Isolation and primary structures of seven serine proteinase inhibitors from Cyclanthera pedata seeds

Seven new trypsin inhibitors, CyPTI I–VII, were purified from ripe seeds of Cyclanthera pedata by affinity chromatography on immobilized chymotrypsin in the presence of 5 M NaCl followed by preparative native PAGE at pH 8.9. The CyPTIs (Cyclanthera pedata trypsin inhibitors) belong to a well-known squash inhibitor family. They contain 28–30 amino acids and have molecular weights from 3031 to 3367 Da. All the isolated inhibitors strongly inhibit bovine β-trypsin (K_a > 10¹¹ M^− 1) and, more weakly, bovine α-chymotrypsin (K_a ≈ 10⁴–10⁶ M^− 1). In the presence of 3 M NaCl the association constants of CyPTIs with α-chymotrypsin increased a few hundred fold. Taking advantage of this phenomenon, a high concentration of NaCl was used to isolate the inhibitors by affinity chromatography on immobilized chymotrypsin. It was found that although one of them, CyPTI IV, had split the Asn25–Gly26 peptide bond, its inhibitory activity remained unchanged. The hydrolyzed bond is located downstream of the reactive site. Presumably, the inhibitor is a naturally occurring, double-chain protein arising during posttranslational modifications.     

Selenium Improves Physiological Parameters and Alleviates Oxidative Stress in Shoots of Lead-Exposed <Emphasis Type="Italic">Vicia faba</Emphasis> L. <Emphasis Type="Italic">minor</Emphasis> Plants Grown Under Phosphorus-Deficient Conditions

The goal of the study was to investigate the effects of exogenous selenium (Se) on the tolerance of faba bean plants to lead (Pb) stress under P-deficient conditions. The bean plants were grown for 2 weeks on Hoagland solution supplied with Pb (0, 50 μM) and Se (0, 1.5, or 6 μM), separately or simultaneously. It was shown that Pb did not affect shoot growth but caused major damage in the leaves, which was accompanied by Pb accumulation in these tissues. The exposure of the shoots to Pb led to significant changes in the biochemical parameters: the MDA content, glutathione peroxidase (GSH-Px), guaiacol peroxidase (GPOX), and catalase (CAT) activity increased. Furthermore, Pb intensified O ₂ ^?? and H₂O₂ production. Both the Se concentrations used increased the chlorophyll b, chlorophyll a+b, and carotenoid content in the faba bean plants. Selenite also generally enhanced CAT, GPOX, and GSH-Px activities and the T-SH level. Our results imply that the degree of disturbances caused by Pb could be partially ameliorated by Se supplementation. Selenite at a lower dose alleviated Pb toxicity by decreased H₂O₂ and O ₂ ^?? production and decreased the GSH-Px, GPOX, and CAT activities. The beneficial effect of the higher selenite concentration could be related to reduction of lipid peroxidation in the shoots of the Pb-treated plants. However, the effect of Se on the Pb-stressed plants greatly depended on the selenite dose in the nutrient solution.     

Mechanism of actin filament bundling by fascin

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four β-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in β-trefoil domains 1 and 3. The site in β-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in β-trefoil-3 is related by pseudo-2-fold symmetry to that in β-trefoil-1. The two sites are ～5 nm apart, resulting in a distance between actin filaments in the bundle of ～8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.     

Genotyping by REA-PFGE of Listeria monocytogenes isolated from clinical, environmental and food samples

Listeria monocytogenes strains isolated from clinical food and environmental samples were genotyped by Restriction Enzyme Analysis with Pulsed Field Gel Electrophoresis (REA-PFGE) using ApaI and AscI enzymes according to PulseNet Europe procedure. Analysis of DNA fragments profiles obtained by AscI digestion demonstrated presence of 62 REA-PFGE profiles grouped in 2 lineages (FI, FII). Diversity of strains source among both lineages was observed. Statistical analysis showed, that strains isolated from clinical samples more frequently are included to lineage FI, then lineage FII. Non-clinical strains were more frequently included to lineage FII. Combined analysis of REA-PFGE profiles for ApaI and AscI enzymes showed 8 unique pulsotypes characteristic for two or more L. monocytogenes isolates. Moreover researched L. monocytogenes strains were analyzed by multiplex-PCR according Doumith et al methodology. PCR-group 4B was most frequent among strains isolated from clinical samples. Correlation between PCR-group and pulsotype was observed only in few cases.     

Oenothein B's contribution to the anti-inflammatory and antioxidant activity of Epilobium sp

Willow herb tea or preparation are available and relatively popular in the European market, and claimed to be effective inter alia because of their anti-inflammatory activity. The present study is therefore aimed at comparing the anti-inflammatory and antioxidant activity of extracts of the three most popular Epilobium species (E. angustifolium, E. hirsutum and E. parviflorum) and at juxtaposing this activity against the dominating compounds from the following extracts: oenothein B (OeB), quercetin-3-O-glucuronide and myricetin-3-O-rhamnoside. The phytochemical analysis of the extracts has shown that OeB quantities vary between 20% and 35%, while flavonoids content does not exceed 2%. All extracts have inhibited the activity of hyaluronidase and lipoxygenase with IC₅₀ around 5 μg/ml and 25 μg/ml. The inhibition of hyaluronidase is related with the presence of OeB, a strong inhibitor of this enzyme (IC₅₀ 1.1 μM). Additionally, the extracts inhibited myeloperoxidase (MPO) release from stimulated neutrophils. OeB inhibited MPO release similarly to the anti-inflammatory drug indomethacin with IC₅₀ 7.7 μM and 15.4 μM, respectively. Tested extracts significantly reduced the production of reactive oxygen species (ROS) from f-MLP and PMA induced neutrophils with IC₅₀ 5 μg/ml and 25 μg/ml, respectively. The flavonoids content seems to exert little influence on extracts’ activity, contrary to OeB, whose high concentration explains the activity of extract obtained from Epilobium. Tested currently marketed Epilobium preparations are often wrongly assigned, but we should stress that the level of OeB in all tested herbs was high and always exceeded 2% in raw material.     

Analysis of genetic relationships and antimicrobial susceptibility of Escherichia coli isolated from Clethrionomys glareolus

Eleven strains of Escherichia coli were isolated from 54 bank voles living in the LomZa Landscape Park of the Narew River Valley, indicating that E. coli is not common in the alimentary tract of these mammals. On the basis of pulsed-field gel electrophoresis and computer-assisted analysis, the isolates were grouped into six genotypes at similarities of 39%. Chromosome length of E. coli under study differed by as much as 900 kb, ranging 2.7-3.6 Mb. All strains were susceptible to amikacin and ciprofloxacin, whereas, for tetracycline, streptomycin, ampicillin, and cefonicid, different results were noted. No differences were detected among the plasmid complements of eight strains (73%), for which plasmid profiles revealed the presence of two plasmidic bands. One, three and four plasmids were observed in a plasmid pattern of single isolates. The observation from the study indicated the high genetic polymorphism among the isolates recovered from the animals of one species living in the same environment.     

Subspecies of the Eeuropean beaver<Emphasis Type="Italic">Castor fiber</Emphasis> Linnaeus, 1758

Subspecific relationships of the European beaverCastor fiber have been obscured by failure to follow the rules of the International Code of Zoological Nomenclature and remain an open issue. Inconsequence in the use of subspecies names caused a nomenclatural as well as a taxonomic confusion. We discuss these controversial and incorrect nomenclatural decisions, and recognize 9 nominal subspecies of European beaver, bearing 9 available names:C. f. fiber Linnaeus, 1758,C. f. galliae Geoffroy, 1803, C. f. albicus Matschie, 1907,C. f. vistulanus Matschie, 1907,C. f. pohlei Serebrennikov, 1929,C. f. birulai Serebrennikov, 1929,C. f. tuvinicus Lavrov, 1969,C. f. belorussicus Lavrov, 1981, andC. f. orientoeuropaeus Lavrov, 1981.