Dual treatment with kynurenine pathway inhibitors and NAD+ precursors synergistically extends life span in Drosophila

Tryptophan catabolism is highly conserved and generates important bioactive metabolites, including kynurenines, and in some animals, NAD⁺. Aging and inflammation are associated with increased levels of kynurenine pathway (KP) metabolites and depleted NAD⁺, factors which are implicated as contributors to frailty and morbidity. Contrastingly, KP suppression and NAD⁺ supplementation are associated with increased life span in some animals. Here, we used DGRP_229 Drosophila to elucidate the effects of KP elevation, KP suppression, and NAD⁺ supplementation on physical performance and survivorship. Flies were chronically fed kynurenines, KP inhibitors, NAD⁺ precursors, or a combination of KP inhibitors with NAD⁺ precursors. Flies with elevated kynurenines had reduced climbing speed, endurance, and life span. Treatment with a combination of KP inhibitors and NAD⁺ precursors preserved physical function and synergistically increased maximum life span. We conclude that KP flux can regulate health span and life span in Drosophila and that targeting KP and NAD⁺ metabolism can synergistically increase life span.     

Synthesis and anti-inflammatory evaluation of N-sulfonyl anthranilic acids via Ir(III)-catalyzed C–H amidation of benzoic acids

The iridium(III)-catalyzed ortho-C–H amidation of benzoic acids with sulfonyl azides is described. These transformations allow the facile generation of N-sulfonyl anthranilic acids, which are known as crucial scaffolds found in biologically active molecules. In addition, all synthetic products were evaluated for in vitro anti-inflammatory activity against interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) with lipopolysaccharide (LPS)-induced RAW264.7 cells. Notably, compounds 4c and 4d, generated from p-OMe- and p-Br-sulfonyl azides, were found to display potent anti-inflammatory property stronger than that of well-known NSAIDs ibuprofen.     

Expression, purification, refolding and characterization of a putative lysophospholipase from Pyrococcus furiosus: retention of structure and lipase/esterase activity in the presence of water-miscible organic solvents at high temperatures

A putative lysophospholipase (PF0480) encoded by the Pyrococcus furiosus genome has previously been cloned and expressed in Escherichia coli. Studies involving crude extracts established the enzyme to be an esterase; however, owing presumably to its tendency to precipitate into inclusion bodies, purification and characterization have thus far not been reported. Here, we report the overexpression and successful recovery and refolding of the enzyme from inclusion bodies. Dynamic light scattering suggests that the enzyme is a dimer, or trimer, in aqueous solution. Circular dichroism and fluorescence spectroscopy show, respectively, that it has mixed beta/alpha structure and well-buried tryptophan residues. Conformational changes are negligible over the temperature range of 30–80 °C, and over the concentration range of 0–50% (v/v) of water mixtures with organic solvents such as methanol, ethanol and acetonitrile. The enzyme is confirmed to be an esterase (hydrolyzing p-NP-acetate and p-NP-butyrate) and also shown to be a lipase (hydrolyzing p-NP-palmitate), with lipolytic activity being overall about 18- to 20-fold lower than esterase activity. Against p-NP-palmitate the enzyme displays optimally activity at pH 7.0 and 70 °C. Remarkably, over 50% activity is retained at 70 °C in the presence of 25% acetonitrile. The high organic solvent stability and thermal stability suggest that this enzyme may have useful biodiesel-related applications, or applications in the pharmaceutical industry, once yields are optimized.     

Genotypic differences in water status,membrane integrity,ionic content,N2-fixing efficiency and dry matter of mungbean nodules under saline irrigation

A pot experiment was conducted under natural conditions of screen house to evaluate the effect of saline irrigation given at flowering stage (30–35 DAS) on nodule functioning and their tolerance in two mungbean genotypes viz. Asha and Muskan based on various physiological traits. The pots containing sandy soil (Typic Torrispamments) were saturated with Cl-dominated saline irrigation to maintain ECe of 2.5, 5.0, 7.5 dS m⁻¹ as compared to control. In both the genotypes osmotic potential (Ψs) and relative water content (RWC %) of nodules decreased significantly, while a sharp rise in proline and total soluble sugars contents were observed with the increasing level of saline irrigation after 10 and 20 days of treatment. A marked increase in hydrogen peroxide (H₂O₂), lipid peroxidation (MDA content) and relative stress injury (RSI %) was noticed in nodules which were much higher in Muskan. The decrease in Ψs of nodules was more pronounced in Asha than in Muskan, while reverse was true for RWC and proline accumulation. A sharp decline in acetylene reduction assay (ARA) for N₂-fixation, leghemoglobin content and dry matter of the nodules was observed, but was more in Muskan than in Asha. Nitrogen (N) content declined while Na⁺/K⁺ ratio and Cl⁻ content increased significantly. The genotype Asha maintained better N₂-fixing efficiency but lower Na⁺/K⁺ ratio and Cl⁻ content in nodules than Muskan. Though the nodule functioning was further deteriorated at 20 DAT in both the genotypes yet the tolerance capacity of nodules in Asha was better than in Muskan under saline conditions which is correlated with the compensatory mechanism i.e. osmoregulation in nodules.Key words: Leghemoglobin, Lipid peroxidation, Minerals, N₂-ase activity, Vigna radiata, Water relations     

