Detection of Escherichia coli O157:H7 in raw meat by immunomagnetic separation and multiplex PCR

The aim of this research was to elaborate fast and sensitive method ofdetection of E. coli O157:H7 in food samples. Raw ground meat obtained from retail was artificially inoculated with low numbers of E. coli O157:H7. 18 h enrichment culture allowed pathogenic bacteria to multiply to the levels detectable in multiplex PCR. Immunomagnetic separation with magnetic beads coated with an antibody against E. coli O157:H7 were used to concentrate target bacteria and to separate PCR inhibitors. A portion of the bacterial suspension was used in a multiplex PCR to amplify eae (attaching and effacing) gene, stx (shiga toxin) genes and 90 kbp plasmid. The sensitivity of E. coli O157:H7 detection method was shown to be 1 cfu per 25 g of food sample. The total analysis can be completed within 24 h, whilst traditional methods involves enrichment, direct plating and confirmation tests with entire time at least 3 days.     

ENOS,ET-1 and ETB-R immunoreactivities in the porcine mesometrial lymphatics during the estrous cycle

Abstract: Immunohistochemical localization and distribution of nitric oxide synthase (eNOS), endothelin (ET-1) and endothelin beta receptor (ETB-R) were investigated in precollector and collector lymph vessels in the broad ligament of the uterus during different phases of the estrous cycle in pigs. The polyclonal antibody for ET-1 and ETB-R and monoclonal antibody for eNOS isoform were used to perform observations on the light microscopic level. Immunoreactivities to ET-1, ETB-R and eNOS were observed in the endothelium of precollector and collector lymphangions but not in smooth muscle cells of the lymphatics examined. The staining for eNOS in the endothelial cells of all studied lymphatic vessels was stronger comparing to ET-1 and ETB-R. During the estrous cycle, only eNOS showed the correlation with the particular phases of the estrous cycle. The differences between ET-1 and ETB-R immunoreactivities were very slight and rather independent of the size or type of the lymphatic lymphangions and estrous cycle. The highest immunoreactivity level for eNOS was displayed by collector lymphangions with widened lumen in the follicular phase comparing to the precollector ones. During the luteal phase, a slight decrease in the reaction intensity was observed. The immunoreactivities for ET-1 in the endothelium of the studied vessels was not comparable with the presence or with the reactivity level of ETB-R. Optically stronger immunoreaction for ETB-R was observed in the cytoplasm of collector lymphangions in the follicular phase. eNOS, ET-1 and ETB-R were also present in the cytoplasm of the lymphatic valves. These results suggest that ET-1 and eNOS can play a role in the mechanisms regulating the vascular contractile activity, promoting lymph flow during the estrous cycle in the porcine broad ligament.     

Involvement of nitric oxide in inflammation of ovaries in gilts

NADPH-diaphorase (NADPH-d) and an inducible type of nitric oxide synthase (iNOS) were demonstrated in porcine ovaries after unilateral infusion of bacteria into the hilus of an ovary. In group I one ml of saline was infused into the hilus of each ovary from the 15th day to the 19th day of the estrous cycle. In group II one ml of bacterial suspension (10(9) colony forming units of Escherichia coli, Staphylococcus aureus and Corynebacterium pyogenes, in a proportion 1:1:1, respectively) in saline was infused into the hilus of one ovary on days corresponding to those of the control group (gr. I), whereas saline was infused into the contralateral ovary. The ovaries were collected on the 7th day of the next estrous cycle. In the bacteria-treated ovary, the activity of NADPH-d was higher in the endothelium of blood vessels, corpora lutea and follicular walls in comparison to that observed in the respective structures of the contralateral ovary. The highest activity of NADPH-d was found in the vascular endothelium in the bacteria-infused ovary. Vascular smooth muscle cells found in both ovaries of the bacteria-treated gilts were more intensely stained for NADPH-d than those in control animals. After bacteria administration, the intensity of NADPH-d reaction in all the structures of both ovaries in group II was higher than in control group. The strongest immunostaining for iNOS was observed in all structures of the bacteria-infused ovary. In the contralateral ovary, iNOS-immunoreactivity was weaker but still stronger than that in control group. The present results revealed that infusions of bacteria into the hilus of one ovary enhanced the activity of NADPH-d and immunoreactivity for iNOS in both porcine ovaries. However, the activity of both enzymes was higher in the bacteria-infused ovary than in the contralateral one. These data suggest that locally synthesized NO can mediate an inflammatory effect of bacteria in the porcine ovaries.     

Capacitance and resistance of the bilayer lipid membrane formed of phosphatidylcholine and cholesterol

Capacity and electric resistance of lipid membranes composed of lecithin and cholesterol were determined. The components were chosen for the study because they were present in biological membranes. Capacitance of the lecithin and cholesterol membranes amounts to 0.38 and 0.61 microF/cm(2), and resistance to 1.44(10(4)and 2.12(10(6)Omega cm(2), respectively. A 1:1 complex appears as a result of lecithin-cholesterol membrane formation. Parameters of the membrane formed of the lecithin-cholesterol complex were determined: surface concentration (Gamma(3)), capacitance (C(3)), and conductance (R;(3)(-1), as well as the stability constant (K) of the complex. The mean values of those magnitudes are as follows: 4.265(10(-6)mol/m(2), 0.54 microF/cm(2), 1.381(10(-6)Omega(-1)cm(-2)and 3.748(10(7), respectively.     

