends strongly on the electrostatic interactions between Ca(2+) ions and negatively charged residues of these proteins. We have investigated the pH dependence of the binding of Ca(2+) ions by calbindin D(9k). This protein offers a unique possibility for interpretation of such data since the pK(a) values of all ionizable groups are known. The binding is independent of pH between 7 and 9, where maximum calcium affinity is observed. An abrupt decrease in the binding affinity is observed at pH values below 7. This decrease is due to protonation of acidic groups, leading to modification of protein charges. The pH dependence of the product of the two macroscopic Ca(2+)-binding constants can be formally described by the involvement of two acidic groups with pK(a) = 6.6. Monte Carlo calculations show that the reduction of Ca(2+) binding is strictly determined by variable electrostatic interactions due to pH-dependent changes not only in the binding sites, but also of the overall charge of the protein.

Factor X activator from Vipera lebetina snake venom, molecular characterization and substrate specificity.

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants of the hemostatic system in the same venom. We showed that V. lebetina venom contains factor X activator (VLFXA) and factor V activator, fibrinolytic enzymes. VLFXA was separated by gel filtration on Sephadex G-100 superfine and ion exchange chromatography on CM-cellulose and on TSK-DEAE (for HPLC) columns. VLFXA is a glycoprotein composed of a heavy chain (57.5 kDa) and two light chains (17.4 kDa and 14.5 kDa) linked by disulfide bonds. VLFXA has multiple molecular forms distinguished by their isoelectric points. The differences in their pI values may be caused by dissimilarities in the respective charged carbohydrate content or in the primary sequence of amino acids. We synthesized 6-9 amino acid residues containing peptides according to physiological cleavage regions of human factor X and human factor IX. The peptides (Asn-Asn-Leu-Thr-Arg-Ile-Val-Gly-Gly - factor X fragment, and Asn-Asp-Phe-Thr-Arg-Val-Val-Gly-Gly - factor IX fragment) were used as substrates for direct assay of VLFXA. Cleavage products of peptide hydrolysis and the molecular masses of cleavage products of human factor X were determined by MALDI-TOF MS. The MALDI-TOF MS was highly efficient for the recovery and identification of peptides released by VLFXA hydrolysis. We can conclude that VLFXA cleaves the Arg(52)-Ile(53) bond in the heavy chain of human factor X and the Arg(226)-Val(227) bond in human factor IX precursor. VLFXA could not activate prothrombin nor had any effect on fibrinogen, and it had no arginine esterase activity toward benzoylarginine ethyl ester.

Functional complexes of mitochondria with Ca,MgATPases of myofibrils and sarcoplasmic reticulum in muscle cells.

Regulation of mitochondrial respiration in situ in the muscle cells was studied by using fully permeabilized muscle fibers and cardiomyocytes. The results show that the kinetics of regulation of mitochondrial respiration in situ by exogenous ADP are very different from the kinetics of its regulation by endogenous ADP. In cardiac and m. soleus fibers apparent K(m) for exogenous ADP in regulation of respiration was equal to 300-400 microM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 microM when about 70% of maximal rate of respiration was achieved. Respiration rate maintained by intracellular ATPases was suppressed about 20-30% during exogenous trapping of ADP with excess pyruvate kinase (PK, 20 IU/ml) and phosphoenolpyruvate (PEP, 5 mM). ADP flux via the external PK+PEP system was decreased by half by activation of mitochondrial oxidative phosphorylation. Creatine (20 mM) further activated the respiration in the presence of PK+PEP. It is concluded that in oxidative muscle cells mitochondria behave as if they were incorporated into functional complexes with adjacent ADP producing systems - with the MgATPases in myofibrils and Ca,MgATPases of sarcoplasmic reticulum.

Proteases from Vipera lebetina venom affecting coagulation and fibrinolysis.

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants in the same venom. We showed that V. lebetina venom contains: (1) proteases that degrade fibrinogen, but not fibrin; (2) fibrinolytic enzyme (lebetase); (3) factor X activator (VLFXA); (4) factor V activator (VLFVA). Fibrinolytic enzyme and VLFXA are metalloproteases; the other studied enzymes are serine proteases. alpha-Fibrinogenase has no homolog among known serine proteases. Beta-fibrinogenase is a typical thermostable arginine esterase that hydrolyzes esters and amides of arginine and attacks the beta-chain of fibrinogen. Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins. We used the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique for the recovery and identification of peptides released by protease hydrolysis and for the detection of human factor X cleavage products after VLFXA hydrolysis. VLFXA cleaves the Arg(52)-Ile(53 )bond in the heavy chain of human factor X and the Arg(226)-Val(227) bond in human factor IX precursor; VLFVA cleaves Arg(1545)-Ser(1546) in factor V.

MALDI-TOF mass spectrometry analysis of substrate specificity of lebetase, a direct-acting fibrinolytic metalloproteinase from Vipera lebetina snake venom.

Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins which include both hemorrhagic and non-hemorrhagic proteinases. Despite apparent structural similarities, fibrinolytic and hemorrhagic proteinases differ significantly in substrate specificity. In this study, we have examined the activity of lebetase I against biologically active peptides (bradykinin, kallidin, substance P) and 6-10 amino acid residues containing peptides synthesized according to cleavage regions of alpha(2)-macroglobulin, pregnancy zone protein (PZP) and fibrinogen. Lebetase was found to have no activity against studied hexapeptides. Surprisingly, the best substrates for lebetase were substance P, and peptide fragment of PZP, both were cleaved at position Pro-Gln. Identification of the hydrolysis products of 15 peptides by MALDI-TOF mass spectrometry analysis indicates that lebetase possesses broad substrate specificity. The MALDI-TOF MS technique was proven to be highly efficient for the recovery and identification of the peptides released by lebetase hydrolysis.

Hydrolysis of emulsified mixtures of triacylglycerols by pancreatic lipase.

Hydrolysis of the emulsified mixture of short-chain triacylglycerols by porcine pancreatic lipase in the presence of procolipase and micellar sodium taurodeoxycholate has been studied. Increase in the content of tributyrin and trioctanoin in the mixture with triacetin had highly cooperative effects on the formation of the interfacial lipase procolipase complex. Abrupt enhancement of the complex stability was observed in the presence of 0.4-0.6 mol mol-1 of tributyrin or 0.58 mol mol-1 of trioctanoin in the substrate phase. The affinity of lipase towards interfacially bound procolipase for the trioctanoin containing 0.07-0.42 mol mol-1 of triacetin was approximately three times higher than that for pure trioctanoin. The cooperative processes involved in complex formation did not contribute to the affinity of the interfacial lipase/(pro)colipase complex towards substrate molecules and its catalytic activity.