The phosphopantetheinyl transferase KirP activates the ACP and PCP domains of the kirromycin NRPS/PKS of Streptomyces collinus Tü 365

The main steps in the biosynthesis of complex secondary metabolites such as the antibiotic kirromycin are catalyzed by modular polyketide synthases (PKS) and/or nonribosomal peptide synthetases (NRPS). During antibiotic assembly, the biosynthetic intermediates are attached to carrier protein domains of these megaenzymes via a phosphopantetheinyl arm. This functional group of the carrier proteins is attached post-translationally by a phosphopantetheinyl transferase (PPTase). No experimental evidence exists about how such an activation of the carrier proteins of the kirromycin PKS/NRPS is accomplished. Here we report on the characterization of the PPTase KirP, which is encoded by a gene located in the kirromycin biosynthetic gene cluster. An inactivation of the kirP gene resulted in a 90% decrease in kirromycin production, indicating a substantial role for KirP in the biosynthesis of the antibiotic. In enzymatic assays, KirP was able to activate both acyl carrier protein and petidyl carrier domains of the kirromycin PKS/NRPS. In addition to coenzyme A (CoA), which is the natural substrate of KirP, the enzyme was able to transfer acyl-phosphopantetheinyl groups to the apo forms of the carrier proteins. Thus, KirP is very flexible in terms of both CoA substrate and carrier protein specificity. Our results indicate that KirP is the main PPTases that activates the carrier proteins in kirromycin biosynthesis.     

Occurrence and Identification of Yeast Species in Fermented Liquid Feed for Piglets

The major objective of the present study was to investigate the occurrence and identity of yeast species in fermented liquid feed (FLF) used for feeding piglets. In total, 40 different Danish farms were included in the analysis. The preparation and composition of FLF was found to be very heterogeneous with high variations in both yeast counts and yeast species composition. The yeast population varied between 6.0?×?10³ and 4.2?×?10⁷?cfug^?1 with an average yeast count of 8.7?×?10⁶?±?1.1?×?10⁷?cfug^?1. A total of 766 yeasts were isolated and identified by conventional and/or molecular typing techniques. The predominant yeast species in the FLF samples were found to be Candida milleri (58.4%), Kazachstania exigua (17.5%), Candida pararugosa (6.40%) and Kazachstania bulderi (5.09%). No clear separation between isolates of C. milleri and Candida humilis could be obtained based on sequencing of the D1/D2 region of the 26S rRNA gene. The combined use of ITS-RFLP analysis and phenotypic criteria did meanwhile suggest a closer relationship with C. milleri than C. humilis.     

3D hydrogen bonded heteronuclear Co, Ni, Cu and Zn aqua complexes derived from dipicolinic acid

The heteronuclear water-soluble and air-stable compounds [M(H₂O)₅M′(dipic)₂] · mH₂O (M/M′ = Cu^II/Co^II (1), Cu^II/Ni^II (2), Cu^II/Zn^II (3), Zn^II/Co^II (4), Ni^II/Co^II (5), m = 2-3; H₂dipic = dipicolinic acid) have been prepared by self-assembly synthesis in aqueous solution at room temperature, and characterized by IR, UV-Vis and atomic absorption spectroscopies, elemental and X-ray diffraction single crystal (for 1 and 2) analyses. 1-5 represent the first examples of heteronuclear dipicolinate compounds with 3d metals. Extensive H-bonding interactions involving all aqua ligands, dipicolinate oxygens and lattice water molecules further stabilize the dimetallic units by linking them to form three-dimensional polymeric networks.     

NO donor induces Nec-1-inhibitable, but RIP1-independent, necrotic cell death in pancreatic β-cells

Nitric oxide (NO) has been implicated in pancreatic β-cell death in the development of diabetes. The mechanisms underlying NO-induced β-cell death have not been clearly defined. Recently, receptor-interacting protein-1 (RIP1)-dependent necrosis, which is inhibited by necrostatin-1, an inhibitor of RIP1, has emerged as a form of regulated necrosis. Here, we show that NO donor-induced β-cell death was inhibited by necrostatin-1. Unexpectedly, however, RIP1 knockdown neither inhibited cell death nor altered the protective effects of necrostatin-1 in NO donor-treated β-cells. These results indicate that NO donor induces necrostatin-1-inhibitable necrotic β-cell death independent of RIP1. Our findings raise the possibility that NO-mediated β-cell necrosis may be a novel form of signal-regulated necrosis, which play a role in the progression of diabetes.     

Explaining geographical variation in the isotope composition of mouse lemurs (Microcebus)

Aim We sought to quantify geographical variation in the stable isotope values of mouse lemurs (Microcebus) and to determine whether this variation reflects trophic differences among populations or baseline isotopic differences among habitats. If the latter pattern is demonstrated, then Microcebus can become a proxy for tracking baseline habitat isotopic variability. Establishing such a baseline is crucial for identifying niche partitioning in modern and ancient communities. Location We studied five species of Microcebus from eight distinct habitats across Madagascar. Methods We compared isotopic variation in C₃ plants and Microcebus fur within and among localities. We predicted that carbon and nitrogen isotope values of Microcebus should: (1) vary as a function of abiotic variables such as rainfall and temperature, and (2) covary with isotopic values in plants. We checked for trophic differences among Microcebus populations by comparing the average difference between mouse lemur and plant isotope values for each locality. We then used multiple regression models to explain spatial isotope variation in mouse lemurs, testing a suite of explanatory abiotic variables. Results We found substantial isotopic variation geographically. Ranges for mean isotope values were similar for both Microcebus and plants across localities (carbon 3.5–4.0‰; nitrogen 10.5–11.0‰). Mean mouse lemur and plant isotope values were lowest in cool, moist localities and highest in hot, dry localities. Rainfall explained 58% of the variation in Microcebus carbon isotope values, and mean plant nitrogen isotope values explained 99.7% of the variation in Microcebus nitrogen isotope values. Average differences between mouse lemur and plant isotope values (carbon 5.0‰; nitrogen 5.9‰) were similar across localities. Main conclusions Isotopic data suggest that trophic differences among Microcebus populations were small. Carbon isotope values in mouse lemurs were negatively correlated with rainfall. Nitrogen isotope values in Microcebus and plants covaried. Such findings suggest that nitrogen isotope values for Microcebus are a particularly good proxy for tracking baseline isotopic differences among habitats. Our results will facilitate future comparative research on modern mouse lemur communities, and ecological interpretations of extinct Holocene communities.     

