Neuroprotective peptide ADNF-9 in fetal brain of C57BL/6 mice exposed prenatally to alcohol

<p>Abstract</p> <p>Background</p> <p>A derived peptide from activity-dependent neurotrophic factor (ADNF-9) has been shown to be neuroprotective in the fetal alcohol exposure model. We investigated the neuroprotective effects of ADNF-9 against alcohol-induced apoptosis using TUNEL staining. We further characterize in this study the proteomic architecture underlying the role of ADNF-9 against ethanol teratogenesis during early fetal brain development using liquid chromatography in conjunction with tandem mass spectrometry (LC-MS/MS).</p> <p>Methods</p> <p>Pregnant C57BL/6 mice were exposed from embryonic days 7-13 (E7-E13) to a 25% ethanol-derived calorie [25% EDC, Alcohol (ALC)] diet, a 25% EDC diet simultaneously administered i.p. ADNF-9 (ALC/ADNF-9), or a pair-fed (PF) liquid diet. At E13, fetal brains were collected from 5 dams from each group, weighed, and frozen for LC-MS/MS procedure. Other fetal brains were fixed for TUNEL staining.</p> <p>Results</p> <p>Administration of ADNF-9 prevented alcohol-induced reduction in fetal brain weight and alcohol-induced increases in cell death. Moreover, individual fetal brains were analyzed by LC-MS/MS. Statistical differences in the amounts of proteins between the ALC and ALC/ADNF-9 groups resulted in a distinct data-clustering. Significant upregulation of several important proteins involved in brain development were found in the ALC/ADNF-9 group as compared to the ALC group.</p> <p>Conclusion</p> <p>These findings provide information on potential mechanisms underlying the neuroprotective effects of ADNF-9 in the fetal alcohol exposure model.</p

Characterization of a Glycyl Radical Enzyme Bacterial Microcompartment Pathway in Rhodobacter capsulatus

Bacterial microcompartments (BMCs) are large (∼100-nm) protein shells that encapsulate enzymes, their substrates, and cofactors for the purposes of increasing metabolic reaction efficiency and protecting cells from toxic intermediates. The best-studied microcompartment is the carbon-fixing carboxysome that encapsulates ribulose-1,5-bisphosphate carboxylase and carbonic anhydrase. Other well-known BMCs include the Pdu and Eut BMCs, which metabolize 1,2-propanediol and ethanolamine, respectively, with vitamin B$_{12}$-dependent diol dehydratase enzymes. Recent bioinformatic analyses identified a new prevalent type of BMC, hypothesized to utilize vitamin B$_{12}$-independent glycyl radical enzymes to metabolize substrates. Here we use genetic and metabolic analyses to undertake in vivo characterization of the newly identified glycyl radical enzyme microcompartment 3 (GRM3) class of microcompartment clusters. Transcriptome sequencing analyses showed that the microcompartment gene cluster in the genome of the purple photosynthetic bacterium Rhodobacter capsulatus was expressed under dark anaerobic respiratory conditions in the presence of 1,2-propanediol. High-performance liquid chromatography and gas chromatography-mass spectrometry analyses showed that enzymes coded by this cluster metabolized 1,2-propanediol into propionaldehyde, propanol, and propionate. Surprisingly, the microcompartment pathway did not protect these cells from toxic propionaldehyde under the conditions used in this study, with buildup of this intermediate contributing to arrest of cell growth. We further show that expression of microcompartment genes is regulated by a two-component system located downstream of the microcompartment cluster.IMPORTANCE BMCs are protein shells that are designed to compartmentalize enzymatic reactions that require either sequestration of a substrate or the sequestration of toxic intermediates. Due to their ability to compartmentalize reactions, BMCs have also become attractive targets for bioengineering novel enzymatic reactions. Despite these useful features, little is known about the biochemistry of newly identified classes of BMCs. In this study, we have undertaken genetic and in vivo metabolic analyses of the newly identified GRM3 gene cluster

MpeV is a lyase isomerase that ligates a doubly linked phycourobilin on the β-subunit of phycoerythrin I and II in marine Synechococcus

International audienceSynechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within theirlight-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores,green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated tospecific cysteines via bilin lyases, and some of these enzymes, called lyase isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy, and tandem mass spectrometry. Our results show that MpeV is the lyase isomerase that covalently attaches a doubly linked phycourobilin to twocysteine residues (C50, C61) on the β-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB orMpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced byCpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectableactivity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step inchromophore attachment, isomerization, and stability. These data provide novel insights into β-PE biosynthesis and advanceour understanding of the mechanisms guiding lyase isomerases

Lesions in Phycoerythrin Chromophore Biosynthesis in Fremyella diplosiphon Reveal Coordinated Light Regulation of Apoprotein and Pigment Biosynthetic Enzyme Gene Expression

We have characterized the regulation of the expression of the pebAB operon, which encodes the enzymes required for phycoerythrobilin synthesis in the filamentous cyanobacterium Fremyella diplosiphon. The expression of the pebAB operon was found to be regulated during complementary chromatic adaptation, the system that controls the light responsiveness of genes that encode several light-harvesting proteins in F. diplosiphon. Our analyses of pebA mutants demonstrated that although the levels of phycoerythrin and its associated linker proteins decreased in the absence of phycoerythrobilin, there was no significant modulation of the expression of pebAB and the genes that encode phycoerythrin. Instead, regulation of the expression of these genes is coordinated at the level of RNA accumulation by the recently discovered activator CpeR

