Evaluation of the physiological activity of venom from the Eurasian water shrew <Emphasis Type="Italic">Neomys fodiens</Emphasis>

Background

Animal toxins can have medical and therapeutic applications. Principally, toxins produced by insects, arachnids, snakes and frogs have been characterized. Venomous mammals are rare, and their venoms have not been comprehensively investigated. Among shrews, only the venom of Blarina brevicauda has been analysed so far, and blarina toxin has been proven to be its main toxic component. It is assumed that Neomys fodiens employs its venom to hunt larger prey. However, the toxic profile, properties and mode of action of its venom are largely unknown. Therefore, we analysed the cardio-, myo- and neurotropic properties of N. fodiens venom and saliva of non-venomous Sorex araneus (control tests) in vitro in physiological bioassays carried out on two model organisms: beetles and frogs. For the first time, we fractionated N. fodiens venom and S. araneus saliva by performing chromatographic separation. Next, the properties of selected compounds were analysed in cardiotropic bioassays in the Tenebrio molitor heart.

Results

The venom of N. fodiens caused a high decrease in the conduction velocity of the frog sciatic nerve, as well as a significant decrease in the force of frog calf muscle contraction. We also recorded a significant decrease in the frog heart contractile activity. Most of the selected compounds from N. fodiens venom displayed a positive chronotropic effect on the beetle heart. However, one fraction caused a strong decrease in the T. molitor heart contractile activity coupled with a reversible cardiac arrest. We did not observe any responses of the insect heart and frog organs to the saliva of S. araneus. Preliminary mass spectrometry analysis revealed that calmodulin-like protein, thymosin β-10, hyaluronidase, lysozyme C and phospholipase A2 are present in the venom of N. fodiens, whereas thymosin β4, lysozyme C and β-defensin are present in S. araneus saliva.

Conclusion

Our results showed that N. fodiens venom has stronger paralytic properties and lower cardioinhibitory activity. Therefore, it is highly probable that N. fodiens might use its venom as a prey immobilizing agent. We also confirmed that S. araneus is not a venomous mammal because its saliva did not exhibit any toxic effects.

     

Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences

Circularizable oligonucleotide probes can detect short DNA sequences with single-base resolution at the site of ligation and can be amplified by rolling circle amplification (RCA) using strand displacing polymerases. A secondary amplification scheme was developed that uses the loop-mediated amplification reaction concurrent with RCA to achieve rapid signal development from the starting circular molecules. This isothermal reaction was found to be significantly faster than the comparable hyperbranching amplification method and could detect 100 circular copies in less than 1 h.     

Typing of methicillin resistant strains of S. haemolyticus isolated from patients and from the hospital ward environment

Biotyping, antibiograms and fingerprinting were used to determine the relation of 16 methicillin-resistant S. haemolyticus isolated from drains in patients who underwent intraabdominal surgery to 9 methicillin-resistant strains of S. haemolyticus isolated at the same time from hospital environment. The comparison of biochemical properties of the examined strains showed a large variety of biochemical profiles as well as antibiotic patterns of susceptibility. The differences in sensitivity to the antibiotics used were not distinct. Biotyping and antibiograms did not permit determination of the relation of the investigated strains. Only the results of fingerprinting allowed for the division of the 25 examined strains into three genotypes demonstrating three main patterns of PCR products. 16 strains of 25 showed the same pattern of PCR products. This results suggests the presence of a source of infection on the clinical ward. A nurse may have been the source of infection because the same genotype of S. haemolyticus was isolated from her nasal anterior as from the majority of patients.     

Description and interpretation of adaptive evolution of Escherichia coli K-12 MG1655 by using a genome-scale in silico metabolic model

Genome-scale in silico metabolic networks of Escherichia coli have been reconstructed. By using a constraint-based in silico model of a reconstructed network, the range of phenotypes exhibited by E. coli under different growth conditions can be computed, and optimal growth phenotypes can be predicted. We hypothesized that the end point of adaptive evolution of E. coli could be accurately described a priori by our in silico model since adaptive evolution should lead to an optimal phenotype. Adaptive evolution of E. coli during prolonged exponential growth was performed with M9 minimal medium supplemented with 2 g of alpha-ketoglutarate per liter, 2 g of lactate per liter, or 2 g of pyruvate per liter at both 30 and 37 degrees C, which produced seven distinct strains. The growth rates, substrate uptake rates, oxygen uptake rates, by-product secretion patterns, and growth rates on alternative substrates were measured for each strain as a function of evolutionary time. Three major conclusions were drawn from the experimental results. First, adaptive evolution leads to a phenotype characterized by maximized growth rates that may not correspond to the highest biomass yield. Second, metabolic phenotypes resulting from adaptive evolution can be described and predicted computationally. Third, adaptive evolution on a single substrate leads to changes in growth characteristics on other substrates that could signify parallel or opposing growth objectives. Together, the results show that genome-scale in silico metabolic models can describe the end point of adaptive evolution a priori and can be used to gain insight into the adaptive evolutionary process for E. coli.     

