Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite

The liver stages of malaria are clinically silent but have a central role in the Plasmodium life cycle. Liver stages of the parasite containing thousands of merozoites grow inside hepatocytes for several days without triggering an inflammatory response. We show here that Plasmodium uses a PEXEL/VTS motif to introduce the circumsporozoite (CS) protein into the hepatocyte cytoplasm and a nuclear localization signal (NLS) to enter its nucleus. CS outcompetes NFkappaB nuclear import, thus downregulating the expression of many genes controlled by NFkappaB, including those involved in inflammation. CS also influences the expression of over one thousand host genes involved in diverse metabolic processes to create a favorable niche for the parasite growth. The presence of CS in the hepatocyte enhances parasite growth of the liver stages in vitro and in vivo. These findings have far reaching implications for drug and vaccine development against the liver stages of the malaria parasite.     

Antibmicrobial susceptibility of Serratia marcescens isolated from hospitalized patients in 2003 - 2005

500 strains of Serratia marcescens isolated in 2003-2005 were examined for drug susceptibility. By using several phenotypic methods it was shown that 67.6% of these strains produced ESBLs. Strains ESBL(-) and ESBL(+) were compared, paying special attention to their susceptibility to various antibiotics. It was revealed that strains ESBL(+) were much more resistant to majority of the investigated drugs. The biggest differences were in the case of amikacin and gentamicin, sensitive about 50% of ESBL(-) and 10% of ESBL(+), ciprofloxacin, sensitive 42% of ESBL(-) and 6.3% of ESBL(+) and trimethoprim/ sulphametoxazole, sensitive 45.8% of ESBL(-) and 9.4% of ESBL(+). Strains ESBL(-) retained a high susceptibility to ceftazidime (68.9%) and cefepime (71%). All strains ESBL(-) as well as ESBL(+) were susceptible to imipenem and meropenem. 78.9% of ESBL(-) and 67.3% of investigated ESBL(+) were susceptible to piperacillin/ tazobactam.     

Spectral analysis of Delayed Luminescence from human skin as a possible non-invasive diagnostic tool

In vivo measurements of Delayed Luminescence (DL), the low-level photo-induced emission which lasts for a longer time after switching off the excitation light, have been performed on human skin, with the aim to develop a technique for optical biopsy. Preliminary tests have been performed on healthy volunteers, measuring the time decays of the spectral components (λ_emiss = 400–800 nm) starting 10 μs after switching off the excitation (λ_exc = 337 nm). Significant differences in the decay trends of DL from different subjects were revealed and quite a good reproducibility for the same subject was observed. The modeling of experimental data has been examined in detail in order to get parameters, characterizing the theoretical fit, whose changes may be correlated with age differences and seasonal variations. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

RRM proteins interacting with the cap region of topoisomerase I

RNA recognition motif (RRM) domains bind both nucleic acids and proteins. Several proteins that contain two closely spaced RRM domains were previously found in protein complexes formed by the cap region of human topoisomerase I, a nuclear enzyme responsible for DNA relaxation or phosphorylation of SR splicing proteins. To obtain molecular insight into specific interactions between the RRM proteins and the cap region of topo I we examined their binary interactions using the yeast two-hybrid system. The interactions were established for hnRNP A1, p54(nrb) and SF2/ASF, but not for hnRNP L or HuR. To identify the amino acid pattern responsible for binding, experimental mutagenesis was employed and computational modelling of these processes was carried out. These studies revealed that two RRM domains and six residues of the consensus sequence are required for the binding to the cap region. On the basis of the above data, a structural model for the hnRNP A1-topoisomerase I complex was proposed. The main component of the hnRNP A1 binding site is a hydrophobic pocket on the beta-surface of the first RRM domain, similar to that described for Y14 protein interacting with Mago. We demonstrated that the interaction between RRM domains and the cap region was important for the kinase reaction catalyzed by topoisomerase I. Together with the previously described inhibitory effect of RRM domains of SF2/ASF on DNA cleavage, the above suggests that the binding of RRM proteins could regulate the activity of topoisomerase I.     

Phylogenetic Analysis of Orgyia pseudotsugata Single-nucleocapsid Nucleopolyhedrovirus

The Douglas-fir tussock moth Orgyia pseudotsugata （Lepidoptera： Lymantriidae） is a frequent defoliator of Douglas-fir and true firs in western USA and Canada. A single nucleopolyhedrovirus （SNPV） isolated from O. pseudotsugata larvae in Canada （OpSNPV） was previously analyzed via its polyhedrin gene, but is phylogenetic status was ambiguous. Sequences of four conserved baculovirus genes, polyhedrin, lef-8, pif-2 and dpol, were amplified from OpSNPV DNA in polymerase chain reactions using degenerate primer sets and their sequences were analyzed phylogenetically. The analysis revealed that OpSNPV belongs to group Ⅱ NPVs and is most closely related to SNPVs that infect O. ericae and O. anartoides, respectively. These results show the need for multiple, concatenated gene phylogenies to classify baculoviruses.     