Metabolomic Analysis Reveals That the Drosophila Gene lysine Influences Diverse Aspects of Metabolism

The fruit fly Drosophila melanogaster has emerged as a powerful model for investigating the molecular mechanisms that regulate animal metabolism. However, a major limitation of these studies is that many metabolic assays are tedious, dedicated to analyzing a single molecule, and rely on indirect measurements. As a result, Drosophila geneticists commonly use candidate gene approaches, which, while important, bias studies toward known metabolic regulators. In an effort to expand the scope of Drosophila metabolic studies, we used the classic mutant lysine (lys) to demonstrate how a modern metabolomics approach can be used to conduct forward genetic studies. Using an inexpensive and well-established gas chromatography-mass spectrometry-based method, we genetically mapped and molecularly characterized lys by using free lysine levels as a phenotypic readout. Our efforts revealed that lys encodes the Drosophila homolog of Lysine Ketoglutarate Reductase/Saccharopine Dehydrogenase, which is required for the enzymatic degradation of lysine. Furthermore, this approach also allowed us to simultaneously survey a large swathe of intermediate metabolism, thus demonstrating that Drosophila lysine catabolism is complex and capable of influencing seemingly unrelated metabolic pathways. Overall, our study highlights how a combination of Drosophila forward genetics and metabolomics can be used for unbiased studies of animal metabolism, and demonstrates that a single enzymatic step is intricately connected to diverse aspects of metabolism

Chelation-induced diradical formation as an approach to modulation of the amyloid-?? aggregation pathway

Current approaches toward modulation of metal-induced A beta aggregation pathways involve the development of small molecules that bind metal ions, such as Cu(II) and Zn(II), and interact with Ab. For this effort, we present the enediyne-containing ligand (Z)-N, N&apos;-bis[1-pyridin-2-yl-meth(E)-ylidene]oct-4-ene- 2,6-diyne-1,8-diamine (PyED), which upon chelation of Cu(II) and Zn(II) undergoes Bergmancyclization to yield diradical formation. The ability of this chelation-triggered diradical to modulate Ab aggregation is evaluated relative to the non-radical generating control pyridine-2-ylmethyl(2-{[(pyridine-2-ylmethylene)-amino]-methyl}-benzyl)-amine (PyBD). Variable-pH, ligand UV-vis titrations reveal pK(a) = 3.81(2) for PyBD, indicating it exists mainly in the neutral form at experimental pH. Lipinski&apos;s rule parameters and evaluation of blood-brain barrier (BBB) penetration potential by the PAMPA-BBB assay suggest that PyED may be CNS+ and penetrate the BBB. Both PyED and PyBD bind Zn(II) and Cu(II) as illustrated by bathochromic shifts of their UV-vis features. Speciation diagrams indicate that Cu(II)PyBD is the major species at pH 6.6 with a nanomolar K-d, suggesting the ligand may be capable of interacting with Cu(II)-A beta species. In the presence of A beta(40/42) under hyperthermic conditions (43 degrees C), the radical-generating PyED demonstrates markedly enhanced activity (2-24 h) toward the modulation of A beta species as determined by gel electrophoresis. Correspondingly, transmission electron microscopy images of these samples show distinct morphological changes to the fibril structure that are most prominent for Cu(II)-A beta cases. The loss of CO2 from the metal binding region of A beta in MALDI-TOF mass spectra further suggests that metal-ligand-A beta interaction with subsequent radical formation may play a role in the aggregation pathway modulation.close

Selective and Reversible Polysulfide-macrocycle Binding at Modified Membranes Suppress Shuttling in Lithium Sulfur Batteries

Lithium sulfur (LiS) batteries are among the next generation of rechargeable batteries offering high energy densities. Obstacles remain for their practical application, such as capacity fading and low Coulombic efficiency resulting from shuttling and reaction of polysulfides with the Li anode. A new supramolecular approach to suppress shuttling using reversible binding of anionic polysulfides, e.g., S3•–, S62–, S72–, S82–, by anion-selective receptors, called cyanostar (CS) macrocycles is reported. Standard separators were coated with the macrocycles and formed chemically selective membranes. Unlike adsorption materials and non-selective supramolecular approaches, cyanostar provides a well-defined molecule-to-molecule mechanism to capture polysulfides as host-guest complexes like (CS)2•S3•– and (CS)4•S72–. Permselectivity emerges from reversibly binding polysulfides inside the membrane to prevent anions passing while allowing cations to pass. Controls using macrocycles that are not selective for anions do not stop shuttling. Cyanostar-coated membranes turn on charging, reduce capacity fading from 0.51 to 0.36% per cycle and improve Columbic efficiency to 85%. These improvements occur even without addition of lithium nitrate used almost universally to inhibit reactions with the Li anode. This new strategy showcases the benefits of using selective receptors to manage movement of ions in sulfur-based batteries