Highly Invasive Listeria monocytogenes Strains Have Growth and Invasion Advantages in Strain Competition

Multiple Listeria monocytogenes strains can be present in the same food sample; moreover, infection with more than one L. monocytogenes strain can also occur. In this study we investigated the impact of strain competition on the growth and in vitro virulence potential of L. monocytogenes. We identified two strong competitor strains, whose growth was not (or only slightly) influenced by the presence of other strains and two weak competitor strains, which were outcompeted by other strains. Cell contact was essential for growth inhibition. In vitro virulence assays using human intestinal epithelial Caco2 cells showed a correlation between the invasion efficiency and growth inhibition: the strong growth competitor strains showed high invasiveness. Moreover, invasion efficiency of the highly invasive strain was further increased in certain combinations by the presence of a low invasive strain. In all tested combinations, the less invasive strain was outcompeted by the higher invasive strain. Studying the effect of cell contact on in vitro virulence competition revealed a complex pattern in which the observed effects depended only partially on cell-contact suggesting that competition occurs at two different levels: i) during co-cultivation prior to infection, which might influence the expression of virulence factors, and ii) during infection, when bacterial cells compete for the host cell. In conclusion, we show that growth of L. monocytogenes can be inhibited by strains of the same species leading potentially to biased recovery during enrichment procedures. Furthermore, the presence of more than one L. monocytogenes strain in food can lead to increased infection rates due to synergistic effects on the virulence potential.     

Accessibility of an epitope common to all histone H3 variants in folded and unfolded chromatin as studied by a monoclonal antibody

In a recent publication the isolation and some characteristics of an anti-histone 3 monoclonal antibody, 1GB3 were described (Muller et al. FEBS Lett. 182: 459–464, 1985). We now report that the epitope recognized is phylogenetically conserved and located in the N-terminal part of H3, most likely between residues 40 and 50. Using the ELISA technique we found this region to be accessible in chromatin to the monoclonal antibody. The effect of non-ionic detergents on the adsorbtion of chromatin on microtiter plates was studied in this context.Immunological analysis of the reaction of the monoclonal antibody with chromatin by immunoinhibition and immunosedimentation shows that the H3 epitope is accessible in both folded and unfolded chromatin fibre as well as in high- and low-molecular weight oligonucleosomes.Abbreviations BSA Bovine srum albumin - mab Monoclonal antibody - PBS Phosphate buffered saline - PMSF Phenylmethyl sulfonyl fluoride     

Regulation of Ca2+ handling by phosphorylation status in mouse fast- and slow-twitch skeletal muscle fibers

The effects ofphosphorylation status on Ca²⁺release and Ca²⁺ removal werestudied in fast-twitch flexor digitorum brevis and slow-twitch soleusskeletal muscle fibers enzymatically isolated from wild-type andphospholamban knockout (PLBko) mice. In all fibers the adenosine3',5'-cyclic monophosphate-dependent protein kinase (PKA)inhibitor H-89 decreased the peak amplitude of the intracellularCa²⁺ concentration([Ca²⁺]) transient fora single action potential, and the PKA activator dibutyryl adenosine3',5'-cyclic monophosphate (DBcAMP) reversed this effect,indicating modulation of Ca²⁺release by phosphorylation status in all fibers. H-89 decreased thedecay rate constant of the[Ca²⁺] transient andDBcAMP reversed this effect only in phospholamban-expressing fibers(wild-type soleus), indicating modulation ofCa²⁺ removal only in the presenceof phospholamban. A high basal level of PKA phosphorylation in soleusfibers maintained under our control conditions was indicated bythe lack of effect of direct application of DBcAMP onCa²⁺ release orCa²⁺ removal in wild-type or PLBkosoleus fibers and was confirmed by analysis of phospholamban fromwild-type soleus fibers.

     

Microwave-assisted solid-phase peptide synthesis of the 60–110 domain of human pleiotrophin on 2-chlorotrityl resin

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60–110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N′-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60–110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys⁷⁷–Cys¹⁰⁹ bond, followed by iodine oxidation to form the Cys⁶⁷–Cys⁹⁹ bond.     

Efficient synthesis of exomethylene- and keto-exomethylene-d-glucopyranosyl nucleoside analogs as potential cytotoxic agents

A novel series of exomethylene- and keto-exomethylene-d-glucopyranonucleosides with thymine, uracil, and 5-fluorouracil as heterocyclic bases have been designed and synthesized. Wittig condensation of the 3-keto glucoside 1 gave the corresponding 1,2:5,6-di-O-isopropylidene-3-deoxy-3-methylene-d-glucofuranose (2), which after hydrolysis and acetylation led to the precursor 1,2,4,6-tetra-O-acetyl-3-deoxy-3-methylene-d-glucopyranose (4).Compound 4 was condensed with silylated thymine, uracil, and 5-fluorouracil, respectively, deacetylated and acetalated to afford 1-(3′-deoxy-4′,6′-O-isopropylidene-3′-methylene-β-d-glucopyranosyl)pyrimidines 7a–c. Oxidation of the free hydroxyl group in the 2′-position of the sugar moiety led to the formation of the labile 1-(3′-deoxy-4′,6′-O-isopropylidene-3′-methylene-β-d-glucopyranosyl-2′-ulose)pyrimidines 8a–c. Finally, deisopropylidenation of the resulted derivatives 8a–c afforded the diol nucleosides 9a–c. The target keto-exomethylene analogs 9a–c were more cytostatic against a variety of tumor cell lines than the corresponding saturated-hydroxy-exomethylene derivatives 6. In particular, the 5-fluorouracil derivative 9c was highly cytostatic at an IC₅₀ (50% inhibitory concentration) ranging between 0.56 and 9.4 μg/mL, which was comparable to the free parental 5-fluorouracil base.     

Growth and biomass production with enhanced β‐glucan and dietary fibre contents of Ganoderma australe ATHUM 4345 in a batch‐stirred tank bioreactor

In this study we maximized biomass production by the basidiomycete Ganoderma australe ATHUM 4345, a species of pharmaceutical interest as it is a valuable source of nutraceuticals, including dietary fibers and glucans. We used the Biolog FF MicroPlate to screen 95 different carbon sources for growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint, which is useful in selecting components for media optimization of maximum biomass production. Response surface methodology, based on the central composite design was applied to explore the optimum concentrations of carbon and nitrogen sources of culture medium in shake flask cultures. When the improved culture medium was tested in a 20‐L stirred tank bioreactor, using 13.7 g/L glucose and 30.0 g/L yeast extract, high biomass yields (10.1±0.4 g/L) and productivity of 0.09 g L^?1 h^?1 were obtained. The yield coefficients for total glucan and dietary fibers on biomass formed were 94.82±6 and 341.15±12.3 mg/g mycelium dry weight, respectively.     

Insecticide resistance in Bemisia tabaci from Cyprus

Abstract A comprehensive study on the Bemisia tabaci (biotype B) resistance to neonicotinoid insecticides imidacloprid, acetamiprid and thiamethoxam, and pyrethroid bifenthrin was conducted in Cyprus. The resistance level to eight field‐collected B. tabaci populations was investigated. The activities of enzymes involved in metabolic detoxification and the frequencies of pyrethroid and organophosphates target site resistance mutations were determined. Moderate to high levels of resistance were detected for imidacloprid (resistance factor [RF] 77–392) and thiamethoxam (RF 50–164) while low resistance levels were observed for acetamiprid (RF 7–12). Uniform responses by the Cypriot whiteflies could be observed against all neonicotinoid insecticides. No cross‐resistance between the neonicotinoids was detected as well as no association with the activity of the P450 microsomal oxidases. Only imidacloprid resistance correlated with carboxylesterase activity. Low to extremely high resistance was observed for insecticide bifenthrin (RF 49–1 243) which was associated with the frequency of the resistant allele in the sodium channel gene but not with the activity of the detoxification enzymes. Finally, the F331W mutation in the acetylcholinesterase enzyme ace1 gene was fixed in all B. tabaci populations from Cyprus.     

Seasonal vertical distribution and diel migration of zooplankton in a temperate stratified lake

The investigation of the vertical distribution of the zooplankton community in the temperate Lake Trichonis during four seasons in 2005, showed the existence of vertical segregation among species, ontogenetic stages and sexes within and between the major groups. In each season, the two or three more abundant rotifer species distributed at separate depth layers, while this feature was maintained during the entire 24 h period, since no diel vertical migrations (DVM) were performed. In contrast, the crustacean community, comprised mainly by the calanoid copepod Eudiaptomus drieschi and the cladoceran Diaphanosoma orghidani, showed various patterns of DVM, being more pronounced in spring and summer. Females of E. drieschi distributed deeper than males, while the copepod nauplii were found mainly in the surface layer in all four seasons. Temperature was the most important abiotic factor affecting directly and indirectly the vertical distribution and migration of various species. During stratification, the metalimnion was the most productive layer in Lake Trichonis, having maximum values of dissolved oxygen and low transparency due to high concentration of organic matter and phytoplankton. The DVM patterns of the crustaceans indicate that the metalimnion acts probably as a daylight refuge against predation by Atherina boyeri, which is the dominant planktivorous fish in the lake.     

Embryoid bodies from mouse stem cells express oxytocin receptor, Oct-4 and DAZL

Oxytocin is a nine amino acid peptide involved in a wide spectrum of physiological functions; predominantly those concerning reproduction and differentiation are of interest. Oxytocin receptors are expressed at early developmental stages of mammals, suggesting that oxytocin might be involved in the determination of the germ stem cell line, at the very early stages of mammalian development. In this respect, the proximate aim of the present study was to confirm and further analyze the existence of oxytocin receptors at a very early level of cell commitment, that is, the determination of germ cells derived from embryoid bodies. To achieve our purpose we have cultured mouse embryonic stem cells under conditions inducing formation of embryoid bodies. In this work, ES cells were allowed to aggregate in a novel medium, “Stefanidis medium” from day 0 to day 14 until formed EBs. RNA was isolated from EBs and using RT-PCR we showed that EBs expressed Oct-4, OTR, OT, and DAZL. To demonstrate simultaneous expression immunocytochemistry was preformed, in which EBs showed strong immunoreactivity for both, OTR and DAZL molecular markers. We found that 35% of the cells displayed OTR, using flow cytometry. In addition, this novel medium showed to increase OTR mRNA. We propose, that at least in murine induced embryoid bodies there is simultaneous expression of oxytocin receptors and germ cell markers (DAZL) in many cells (expressing Oct-4). We thus conclude that, the oxytocin might indeed be a molecule playing a leading role in germ cell determination.