Developmental exposure to xenoestrogen enhances spatial learning in male rats

Steroid hormones have profound effects on the development and function of the nervous system. Environmental estrogens or xenoestrogens are manmade or are natural compounds, which mimics the action of estrogen hormones. The experimental evidence for impairment of cognitive functions in humans and mammals following exposure to xenoestrogens has been fiercely debated. The strongest arguments against such studies have been that the route, time course, and intensity of exposure did not simulate environmental exposure, and that the chemicals tested have additional, non-estrogenic toxic effects, hindering a generalization of actual "xenoestrogenic" effects. Here we show that an environmental-like exposure to the pure estrogen, 17alpha-ethynylestradiol (EE2) during development enhances spatial learning abilities in adult male Sprague-Dawley rats. To simulate an environmental exposure, we used a very low dose (4 ng/kg/day) of EE2 equivalent to concentrations measured in European and US streams which was given orally with a non-invasive method, and we extended the treatment for the entire course of development, from conception to puberty. The animals were tested in a Morris water maze protocol at 6 months of age. Male rats treated with EE2 during development showed a faster learning during the training phase, and remembered better the position of the hidden platform in the short term. Our study demonstrates that actual levels of exposure to xenoestrogens can permanently alter cognitive abilities of a mammalian species.     

Differential pathways govern CD4+ CD28- T cell proinflammatory and effector responses in patients with coronary artery disease

Patients with acute coronary syndromes experience circulatory and intraplaque expansion of an aggressive and unusual CD4(+) lymphocyte subpopulation lacking the CD28 receptor. These CD4(+)CD28(-) cells produce IFN-gamma and perforin, and are thought to play an important role in coronary atheromatous plaque destabilization. Aberrant expression of killer Ig-like receptors (KIRs) in CD4(+)CD28(-) cells is broadly thought to be responsible for their cytotoxicity, but the mechanisms involved remain poorly defined. We therefore sought to investigate the mechanism and regulation of CD4(+)CD28(-) cell functionality using T cell clones (n = 536) established from patients with coronary artery disease (n = 12) and healthy volunteers (n = 3). Our functional studies demonstrated that KIR2DS2 specifically interacted with MHC class I-presenting human heat shock protein 60 (hHSP60) inducing cytotoxicity. Further investigations revealed the novel finding that hHSP60 stimulation of TCR alone could not induce a cytotoxic response, and that this response was specific and KIR dependent. Analysis of CD4(+)CD28(-)2DS2(+) clones (n = 162) showed that not all were hHSP60 cytotoxic; albeit, their prevalence correlated with coronary disease status (p = 0.017). A higher proportion of clones responded to hHSP60 by IFN-gamma compared with perforin (p = 0.008). In this study, for the first time, we define the differential regulatory pathways involved in CD4(+)CD28(-) cell proinflammatory and effector responses. We describe in this study that, contrary to previous reports, CD4(+)CD28(-) cell recognition and killing can be specific and discriminate. These results, in addition to contributing to the understanding of CD4(+)CD28(-) cell functionality, may have implications for the monitoring and management of coronary artery disease progression.     

Ferredoxin-thioredoxin reductase, an iron-sulfur enzyme linking light to enzyme regulation in oxygenic photosynthesis: purification and properties of the enzyme from C3, C4, and cyanobacterial species

Ferredoxin-thioredoxin reductase (FTR), an enzyme involved in the light regulation of chloroplast enzymes, was purified to homogeneity from leaves of spinach (a C3 plant) and corn (a C4 plant) and from cells of a cyanobacterium (Nostoc muscorum). The enzyme is a yellowish brown iron-sulfur protein, containing four nonheme iron and labile sulfide groups, that catalyzes the activation of NADP-malate dehydrogenase and fructose 1,6-bisphosphatase in the presence of ferredoxin and of thioredoxin m and f, respectively. FTR is synonymous with the protein earlier called ferralterin. FTR showed an Mr of about 30,000 (determined by sedimentation equilibrium ultracentrifugation, amino acid composition, gel filtration, and gradient gel electrophoresis) and was composed of two dissimilar subunits (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the FTR subunits from each source was similar both in Mr (about 13,000) and immunological properties, while the other subunit (of variable molecular weight) was characteristic of a particular organism. The similar subunit contained a disulfide group that was rapidly reduced by a dithiol (dithiothreitol) but not by monothiols (2-mercaptoethanol or reduced glutathione). Homogeneous FTR formed a tight noncovalent complex with ferredoxin on affinity columns. The basis for the structural variation in the different FTR enzymes remains to be determined.     

Solution structures and model membrane interactions of Ctriporin,an anti‐methicillin‐resistant Staphylococcus aureus Peptide from Scorpion Venom

Ctriporin peptide (Ctr), a novel antimicrobial peptide isolated from the venom of the scorpion Chaerilus tricostatus, shows a broad‐spectrum of antimicrobial activity and is able to inhibit antibiotic resistant pathogens, including Methicillin resistant Staphylococcus aureus, Methicillin Resistant Coagulase‐negative Staphylococcus, and Penicillin Resistant Staphylococcus epidermidis strains. To understand the active conformation of the Ctr peptide in membranes, we have investigated the interaction of Ctr with the negatively charged and zwitterionic membrane‐mimetic micelles such as sodium dodecyl sulphate (SDS) and n‐dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N‐terminus tryptophan residue of Ctr interacted with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane‐mimetic micelles induce an α‐helix conformation in Ctr. Moreover, we have determined the solution structures of Ctr in SDS and DPC micelles using nuclear magnetic resonance (NMR) spectroscopy. The structural comparison of Ctr in the presence of SDS and DPC micelles showed significant conformational changes. The observed structural differences of Ctr in anionic versus zwitterionic membrane‐mimetic micelles suggest that the mode of interaction of this peptide may be different in two environments which may account for its ability to differentiate bacterial and eukaryotic cell membrane. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1143–1153, 2014.     