Species- and substrate-specific stimulation of human plasma paraoxonase 1 (PON1) activity by high chloride concentration

Paraoxonase 1 (PON1), contained in plasma high-density lipoproteins, plays an important role in the protection of plasma lipoproteins and cell membranes from oxidative damage. Previous studies indicate that human PON1 is stimulated by high NaCl concentrations. The aim of this study was to characterize in more detail the effect of salts on serum PON1. Paraoxon-hydrolyzing activity of human serum was stimulated by 81.6% following the addition of 1 M NaCl. The effect of NaCl was dose-dependent between 0.5 and 2 M. PON1 activity toward phenyl acetate was reduced by 1 M NaCl by 55.2%. Both the paraoxon- and phenyl acetate-hydrolysing activity was slightly lower in heparinized plasma than in serum, but NaCl had similar stimulatory and inhibitory effects on these activities, respectively. In rat, rabbit, and mouse, NaCl reduced PON1 activity. KCl had a similar effect on human PON1 as NaCl. Sodium nitrite also stimulated human PON1 but much less effectively than chloride salts. In contrast, sucrose, sodium acetate and sodium lactate had no significant effect. NaBr was a less effective PON1 activator than NaCl, whereas the effect of NaJ was non-significant. The activity of human PON1 toward homogentisic acid lactone and gamma-decanolactone was unaltered by NaCl. These data indicate that: 1) high concentrations of chlorides stimulate human PON1 activity toward paraoxon but not other substrates, 2) PON1 is inhibited by Cl(-) in other mammalian species, 3) the potency of human PON1 activation by halogene salts increases with decreasing atomic mass of the halide anion.     

Disruption in gastric mucin synthesis by Helicobacter pylori lipopolysaccharide involves ERK and p38 mitogen-activated protein kinase participation

Helicobacter pylori is a primary factor in the etiology of gastric disease, and its early pathogenic effects are manifested by up-regulation of inflammatory processes and the loss of mucus coat continuity. We investigated the role of extracellular signal-regulated kinase (ERK) and p38 mitogen activated protein kinase (MAPK) in the disturbances in gastric mucin synthesis and apoptotic processes evoked by H. pylori lipopolysaccharide (LPS). Exposure of gastric mucosal cells to the LPS led to a dose-dependent decrease (up to 59.5%) in mucin synthesis, accompanied by a marked increase in caspase-3 activity and apoptosis. Inhibition of ERK with PD98059 accelerated (up to 36.1%) the LPS-induced decrease in mucin synthesis, and caused further enhancement in caspase-3 activity and apoptosis. Blockade of p38 kinase with SB203580 produced reversal in the LPS-induced reduction in mucin synthesis, and substantially countered the LPS-induced increases in caspas-3 activity and apoptosis. Moreover, inhibition of caspase-3 blocked the LPS-induced increase in caspse-3 activity and produced an increase in mucin synthesis. Thus the detrimental influence of H. pylori LPS on gastric mucin synthesis is closely linked to caspase-3 activation and apoptosis, and involves ERK and p38 kinase participation.     

Myotropic activity and immunolocalization of selected neuropeptides of the burying beetle Nicrophorus vespilloides (Coleoptera: Silphidae)

Burying beetles (Nicrophorus sp.) are necrophagous insects with developed parental care. Genome of Nicrophorus vespilloides has been recently sequenced, which makes them interesting model organism in behavioral ecology. However, we know very little about their physiology, including the functioning of their neuroendocrine system. In this study, one of the physiological activities of proctolin, myosuppressin (Nieve? MS), myoinhibitory peptide (Trica-MIP-5) and the short neuropeptide F (Nicve-sNPF) in N. vespilloides have been investigated. The tested neuropeptides were myoactive on N. vespilloides hindgut. After application of the proctolin increased hindgut contraction frequency was observed (EC50 value was 5.47 x 10-8 mol/L). The other tested neuropeptides led to inhibition of N. vespilloides hindgut contractions (Nicve-MS: IC50 = 5.20 x 10~5 mol/L;Trica-MIP-5: IC50 = 5.95 x 10-6 mol/L;Nicvc-sNPF: IC50 = 4.08 x 10-5 mol/L). Moreover, the tested neuropeptides were immunolocalized in the nervous system of N. vespilloides. Neurons containing sNPF and MIP in brain and ventral nerve cord (VNC) were identified. Proctolin-immunolabeled neurons only in VNC were observed. Moreover, MIP-immunolabeled varicosities and fibers in retrocerebral complex were observed. In addition, our results have been supplemented with alignments of amino acid sequences of these neuropeptides in beetle species. This alignment analysis clearly showed amino acid sequence similarities between neuropeptides. Moreover, this allowed to deduce amino acid sequence of N. vespilloides proctolin (RYLPTa), Nicve-MS (QDVDHVFLRFa) and six isoforms ofNicve-MIP (Nicve-MIP-1一 DWNRNLHSWa;Nicve-MIP-2—AWQNLQGGWa;Nicve-MIP-3—AWQNLQGGWa;Nicve-MlP-4—AWKNLNNAGWa;Nicve-MIP-5—SEWGNFRGSWa;Nicve-MIP-6— DPAWTNLKGIWa;and Nicve-sNPF—SGRSPSLRLRFa).     

Ion Exclusion Chromatography: Parameters Influencing Retention

Ion Exclusion Chromatography (IEC) finds application in the separation of a wide range of small, neutral or partially ionized molecules. In IEC, the strong as well as weak electrolytes are eluted unseparated, the first at the beginning and the latter at the end of the elution. The retention volumes of the remaining electrolytes are found to be proportional to their dissociation constant values. The dead and inner volumes of the chromatographic column can be determined from the observed dependence of retention volumes on dissociation constant values. The retention mechanism is described by the analytical equations and by the results obtained from the computer simulation of the column performance (using global thermodynamic and chromatographic equations or the Craig method). The mixed retention mechanism involving hydrophobic adsorption and screening effect is observed for weak electrolytes and aromatic compounds. Aromatic compounds are found to be retained almost solely by a reverse-phase mechanism involving interaction of the solute with the unfunctionalized regions of the stationary phase. The purpose of this paper is to survey the field. Using theoretical and experimental approaches, I show how different parameters can influence ion-exclusion solute retention. Although this retention is affected by the physicochemical parameters of the sorbent, stationary and mobile phases especially, this study primarily deals with the structural solute parameters, stationary phase form and column temperature, that have had little or no discussion in literature.