Novel voltammetric biosensor for determining acrylamide in food samples

Recent findings showing that acrylamide is formed in heat-treated foods rich in asparagine and reducing sugars such as glucose, have accelerated the needs for the development of new analytical methods to determine this potential human carcinogen. Acrylamide forms adduct with hemoglobin (Hb) as a result of the reaction with the alpha-NH2 group of N-terminal valine of Hb. This interaction is the basis of a new voltammetric biosensor to detect acrylamide. The biosensor was constructed using a carbon-paste electrode modified with hemoglobin (Hb), which contains four prosthetic groups of heme--Fe(III). Such an electrode displays a reversible reduction/oxidation process of Hb-Fe(III)/Hb-Fe(II). Interaction between Hb and acrylamide was observed through decreasing of the peak current of Hb-Fe(III) reduction. The electrodes presented a very low detection limit (1.2 x 10(-10)M). The validation made in the matrix obtained by water extraction of potato chips showed that the electrodes presented are suitable for the direct determination of acrylamide in food samples.     

Effect of the phytoestrogen, genistein-8-C-glucoside, on Chinese hamster ovary cells in vitro

Genistein-8-C-glucoside (G8CG) belongs to isoflavones, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants. A number of studies on flavonoids show their cardioprotective and antiosteoporosis properties in in vitro and in vivo models. As a phytoestrogen, genistein has recently generated interest as a potential anticancer and antiatherogenic agent. Several flavonoids are known as antioxidants and scavengers of free oxygen radicals. In the current investigation we used glycosylated genistein (genistein-8-C-glucoside) from flowers of lupine (Lupinus luteus L.). Many authors have found that the action of genistein is not so simple, although many reports conducted in vitro have demonstrated that it is cytotoxic and genotoxic. Therefore, the cytotoxic and genotoxic effects of this compound in Chinese hamster ovary cells (line CHO) were studied. A colorimetric MTT assay to assess cytotoxicity and a Comet assay for the detection of DNA damage were used. Apoptosis was determined by the Hoechst 33258/propidium iodide staining technique. We have also demonstrated antioxidant properties of G8CG. The level of reactive oxygen species generated by G8CG alone and/or H2O2 was evaluated with fluorescence probes: dichlorofluorescein-diacetate (DCFDA) by flow cytometry. The cells were exposed to various concentrations of genistein-8-C-glucoside (1-290 microM) and hydrogen peroxide (10-130 microM) and the effect of G8CG alone or in combination with H2O2 was determined. The results reveal that G8CG at concentrations higher than 10 microM significantly reduced cell viability, induced apoptosis and DNA damage. However at lower concentrations (5 and 7.5 microM), G8CG showed antioxidant properties, but had no cytotoxic or genotoxic activity.     

Molecular mechanisms underlying the probiotic effects of Escherichia coli Nissle 1917 involve ZO-2 and PKCzeta redistribution resulting in tight junction and epithelial barrier repair

The probiotic Escherichia coli strain Nissle 1917 (EcN) has been used for decades in human medicine in Central Europe for the treatment and prevention of intestinal disorders and diseases. However, the molecular mechanisms underlying its beneficial effects are only partially understood. To identify molecular responses induced by EcN that might contribute to its probiotic properties polarized T84 cells were investigated employing DNA microarrays, quantitative RT-PCR, Western blotting, immunofluorescence and specific protein kinase C (PKC) inhibitors. Polarized T84 epithelial cell monolayers were used as a model to monitor barrier disruption by infection with the enteropathogenic E. coli (EPEC) strain E2348/69. Co-incubation of EPEC with EcN or addition of EcN following EPEC infection abolished barrier disruption and, moreover, restored barrier integrity as monitored by transepithelial resistance. DNA-microarray analysis of T84 cells incubated with EcN identified 300+ genes exhibiting altered expression. EcN altered the expression, distribution of zonula occludens-2 (ZO-2) protein and of distinct PKC isotypes. ZO-2 expression was enhanced in parallel to its redistribution towards the cell boundaries. This study provides evidence that EcN induces an overriding signalling effect leading to restoration of a disrupted epithelial barrier. This is transmitted via silencing of PKCzeta and the redistribution of ZO-2. We suggest that these properties contribute to the reported efficacy in the treatment of inflammatory bowel diseases and in part rationalize the probiotic nature of EcN.