Effects of pyrroline and pyrrolidine nitroxides on lipid peroxidation in heart tissue of rats treated with doxorubicin

Protection from doxorubicin-induced lipid peroxidation in vivo by two pyrroline and pyrrolidine nitroxides, Pirolin, PL, and Pirolid, PD, was examined in the heart tissue of rats treated with this drug. The level of lipid peroxidation was estimated on the basis of MDA content. A considerable (three-fold) increase in the MDA amount was found in heart homogenates from rats injected with doxorubicin, whereas no significant changes in MDA content compared to control were observed in cardiomyocytes treated with the nitroxides (Pirolin or Pirolid) only. Pirolin injected simultaneously with doxorubicin showed antioxidative effect and markedly attenuated lipid peroxidation in the heart tissue caused by this drug. In contrast to Pirolin, structurally related Pirolid was ineffective in the protection of heart myocytes from DOX-induced lipid peroxidation.     

Enhancing phytoremediative ability of Pisum sativum by EDTA application

The aim of our research was to demonstrate how the presence of EDTA affects resistance of pea plants to Pb and Pb-EDTA presence, and to show the effectivity of lead ions accumulation and translocation. It was determined that EDTA not only increased the amount of Pb taken up by plants but also Pb ion transport through the xylem and metal translocation from roots to stems and leaves. It can be seen in the presented research results that addition of the chelator with Pb limited metal phytotoxicity. We also demonstrated a significant effect of EDTA not only on Pb accumulation and metal transport to the aboveground parts but also on the profile and amount of thiol compounds: glutathione (GSH), homoglutathione (hGSH) or phytochelatins (PCs), synthesized by the plants. We observed a significant effect of the synthetic chelator on increasing the level of Pb accumulation in roots of plants treated with Pb including EDTA (0.5 and 1 mM). Pisum sativum plants treated only with 1 mM Pb(NO3)2 accumulated over 50 mg Pb x g(-1) dry wt during 4 days of cultivation. Whereas in roots of pea plants exposed to Pb+0.5 mM EDTA 35% more Pb was observed. When 1 mM EDTA was applied roots of pea accumulated over 67% more metal. The presence of EDTA also increased metal uptake and transport to the aboveground parts. In pea plants treated only with 1 mM lead nitrate less than 3 mg Pb x g(-1) dry wt was transported, whereas in P. sativum treated with Pb-EDTA doubled amount of Pb was observed in stems and leaves.     

Acid-base equilibria at interface separating electrolyte solution and lipid bilayer formed from phosphatidylcholine

The dependence of the interfacial tension of a lipid membrane formed from phosphatidylcholine on the pH of the aqueous solution has been studied. The model describing the H(+) and OH(-) ions adsorption in the bilayer lipid surface has been presented in this work. We take suitable equations to describe the dependence of interfacial tension of a lipid bilayer membrane on H(+) and OH(-) ion concentrations. A theoretical equation is derived to describe this dependence in the whole pH range.     

Septal and lateral wall localization of PBP5, the major D,D‐carboxypeptidase of Escherichia coli,requires substrate recognition and membrane attachment

The distribution of PBP5, the major D,D‐carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild‐type cells and in mutants lacking one or more D,D‐carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane‐bound form localized to the developing septum and restored wild‐type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.     

Novel TNF-α converting enzyme (TACE) inhibitors as potential treatment for inflammatory diseases

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors has led to an acetylene containing series that demonstrates sub-nanomolar potency (K(i)) as well as excellent activity in human whole blood. These studies led to the discovery of highly potent TACE inhibitors with good DMPK profiles.     

The AAA‐type ATPase AtSKD1 contributes to vacuolar maintenance of Arabidopsis thaliana

The vacuole is the most prominent organelle of plant cells. Despite its importance for many physiological and developmental aspects of plant life, little is known about its biogenesis and maintenance. Here we show that Arabidopsis plants expressing a dominant‐negative version of the AAA (ATPase associated with various cellular activities) ATPase AtSKD1 (SUPPRESSOR OF K⁺ TRANSPORT GROWTH DEFECT1) under the control of the trichome‐specific GLABRA2 (GL2) promoter exhibit normal vacuolar development in early stages of trichome development. Shortly after its formation, however, the large central vacuole is fragmented and finally disappears completely. Secretion assays with amylase fused to the vacuolar sorting signal of Sporamin show that dominant‐negative AtSKD1 inhibits vacuolar trafficking of the reporter that is instead secreted. In addition, trichomes expressing dominant‐negative AtSKD1 frequently contain multiple nuclei. Our results suggest that AtSKD1 contributes to vacuolar protein trafficking and thereby to the maintenance of the large central vacuole of plant cells, and might play a role in cell‐cycle regulation.