A Shared Endoplasmic Reticulum-associated Degradation Pathway Involving the EDEM1 Protein for Glycosylated and Nonglycosylated Proteins

Studies of misfolded protein targeting to endoplasmic reticulum-associated degradation (ERAD) have largely focused on glycoproteins, which include the bulk of the secretory proteins. Mechanisms of targeting of nonglycosylated proteins are less clear. Here, we studied three nonglycosylated proteins and analyzed their use of known glycoprotein quality control and ERAD components. Similar to an established glycosylated ERAD substrate, the uncleaved precursor of asialoglycoprotein receptor H2a, its nonglycosylated mutant, makes use of calnexin, EDEM1, and HRD1, but only glycosylated H2a is a substrate for the cytosolic SCF^Fbs2 E3 ubiquitin ligase with lectin activity. Two nonglycosylated BiP substrates, NS-1κ light chain and truncated Igγ heavy chain, interact with the ERAD complex lectins OS-9 and XTP3-B and require EDEM1 for degradation. EDEM1 associates through a region outside of its mannosidase-like domain with the nonglycosylated proteins. Similar to glycosylated substrates, proteasomal inhibition induced accumulation of the nonglycosylated proteins and ERAD machinery in the endoplasmic reticulum-derived quality control compartment. Our results suggest a shared ERAD pathway for glycosylated and nonglycosylated proteins composed of luminal lectin machinery components also capable of protein-protein interactions.     

Rhesus macaques build new social connections after a natural disaster

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BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.     

AKAP79/150 interacts with the neuronal calcium-binding protein caldendrin

The A kinase-anchoring protein AKAP79/150 is a postsynaptic scaffold molecule and a key regulator of signaling events. At the postsynapse it coordinates phosphorylation and dephosphorylation of receptors via anchoring kinases and phosphatases near their substrates. Interactions between AKAP79 and two Ca(2+) -binding proteins caldendrin and calmodulin have been investigated here. Calmodulin is a known interaction partner of AKAP79/150 that has been shown to regulate activity of the kinase PKC in a Ca(2+) -dependent manner. Pull-down experiments and surface plasmon resonance biosensor analyses have been used here to demonstrate that AKAP79 can also interact with caldendrin, a neuronal calcium-binding protein implicated in regulation of Ca(2+) -influx and release. We demonstrate that calmodulin and caldendrin compete for a partially overlapping binding site on AKAP79 and that their binding is differentially dependent on calcium. Therefore, this competition is regulated by calcium levels. Moreover, both proteins have different binding characteristics suggesting that the two proteins might play complementary roles. The postsynaptic enrichment, the complex binding mechanism, and the competition with calmodulin, makes caldendrin an interesting novel player in the signaling toolkit of the AKAP interactome.     

Cigarette smoke metabolically promotes cancer,via autophagy and premature aging in the host stromal microenvironment

Cigarette smoke has been directly implicated in the disease pathogenesis of a plethora of different human cancer subtypes, including breast cancers. The prevailing view is that cigarette smoke acts as a mutagen and DNA damaging agent in normal epithelial cells, driving tumor initiation. However, its potential negative metabolic effects on the normal stromal microenvironment have been largely ignored. Here, we propose a new mechanism by which carcinogen-rich cigarette smoke may promote cancer growth, by metabolically “fertilizing” the host microenvironment. More specifically, we show that cigarette smoke exposure is indeed sufficient to drive the onset of the cancer-associated fibroblast phenotype via the induction of DNA damage, autophagy and mitophagy in the tumor stroma. In turn, cigarette smoke exposure induces premature aging and mitochondrial dysfunction in stromal fibroblasts, leading to the secretion of high-energy mitochondrial fuels, such as L-lactate and ketone bodies. Hence, cigarette smoke induces catabolism in the local microenvironment, directly fueling oxidative mitochondrial metabolism (OXPHOS) in neighboring epithelial cancer cells, actively promoting anabolic tumor growth. Remarkably, these autophagic-senescent fibroblasts increased breast cancer tumor growth in vivo by up to 4-fold. Importantly, we show that cigarette smoke-induced metabolic reprogramming of the fibroblastic stroma occurs independently of tumor neo-angiogenesis. We discuss the possible implications of our current findings for the prevention of aging-associated human diseases and, especially, common epithelial cancers, as we show that cigarette smoke can systemically accelerate aging in the host microenvironment. Finally, our current findings are consistent with the idea that cigarette smoke induces the “reverse Warburg effect,” thereby fueling “two-compartment tumor metabolism” and oxidative mitochondrial metabolism in epithelial cancer cells.