Short-term regulation of multidrug resistance-associated protein 3 in rat and human hepatocytes

The short-term regulation of multidrug resistance-associated protein 3 (Mrp3/MRP3) by cAMP and PKC was investigated in sandwich-cultured rat and human hepatocytes and isolated perfused rat livers. The modulator glucagon (500 nM) and the phorbol ester PMA (0.1 muM) were utilized to increase intracellular cAMP and PKC levels, respectively. In glucagon-treated rat hepatocytes, efflux of the Mrp3 substrate 5-(6)-carboxy-2',7'-dichlorofluorescein (CDF) increased approximately 1.5-fold, even in hepatocytes treated with the organic anion transporter (Oatp) inhibitor sulfobromophthalein (BSP). Confocal microscopy revealed more concentrated Mrp3 fluorescence in the basolateral membrane (less diffuse staining pattern) with glucagon treatment. PMA had no effect on Mrp3 activity or localization in sandwich-cultured rat hepatocytes. Glucagon and PMA treatment in isolated perfused rat livers resulted in a threefold increase (14 +/- 4.6 mul.min(-1).g liver(-1)) and a fourfold decrease (1.3 +/- 0.3 mul.min(-1).g liver(-1)) in CDF basolateral clearance compared with control livers (4.7 +/- 2.3 mul.min(-1).g liver(-1)), whereas CDF biliary clearance was not statistically different. In sandwich-cultured human hepatocytes, glucagon treatment resulted in a 1.3-fold increase in CDF efflux and a concomitant increase in MRP3 fluorescence in the basolateral membrane. In summary, cAMP and PKC appear to be involved in the short-term regulation of Mrp3/MRP3, as demonstrated by alterations in activity and localization in rat and human hepatocytes.     

Optimisation of the fluorescein diacetate antibacterial assay

The fluorescein diacetate (FDA) antibacterial assay relies on the cleavage of fluorescein diacetate by metabolically active bacteria. The recent finding that microbiological media can lead to significant levels of cleavage has reduced the reliability of the assay. Using the nucleophilic scavengers N-ethylmaleimide and maleic anhydride, we have demonstrated that this abiotic cleavage is most likely due to nucleophiles such as cysteine and histidine commonly present in the media. To increase the reliability of the assay we have modified the original assay conditions to include use of dilute medium (peptone 0.2% w/v, yeast extract 0.1% w/v and NaCl 0.1% w/v) in a non-nucleophilic buffer and overnight incubation of the medium after addition of antibacterial agents. The optimised fluorescein diacetate assay has been used to determine the MIC of gentamicin, tetracycline and chloramphenicol for Escherichia coli, Staphyloccocus aureus and Pseudomonas aeruginosa and gave quantitative results that were reproducible and consistent with published data.     

Evidence that complement protein C1q interacts with C-reactive protein through its globular head region

C1q acts as the recognition unit of the first complement component, C1, and binds to immunoglobulins IgG and IgM, as well as to non-Ig ligands, such as C-reactive protein (CRP). IgG and IgM are recognized via the globular head regions of C1q (C1qGR), whereas CRP has been postulated to interact with the collagen-like region (C1qCLR). In the present study, we used a series of nine mAbs to C1q, five directed against C1qGR and four against C1qCLR, to inhibit the interaction of C1q with CRP. The F(ab')(2) of each of the five mAbs directed against C1qGR inhibited binding of C1q to polymerized IgG. These five mAbs also successfully inhibited the interaction of C1q with CRP. Moreover, these five mAbs inhibited C1 activation by CRP as well as by polymerized IgG in vitro. In contrast, none of the four mAbs against C1qCLR inhibited C1q interaction with CRP or IgG, or could reduce activation of complement by CRP or polymerized IgG. These results provide the first evidence that the interaction of C1q with CRP or IgG involves sites located in the C1qGR, whereas sites in the CLR do not seem to be involved in the physiological interaction of C1q with CRP.     

Helpers at the nest improve late-life offspring performance: evidence from a long-term study and a cross-foster experiment

Background

Conditions during an individual''s rearing period can have far reaching consequences for its survival and reproduction later in life. Conditions typically vary due to variation in parental quality and/or the environment, but in cooperative breeders the presence of helpers adds an important component to this. Determining the causal effect of helpers on offspring fitness is difficult, since high-quality breeders or territories are likely to produce high-quality offspring, but are also more likely to have helpers because of past reproductive success. This problem is best resolved by comparing the effect of both helping and non-helping subordinates on offspring fitness, however species in which both type of subordinates commonly occur are rare.

Methodology/Principal Findings

We used multi-state capture-recapture models on 20 years of data to investigate the effect of rearing conditions on survival and recruitment in the cooperatively breeding Seychelles warbler (Acrocephalus sechellensis), with both helping and non-helping subordinates. The number of helpers in the rearing territory, but not territory quality, group- or brood size, was positively associated with survival of offspring in their first year, and later in life. This was not a result of group size itself since the number of non-helpers was not associated with offspring survival. Furthermore, a nestling cross-foster experiment showed that the number of helpers on the pre-foster territory was not associated with offspring survival, indicating that offspring from territories with helpers do not differ in (genetic) quality.