A Five-Year Survey of Dematiaceous Fungi in a Tropical Hospital Reveals Potential Opportunistic Species

Dematiaceous fungi (black fungi) are a heterogeneous group of fungi present in diverse environments worldwide. Many species in this group are known to cause allergic reactions and potentially fatal diseases in humans and animals, especially in tropical and subtropical climates. This study represents the first survey of dematiaceous fungi in Malaysia and provides observations on their diversity as well as in vitro response to antifungal drugs. Seventy-five strains isolated from various clinical specimens were identified by morphology as well as an internal transcribed spacer (ITS)-based phylogenetic analysis. The combined molecular and conventional approach enabled the identification of three classes of the Ascomycota phylum and 16 genera, the most common being Cladosporium, Cochliobolus and Neoscytalidium. Several of the species identified have not been associated before with human infections. Among 8 antifungal agents tested, the azoles posaconazole (96%), voriconazole (90.7%), ketoconazole (86.7%) and itraconazole (85.3%) showed in vitro activity (MIC ≤1 µg/mL) to the largest number of strains, followed by anidulafungin (89.3%), caspofungin (74.7%) and amphotericin B (70.7%). Fluconazole appeared to be the least effective with only 10.7% of isolates showing in vitro susceptibility. Overall, almost half (45.3%) of the isolates showed reduced susceptibility (MIC >1 µg/mL) to at least one antifungal agent, and three strains (one Pyrenochaeta unguis-hominis and two Nigrospora oryzae) showed potential multidrug resistance.     

Effect of non-Legionellaceae bacteria on the multiplication of Legionella pneumophila in potable water.

A naturally occurring suspension of Legionella pneumophila and associated microbiota contained three unidentified non-Legionellaceae bacteria which supported satellite growth of a subculture of L. pneumophila on an L-cysteine-deficient medium and another bacterium which did not support growth of the subculture. Washed suspensions containing 10(3), 10(5), 10(7), or 10(8) CFU of a mixture of isolates of these non-Legionellaceae bacteria failed to support the multiplication of an isolate of agar-grown L. pneumophila which had been washed and seeded into the suspensions. The suspensions which contained 10(3), 10(5), or 10(7) CFU of the non-Legionellaceae bacteria per ml appeared to enhance survival or cryptic growth of agar-grown L. pneumophila. A decline of 1.3 log CFU of L. pneumophila per ml occurred within the first week of incubation in the sample which contained 10(8) CFU of the non-Legionellaceae bacteria per ml. In contrast to these results, naturally occurring L. pneumophila multiplied in the presence of associated microbiota. The necessity to subculture L. pneumophila and the non-Legionellaceae bacteria on artificial medium to obtain pure cultures may have affected the multiplication of L. pneumophila in tap water. Alternatively, other microorganisms may be present in the naturally occurring suspension which support the growth of this bacterium.     

The prion-like domain of Fused in Sarcoma is phosphorylated by multiple kinases affecting liquid- and solid-phase transitions

Fused in Sarcoma (FUS) is a ubiquitously expressed protein that can phase-separate from nucleoplasm and cytoplasm into distinct liquid-droplet structures. It is predominantly nuclear and most of its functions are related to RNA and DNA metabolism. Excessive persistence of FUS within cytoplasmic phase-separated assemblies is implicated in the diseases amyotrophic lateral sclerosis and frontotemporal dementia. Phosphorylation of FUS’s prion-like domain (PrLD) by nuclear phosphatidylinositol 3-kinase-related kinase (PIKK)-family kinases following DNA damage was previously shown to alter FUS’s liquid-phase and solid-phase transitions in cell models and in vitro. However, proteomic data suggest that FUS’s PrLD is phosphorylated at numerous additional sites, and it is unknown if other non-PIKK and nonnuclear kinases might be influencing FUS’s phase transitions. Here we evaluate disease mutations and stress conditions that increase FUS accumulation into cytoplasmic phase-separated structures. We observed that cytoplasmic liquid-phase structures contain FUS phosphorylated at novel sites, which occurred independent of PIKK-family kinases. We engineered phosphomimetic substitutions within FUS’s PrLD and observed that mimicking a few phosphorylation sites strongly inhibited FUS solid-phase aggregation, while minimally altering liquid-phase condensation. These effects occurred independent of the exact location of the phosphomimetic substitutions, suggesting that modulation of PrLD phosphorylation may offer therapeutic strategies that are specific for solid-phase aggregation observed in disease.     

Inhibition and Reversal of Microbial Attachment by an Antibody with Parasteric Activity against the FimH Adhesin of Uropathogenic E. coli

Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection.