Bax inhibitor-1 enhances survival and neuronal differentiation of embryonic stem cells via differential regulation of mitogen-activated protein kinases activities

Bax inhibitor-1 (BI-1), a member of the BI-1 family of integral membrane proteins, was originally identified as an inhibitor of stress-induced cell death in mammalian cells. Previous studies have shown that the withdrawal of leukemia inhibitory factor (LIF) results in differentiation of the majority of mouse embryonic stem (mES) cells into various cell lineages, while some ES cells die within 3days. Thus, to investigate the function of BI-1 in ES cell survival and neuronal differentiation, we generated mES cell lines that overexpress BI-1 or a carboxy-terminal BI-1ΔC mutant. Overexpression of BI-1 in mES cells significantly increased cell viability and resistance to apoptosis induced by LIF withdrawal, while the control vector or BI-1ΔC-overexpressing mES cells had no effect. Moreover, overexpression of BI-1 produced significant inhibition of the p38 mitogen-activated protein kinases (MAPK) pathway in response to LIF withdrawal, while activity of the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) MAPK pathway was increased. Interestingly, we found that BI-1-overexpressing cells showed higher expression levels of neuroectodermal markers (Otx1, Lmx1b, En1, Pax2, Wnt1, Sox1, and Nestin) and greater neuronal differentiation efficiency than control or BI-1ΔC-overexpressing mES cells did. Considering these findings, our results indicated that BI-1-modulated MAPK activity plays a key role in protecting mES cells from LIF-withdrawal-induced apoptosis and in promoting their differentiation toward neuronal lineages.     

Identification of mitochondrial cytochrome P450 induced in response to polycyclic aromatic hydrocarbons in the mummichog (Fundulus heteroclitus)

Increasing evidence suggests that polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are localized to the mitochondria. Because the toxic effects of many PAHs are the result of metabolism by cytochrome P4501A (CYP1A), it is important to investigate whether active forms of these enzymes can be identified in the mitochondria. In this study, we identified mitochondrial P450s with a monoclonal antibody against scup (Stenotomus chrysops) CYP1A in the isolated mitochondrial fraction of the liver from adult male mummichog (Fundulus heteroclitus) livers. The size of the protein in the mitochondria was similar to that of microsomal CYP1A. Fish dosed with 10 mg/kg BaP had increased EROD activity in the mitochondrial fraction compared to controls. In mummichog larvae dosed with 100 µg/L BaP and 100 µg/L benzo[k]fluoranthene, CYP1A protein levels as well as enzyme activity were elevated. However, fish from a PAH-polluted Superfund site (Elizabeth River, Portsmouth VA) showed recalcitrant mitochondrial CYP1A protein levels and enzyme activity in a similar manner to microsomal CYP1A.     

The FRB domain of mTOR: NMR solution structure and inhibitor design

The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth. Rapamycin is a natural product that binds and inhibits mTOR function by interacting with its FKBP-rapamycin-binding (FRB) domain. Here we report on the NMR solution structure of FRB and on further studies aimed at the identification and characterization of novel ligands that target the rapamycin binding pocket. The biological activity of the ligands, and that of rapamycin in the absence of FKBP12, was investigated by assaying the kinase activity of mTOR. While we found that rapamycin binds the FRB domain and inhibits the kinase activity of mTOR even in the absence of FKBP12 (in the low micromolar range), our most potent ligands bind to FRB with similar binding affinity but inhibit the kinase activity of mTOR at much higher concentrations. However, we have also identified one low-affinity compound that is also capable of inhibiting mTOR. Hence, we have identified compounds that can directly mimic rapamycin or can dissociate the FRB binding from the inhibition of the catalytic activity of mTOR. As such, these ligands could be useful in deciphering the complex regulation of mTOR in the cell and in validating the FRB domain as a possible target for the development of novel therapeutic compounds.     

A structure-based approach to retinoid X receptor-alpha inhibition

In this paper we describe a structure-based approach designed to identify novel ligands for retinoid X receptor-alpha (RXRalpha). By using a virtual approach based on a modified scoring function, we have selected 200 potential candidates on the basis of their predicted ability of docking into the ligand-binding site of the target. Subsequent experimental verification of the compounds in in vitro and cell-based assays led to the identification of a number of novel high affinity ligands for RXRalpha. The compounds are capable of displacing 9-cis-retinoic acid with IC(50) values in the 10 nm and 5 mum range and exhibit marked antagonistic activity in cellular assays. The inhibitory scaffolds discovered with this method form the basis for the development of novel RXRalpha ligands with potential therapeutic properties.     

Nisaea sediminum sp. nov., a heavy metal resistant bacterium isolated from marine sediment in the East China Sea

A Gram-negative, rod-shaped, motile and strictly aerobic bacterium, designated NBU1469^T, was isolated from marine sediment sampled on Meishan Island located in the East China Sea. Strain NBU1469^T grew optimally at temperature of 40 °C, NaCl concentration of 2.0% (w/v) and pH 7.5. Catalase and oxidase activities, H₂S production, nitrate reduction and hydrolysis of Tween 20 were positive. Indole, methyl red reaction, urease, hydrolysis of gelatin, starch, casein, Tweens 40, 60 and 80 were negative. The major cellular fatty acids were C_16:0, C_19:0 cyclo ω8c and summed feature 8 (C_18:1 ω7c and/or C_18:1 ω6c). The only respiratory quinone was ubiquinone-10 (Q-10). The major polar lipids were phosphatidylglycerol, two unidentified amino-phospholipids and two unidentified phospholipids. Comparative analysis of the 16S rRNA gene sequence showed highest similarities to the species with validated name Nisaea nitritireducens DR41_18^T (98.1%) and Nisaea denitrificans DR41_21^T (97.6%). Phylogenetic analyses indicated that strain NBU1469^T formed a distinct lineage with strains Nisaea nitritireducens DR41_18^T and Nisaea denitrificans DR41_21^T within the genus Nisaea. The average nucleotide identity and digital DNA-DNA hybridization values between strain NBU1469^T and related species of genus Nisaea were well below the threshold limit for prokaryotic species delineation. The DNA G?+?C content was 63.6%. Based on its phenotypic, chemotaxonomic and genotypic data, strain NBU1469^T is considered to be a representative of a novel species in the genus Nisaea, for which the name Nisaea sediminum sp. nov. is proposed. The type strain is NBU1469^T (=KCTC 82224 ^T?=MCCC 1K04763^T).

     

Structure-based discovery of a new class of Bcl-xL antagonists

Apoptosis, or programmed cell death, plays a key role in normal tissue homeostasis ensuring a proper balance between cell production and cell loss. Anti-apoptotic Bcl-2-family proteins are central regulators of the apoptotic pathway and due to their ability to confer tumor resistance to chemotherapy or radiation, have been recently validated as targets for cancer drug discovery. Since the crucial interaction between pro- and anti-apoptotic members occurs via a conserved region located on the surface of the protein, a viable way to inhibit the anti-death activity of Bcl-2 proteins is to design small molecule inhibitors that occupy this cavity. Here, we describe a structure-based approach that led to the identification of four small molecule inhibitors directed at the hydrophobic groove on the surface of the Bcl-2 family protein Bcl-xL. The compounds were characterized in a number of assays including in vitro binding using 15N-labeled protein, a displacement DELFIA assay, and a cell-based viability assay with human cancer cells.     

Characterization of the Paracoccidioides Hypoxia Response Reveals New Insights into Pathogenesis Mechanisms of This Important Human Pathogenic Fungus

Background

Hypoxic microenvironments are generated during fungal infection. It has been described that to survive in the human host, fungi must also tolerate and overcome in vivo microenvironmental stress conditions including low oxygen tension; however nothing is known how Paracoccidioides species respond to hypoxia. The genus Paracoccidioides comprises human thermal dimorphic fungi and are causative agents of paracoccidioidomycosis (PCM), an important mycosis in Latin America.

Methodology/Principal Findings

In this work, a detailed hypoxia characterization was performed in Paracoccidioides. Using NanoUPLC-MS^E proteomic approach, we obtained a total of 288 proteins differentially regulated in 12 and 24 h of hypoxia, providing a global view of metabolic changes during this stress. In addition, a functional characterization of the homologue to the most important molecule involved in hypoxia responses in other fungi, the SREBP (sterol regulatory element binding protein) was performed. We observed that Paracoccidioides species have a functional homologue of SREBP, named here as SrbA, detected by using a heterologous genetic approach in the srbA null mutant in Aspergillus fumigatus. Paracoccidioides srbA (PbsrbA), in addition to involvement in hypoxia, is probable involved in iron adaptation and azole drug resistance responses.

Conclusions/Significance

In this study, the hypoxia was characterized in Paracoccidioides. The first results can be important for a better understanding of the fungal adaptation to the host and improve the arsenal of molecules for the development of alternative treatment options in future, since molecules related to fungal adaptation to low oxygen levels are important to virulence and pathogenesis in human pathogenic fungi.     

Dsc Orthologs Are Required for Hypoxia Adaptation,Triazole Drug Responses,and Fungal Virulence in Aspergillus fumigatus

Hypoxia is an environmental stress encountered by Aspergillus fumigatus during invasive pulmonary aspergillosis (IPA). The ability of this mold to adapt to hypoxia is important for fungal virulence and genetically regulated in part by the sterol regulatory element binding protein (SREBP) SrbA. SrbA is required for fungal growth in the murine lung and to ultimately cause lethal disease in murine models of IPA. Here we identified and partially characterized four genes (dscA, dscB, dscC, and dscD, here referred to as dscA-D) with previously unknown functions in A. fumigatus that are orthologs of the Schizosaccharomyces pombe genes dsc1, dsc2, dsc3, and dsc4 (dsc1-4), which encode a Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. A. fumigatus null dscA-D mutants displayed remarkable defects in hypoxic growth and increased susceptibility to triazole antifungal drugs. Consistent with the confirmed role of these genes in S. pombe, both ΔdscA and ΔdscC resulted in reduced cleavage of the SrbA precursor protein in A. fumigatus. Inoculation of corticosteroid immunosuppressed mice with ΔdscA and ΔdscC strains revealed that these genes are critical for A. fumigatus virulence. Reintroduction of SrbA amino acids 1 to 425, encompassing the N terminus DNA binding domain, into the ΔdscA strain was able to partially restore virulence, further supporting a mechanistic link between DscA and SrbA function. Thus, we have shown for the first time the importance of a previously uncharacterized group of genes in A. fumigatus that mediate hypoxia adaptation, fungal virulence, and triazole drug susceptibility and that are likely linked to regulation of SrbA function.     

The long amplicon quantitative PCR for DNA damage assay as a sensitive method of assessing DNA damage in the environmental model, Atlantic killifish (Fundulus heteroclitus)

DNA damage is an important mechanism of toxicity for a variety of pollutants, and therefore, is often used as an indicator of pollutant effects in ecotoxicological studies. Here, we adapted a PCR-based assay for nuclear and mitochondrial DNA damage for use in an important environmental model, the Atlantic killifish (Fundulus heteroclitus). We refer to this assay as the long amplicon quantitative PCR (LA-QPCR) assay. To validate this method in killifish, DNA damage was measured in liver, brain, and muscle of fish dosed with 10 mg/kg benzo[a]pyrene. This exposure caused 0.4-0.8 lesions/10 kb. We also measured DNA damage in liver and muscle tissues from killifish inhabiting a Superfund site, confirming the utility of this method for biomonitoring. In both cases, damage levels were comparable in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Since extensive nDNA sequence data are not readily available for many environmentally relevant species, but mitochondrial genomes are frequently fully sequenced, this assay can be adapted to examine mtDNA damage in virtually any species with little development. Therefore, we argue that this assay will be a valuable tool in assessing DNA damage in ecotoxicological studies.     

Multi‐Functional Transparent Luminescent Configuration for Advanced Photovoltaics

The conversion and manipulation of light via luminescent down‐shifting (LDS) show promise in numerous applications. An elegant combination of lanthanide‐doped polymer‐derived ceramics incorporated with versatile nanopatterns is demonstrated using direct nanoimprint techniques. The prompt formation of nanoscale photonic structures enhances the fluorescence emission from the LDS while retaining the material's optical transparency. The functionality of this material is further expanded to accommodate surface energy modulation by nanopatterns. The practical applicability of this platform in photovoltaic devices is evaluated, showing distinctively enhanced efficiency and lifetime mainly attributed to the nanopattern assisted strong LDS property. Moreover, to efficiently combine two lanthanide emissions, so called a “double imprint” approach is devised by superpositioning two LDS nanopatterned arrays. Combined with the multi‐functionality such as prominent LDS characteristics, color tunability, and surface energy modulation, the developed LDS platform offers promise for esthetic building‐integrated photovoltaics.