Gastrointestinal absorption and metabolism of apple polyphenols ex vivo by the pig intestinal mucosa in the Ussing chamber

Polyphenols contained in food have various positive effects on human health. The absorption and metabolism of polyphenols in the intestinal tract needs to be studied to estimate these effects. The Ussing chamber technique was used to investigate the transport behavior of apple polyphenols through pig small intestinal mucosa, which served as a model for human gastrointestinal mucosa. The identities and concentrations of polyphenols and their metabolites in the half-chambers (luminal and basolateral) within an incubation period of 4 h were determined by HPLC–MS/MS and HPLC–DAD (DAD = diode-array detection). Flux values were also measured. It was found that 5-caffeoylquinic acid and caffeic acid were absorbed and translocated to the basolateral side (1.9 and 3.7%, respectively), but other compounds, including glycosides of phloretin and quercetin, were observed without translocation. A Ussing chamber utilizing pig small intestinal mucosa is a suitable model for assessing the effect of apple polyphenols on mucosal integrity and nutrition absorption across porcine mucosa.     

Competing for blood: the ecology of parasite resource competition in human malaria–helminth co‐infections

Ecological theory suggests that co‐infecting parasite species can interact within hosts directly, via host immunity and/or via resource competition. In mice, competition for red blood cells (RBCs) between malaria and bloodsucking helminths can regulate malaria population dynamics, but the importance of RBC competition in human hosts was unknown. We analysed infection density (i.e. the concentration of parasites in infected hosts), from a 2‐year deworming study of over 4000 human subjects. After accounting for resource‐use differences among parasites, we find evidence of resource competition, priority effects and a competitive hierarchy within co‐infected individuals. For example reducing competition via deworming increased Plasmodium vivax densities 2.8‐fold, and this effect is limited to bloodsucking hookworms. Our ecological, resource‐based perspective sheds new light into decades of conflicting outcomes of malaria–helminth co‐infection studies with significant health and transmission consequences. Beyond blood, investigating within‐human resource competition may bring new insights for improving human health.     

The lipopolysaccharide of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum

The cell wall lipopolysaccharide of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum was obtained by the phenol-chloroform-petroleum ether and the hot phenol-water methods, respectively. It contained mannose, glucose, galacturonic acid, glucosamine, glycine, and small amounts of rhamnose, galactose and glucuronic acid. In addition to d-glycero-d-mannoheptose, the corespecific constituents 2-keto-3-deoxyoctonate and l-glycero-d-mannoheptose were found. Polyacrylamide gel-electrophoresis in the presence of sodium deoxycholate gave no indication for the presence of O-specific repeating units. Degradation of the lipopolysaccharide required 10% acetic acid (100° C, 2 h). The lipid A moiety contained the total of glucosamine of the lipopolysaccharide as well as small amounts of 2,3-diamino-2,3-dideoxy-glucose. It was phosphate-free. The fatty acid spectrum comprised 3-OH-14:0, 3-OH-16:0, and iso-3-OH-18:0 besides little 12:0, 14:0 and 16:0. Hydroxylaminolysis and sodium methylate treatment revealed all of the three hydroxy fatty acids to be amidebound.Abbreviations DOC sodium deoxycholate - PAGE polyacrylamide gel-electrophoresis     

Bloom forming Alexandrium ostenfeldii (Dinophyceae) in shallow waters of the Åland Archipelago,Northern Baltic Sea

In the past years, late summer blooms of the bioluminescent dinoflagellate Alexandrium ostenfeldii have become a recurrent phenomenon in coastal waters of the central and Northern Baltic Sea. This paper reports exceptionally high cell concentrations (10⁵ to 10⁶ cells L^?1) of the species found during bioluminescent blooms in 2003 and 2004 in a shallow embayment of the Åland archipelago at the SW coast of Finland. Clonal cultures were established for morphological, molecular, toxicological and ecophysiological investigations to characterize the Finnish populations and compare them to other global A. ostenfeldii isolates. The Finnish isolates exhibited typical morphological features of A. ostenfeldii such as large size, a prominent ventral pore and an orthogonally bent first apical plate. However, unambiguous differentiation from closely related Alexandrium peruvianum was difficult due to considerable variation of sulcal anterior plate shapes. The Finnish strains were genetically distinct from other isolates of the species, but phylogenetic analyses revealed a close relationship to isolates from southern England and an A. peruvianum morphotype from the Spanish Mediterranean. Together these isolates formed a distinct clade which was separated from a clade containing other Northern European, North American and New Zealand populations. Toxin analyses confirmed the presence of the PSP toxins GTX2, GTX3 and STX in both Finnish isolates with GTX3 being the dominant toxin. Total relative PSP toxin contents were moderate, ranging from approximately 6 to 15 fmol cell^?1 at local salinities of 5 and 10 psu, respectively. Spirolides were not detected. Salinity tolerance experiments showed that the Finnish isolates were well adapted to grow at the low salinities of the Baltic Sea. With a salinity range of approximately 6 to 20–25 psu, Baltic populations are physiologically distinct from their marine relatives. Vigorous production of different cyst types in the cultures suggest that cysts may play a crucial role in the survival and retainment of A. ostenfeldii populations in the Baltic Sea.     

Extremotolerance and Resistance of Lichens: Comparative Studies on Five Species Used in Astrobiological Research II. Secondary Lichen Compounds

Lichens, which are symbioses of a fungus and one or two photoautotrophs, frequently tolerate extreme environmental conditions. This makes them valuable model systems in astrobiological research to fathom the limits and limitations of eukaryotic symbioses. Various studies demonstrated the high resistance of selected extremotolerant lichens towards extreme, non-terrestrial abiotic factors including space exposure, hypervelocity impact simulations as well as space and Martian parameter simulations. This study focusses on the diverse set of secondary lichen compounds (SLCs) that act as photo- and UVR-protective substances. Five lichen species used in present-day astrobiological research were compared: Buellia frigida, Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, and Pleopsidium chlorophanum. Detailed investigation of secondary substances including photosynthetic pigments was performed for whole lichen thalli but also for axenically cultivated mycobionts and photobionts by methods of UV/VIS-spectrophotometry and two types of high performance liquid chromatography (HPLC). Additionally, a set of chemical tests is presented to confirm the formation of melanic compounds in lichen and mycobiont samples. All investigated lichens reveal various sets of SLCs, except C. gyrosa where only melanin was putatively identified. Such studies will help to assess the contribution of SLCs on lichen extremotolerance, to understand the adaptation of lichens to prevalent abiotic stressors of the respective habitat, and to form a basis for interpreting recent and future astrobiological experiments. As most of the identified SLCs demonstrated a high capacity in absorbing UVR, they may also explain the high resistance of lichens towards non-terrestrial UVR.     

In planta analysis of protein–protein interactions related to light signaling by bimolecular fluorescence complementation

The determination of protein-protein interactions is becoming more and more important in the molecular analysis of signal transduction chains. To this purpose the application of a manageable and simple assay in an appropriate biological system is of major concern. Bimolecular fluorescence complementation (BiFC) is a novel method to analyze protein-protein interactions in vivo. The assay is based on the observation that N- and C-terminal subfragments of the yellow-fluorescent protein (YFP) can only reconstitute a functional fluorophore when they are brought into tight contact. Thus, proteins can be fused to the YFP subfragments and the interaction of the fusion proteins can be monitored by epifluorescence microscopy. Pairs of interacting proteins were tested after transient cotransfection in etiolated mustard seedlings, which is a well characterized plant model system for light signal transduction. BiFC could be demonstrated with the F-box protein EID1 (empfindlicher im dunkelroten Licht 1) and the Arabidopsis S-phase kinase-related protein 1 (ASK1). The interaction of both proteins was specific and strictly dependent on the presence of an intact F-box domain. Our studies also demonstrate that etiolated mustard seedlings provide a versatile transient assay system to study light-induced subcellular localization events.     

Primary infection of mice with high titer inoculum respiratory syncytial virus: characterization and response to antiviral therapy

Intranasal infection of BALB/c mice with respiratory syncytial virus (RSV)-A2 (0.5 x 10(8) - 2.0 x 10(8) plaque-forming units, PFU) produced disease characterized by weight loss (2-3 g) and mortality (60%-100%) with the mean day of death ranging from 6-7 d after infection. The extent of RSV disease was inoculum titer-dependent and required a replication competent virus. Lung titers of virus peaked at 0.5-1 x 10(6) PFU/g wet weight. Bronchoalveolar lavage fluid (BALF) levels of IL-1beta, TNF-alpha, INF-gamma IL-12, IL-6, MIP-1alpha, RANTES, and protein were elevated, whereas IL-2, IL-4, IL-5, IL-13, and IL-10 were unchanged. Histological assessment of lungs revealed marked inflammatory pathology characterized by bronchiolitis, vasculitis, and interstitial pneumonia. Whole-body plethysmography revealed significant disease-associated deficits of respiratory function. Therapy with ribavirin administered either by the intranasal, subcutaneous, or oral route significantly reduced disease in a dose-dependent manner. Delaying the initiation of therapy resulted in a loss of activity for ribavirin. Synagis administered either intramuscularly as a single dose in prophylaxis or intranasally in prophylaxis, followed by therapy, also significantly reduced disease in a dose-dependent manner. Infection of mice with a high titer inoculum of RSV-A2 resulted in severe and fatal pulmonary disease that was responsive to treatment. This model may be useful to characterize the in vivo activity of experimental therapies for RSV infection.     

Composition and functional specificity of SWI2/SNF2 class chromatin remodeling complexes

By regulating the structure of chromatin, ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in the maintenance, transmission and expression of the eukaryotic genome. Although all known chromatin-remodeling complexes contain an ATPase as a central motor subunit, a number of distinct classes have been recognized. Recent studies have emphasized a more extensive functional diversification among closely related chromatin remodeling complexes than previously anticipated. Here, we discuss recent insights in the functional differences between two evolutionary conserved subclasses of SWI/SNF-related chromatin remodeling factors. One subfamily comprises yeast SWI/SNF, fly BAP and mammalian BAF, whereas the other subfamily includes yeast RSC, fly PBAP and mammalian PBAF. We review the subunit composition, conserved protein modules and biological functions of each of these subclasses of SWI/SNF remodelers. In particular, we will focus on the roles of specific subunits in developmental gene control and human diseases. Recent findings suggest that functional diversification among SWI/SNF complexes allows the eukaryotic cell to fine-tune and integrate the execution of diverse biological programs involving the expression, maintenance and duplication of its genome.     

Identifying clades in Asian Annonaceae: monophyletic genera in the polyphyletic Miliuseae

The tribe Miliuseae (Annonaceae) comprises six genera distributed in Asia: Alphonsea, Mezzettia, Miliusa, Orophea, Platymitra, and Phoenicanthus. A phylogenetic study to investigate the putative monophyly of the tribe and the intergeneric relationships is presented here. Nucleotide sequences of the plastid gene rbcL, trnL intron, and trnL-trnF intergenic spacer were analyzed from 114 Annonaceae taxa, including 24 Miliuseae species and two outgroups using maximum parsimony and Bayesian inference. The two data sets (rbcL and the trnL-trnF regions) were analyzed separately and in combination. Miliuseae were found to be polyphyletic due to the position of Mezzettia and are part of a large, predominantly Asian and Central-American clade (miliusoid clade). Although intergeneric relationships were poorly resolved, all genera, except Polyalthia, were monophyletic, supporting previous generic delimitation based on morphology. A group of three Polyalthia species seems the most likely sister group of Miliusa. Several infrageneric groups of Miliusa, Orophea, and Polyalthia are supported by both molecular and morphological data. No morphological synapomorphies have yet been found for the miliusoid clade. Molecular clades within the miliusoid clade, however, can be characterized by size and the shape of the outer petals, number of ovules per carpel, and the size of the fruits.     

Sensitivity of NS3 serine proteases from hepatitis C virus genotypes 2 and 3 to the inhibitor BILN 2061

Hepatitis C virus (HCV) displays a high degree of genetic variability. Six genotypes and more than 50 subtypes have been identified to date. In this report, kinetic profiles were determined for NS3 proteases of genotypes 1a, 1b, 2ac, 2b, and 3a, revealing no major differences in activity. In vitro sensitivity studies with BILN 2061 showed a decrease in affinity for proteases of genotypes 2 and 3 (K(i), 80 to 90 nM) compared to genotype 1 enzymes (K(i), 1.5 nM). To understand the reduced sensitivity of genotypes 2 and 3 to BILN 2061, active-site residues in the proximity of the inhibitor binding site were replaced in the genotype-1b enzyme with the corresponding genotype-2b or -3a residues. The replacement of five residues at positions 78, 79, 80, 122, and 132 accounted for most of the reduced sensitivity of genotype 2b, while replacement of residue 168 alone could account for the reduced sensitivity of genotype 3a. BILN 2061 remains a potent inhibitor of these non-genotype-1 NS3-NS4A proteins, with K(i) values below 100 nM. This in vitro potency, in conjunction with the good pharmacokinetic data reported for humans, suggests that there is potential for BILN 2061 as an antiviral agent for individuals infected with non-genotype-1 HCV.