Conclusions/Significance

Our results suggest that the presence of helpers not only increase survival of offspring in their first year of life, but also subsequent adult survival, and therefore have important fitness consequences later in life. This means that when calculating the fitness benefits of helping not only short-term but also the late-life benefits have to be taken into account to fully understand the evolution of cooperative breeding.     

The human exosome: an autoantigenic complex of exoribonucleases in myositis and scleroderma

The anti-PM/Scl autoantibodies are known to characterize a subset of autoimmune patients with myositis, scleroderma (Scl), and the PM/Scl overlap syndrome. The major autoantigens that are recognized by anti-PM/Scl autoantibodies are designated PM/Scl-100 and PM/Scl-75. These autoantigens have been reported to associate into a large complex consisting of 11 to 16 proteins and to play a role in ribosome synthesis. Recently, it was discovered that the PM/Scl complex is the human counterpart of the yeast (Saccharomyces cerevisiae) exosome, which is an RNA-processing complex consisting of 11 3' → 5' exoribonucleases. To date, 10 human exosome components have been identified, although only some of these were studied in more detail. In this review, we discuss some recent advances in the characterization of the PM/Scl complex.     

Bio-inspired synthetic receptor molecules towards mimicry of vancomycin.

A 512-member library of bio-inspired synthetic receptor molecules was prepared featuring a triazacyclophane scaffold. The purpose of this scaffold was to orient three (identical) peptide 'binding arms' in order to mimic an antibiotic binding cavity as is present in the vancomycin antibiotics. The library was screened with D-Ala-D-Ala and D-Ala-D-Lac containing ligands, which are present in the cell wall precursors of pathogenic bacteria. Screening and validation led to identification of a synthetic receptor capable of binding these ligands.     

Phytophthora gemini sp. nov., a new species isolated from the halophilic plant Zostera marina in the Netherlands

Eight strains belonging to the Oomycete genus Phytophthora were isolated from Zostera marina (seagrass) in The Netherlands over the past 25 y. Based on morphology, isozymes, temperature-growth relationships and ITS sequences, these strains were found to belong to two different Phytophthora species. Five strains, four of them isolated from rotting seeds and one isolated from decaying plants, could not be assigned to a known species and hence belong to a new species for which we propose the name Phytophthora gemini sp. nov. Three strains were isolated from decaying plants and were identified as Phytophthora inundata, thereby expanding the known habitat range of this species from fresh to brackish-saline areas. The possible role of both Phytophthora species in the decline of Z. marina in The Netherlands and the evolutionary significance of the presence of Phytophthora species in marine environments are discussed.     

Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation

The human FMR1 gene contains a CGG repeat in its 5' untranslated region. The repeat length in the normal population is polymorphic (5-55 CGG repeats). Lengths beyond 200 CGGs (full mutation) result in the absence of the FMR1 gene product, FMRP, through abnormal methylation and gene silencing. This causes Fragile X syndrome, the most common inherited form of mental retardation. Elderly carriers of the premutation, defined as a repeat length between 55 and 200 CGGs, can develop a progressive neurodegenerative syndrome: Fragile X-associated tremor/ataxia syndrome (FXTAS). In FXTAS, FMR1 mRNA levels are elevated and it has been hypothesised that FXTAS is caused by a pathogenic RNA gain-of-function mechanism. We have developed a knock in mouse model carrying an expanded CGG repeat (98 repeats), which shows repeat instability and displays biochemical, phenotypic and neuropathological characteristics of FXTAS. Here, we report further repeat instability, up to 230 CGGs. An expansion bias was observed, with the largest expansion being 43 CGG units and the largest contraction 80 CGG repeats. In humans, this length would be considered a full mutation and would be expected to result in gene silencing. Mice carrying long repeats ( approximately 230 CGGs) display elevated mRNA levels and decreased FMRP levels, but absence of abnormal methylation, suggesting that modelling the Fragile X full mutation in mice requires additional repeats or other genetic manipulation.     

Three novel components of the human exosome

The yeast exosome is a complex of 3' --> 5' exoribonucleases. Sequence analysis identified putative human homologues for exosome components, although several were found only as expressed sequence tags. Here we report the cloning of full-length cDNAs, which encode putative human homologues of the Rrp40p, Rrp41p, and Rrp46p components of the exosome. Recombinant proteins were expressed and used to raise rabbit antisera. In Western blotting experiments, these decorated HeLa cell proteins of the predicted sizes. All three human proteins were enriched in the HeLa cells nucleus and nucleolus, but were also clearly detected in the cytoplasm. Size exclusion chromatography revealed that hRrp40p, hRrp41p, and hRrp46p were present in a large complex. This cofractionated with the human homologues of other exosome components, hRrp4p and PM/Scl-100. Anti-PM/Scl-positive patient sera coimmunoprecipitated hRrp40p, hRrp41p, and hRrp46p demonstrating their physical association. The immunoprecipitated complex exhibited 3' --> 5' exoribonuclease activity in vitro. hRrp41p was expressed in yeast and shown to suppress the lethality of genetic depletion of yeast Rrp41p. We conclude that hRrp40p, hRrp41p, and hRrp46p represent novel components of the human exosome complex.