Pch2 Links Chromosome Axis Remodeling at Future Crossover Sites and Crossover Distribution during Yeast Meiosis

Segregation of homologous chromosomes during meiosis I depends on appropriately positioned crossovers/chiasmata. Crossover assurance ensures at least one crossover per homolog pair, while interference reduces double crossovers. Here, we have investigated the interplay between chromosome axis morphogenesis and non-random crossover placement. We demonstrate that chromosome axes are structurally modified at future crossover sites as indicated by correspondence between crossover designation marker Zip3 and domains enriched for axis ensemble Hop1/Red1. This association is first detected at the zygotene stage, persists until double Holliday junction resolution, and is controlled by the conserved AAA+ ATPase Pch2. Pch2 further mediates crossover interference, although it is dispensable for crossover formation at normal levels. Thus, interference appears to be superimposed on underlying mechanisms of crossover formation. When recombination-initiating DSBs are reduced, Pch2 is also required for viable spore formation, consistent with further functions in chiasma formation. pch2Δ mutant defects in crossover interference and spore viability at reduced DSB levels are oppositely modulated by temperature, suggesting contributions of two separable pathways to crossover control. Roles of Pch2 in controlling both chromosome axis morphogenesis and crossover placement suggest linkage between these processes. Pch2 is proposed to reorganize chromosome axes into a tiling array of long-range crossover control modules, resulting in chiasma formation at minimum levels and with maximum spacing.     

DYF-1 Is Required for Assembly of the Axoneme in Tetrahymena thermophila

In most cilia, the axoneme can be subdivided into three segments: proximal (the transition zone), middle (with outer doublet microtubules), and distal (with singlet extensions of outer doublet microtubules). How the functionally distinct segments of the axoneme are assembled and maintained is not well understood. DYF-1 is a highly conserved ciliary protein containing tetratricopeptide repeats. In Caenorhabditis elegans, DYF-1 is specifically needed for assembly of the distal segment (G. Ou, O. E. Blacque, J. J. Snow, M. R. Leroux, and J. M. Scholey. Nature. 436:583-587, 2005). We show that Tetrahymena cells lacking an ortholog of DYF-1, Dyf1p, can assemble only extremely short axoneme remnants that have structural defects of diverse natures, including the absence of central pair and outer doublet microtubules and incomplete or absent B tubules on the outer microtubules. Thus, in Tetrahymena, DYF-1 is needed for either assembly or stability of the entire axoneme. Our observations support the conserved function for DYF-1 in axoneme assembly or stability but also show that the consequences of loss of DYF-1 for axoneme segments are organism specific.Cilia are microtubule-rich cellular extensions that arise from basal bodies near the surfaces of most eukaryotic cell types. Defective cilia cause a wide variety of diseases, including polycystic kidney disease, primary ciliary dyskinesia, and retinal degeneration (3). A typical motile cilium has a microtubule-based framework, the axoneme, which contains nine outer (mostly doublet) microtubules and two central (singlet) microtubules. In most cilia, the axoneme can be subdivided into three segments: proximal (transition zone), middle (containing outer doublet microtubules), and distal (containing singlet extensions of peripheral microtubules). The outer doublet microtubules of the middle segment have a complete tubule A made of 13 protofilaments and an incomplete tubule B made of 11 protofilaments that is fused to the wall of the A tubule (36, 57). The outer microtubules in the distal segment lack the B tubule (32, 49). The distal segment also lacks dynein arms and radial spokes, and its microtubules are terminated by caps that are associated with the plasma membranes at the tips of cilia (11, 50). The distal segments are characterized by a high level of microtubule turnover, which could play a role in the regulation of the length of cilia (31).The mechanisms that establish the segmental subdivision of the axoneme are not well understood. Studies of Caenorhabditis elegans indicate that the distal segment is assembled using a mechanism that differs from the one utilized in the middle and proximal segments (54). In most cell types, ciliogenesis is dependent on the intraflagellar transport (IFT) pathway, a bidirectional motility of protein aggregates, known as IFT particles, that occurs along outer microtubules (10, 28, 29, 42). IFT particles are believed to provide platforms for transport of axonemal precursors (23, 44). The anterograde component of IFT that delivers cargo from the cell body to the tips of cilia is carried out by kinesin-2 motors (28, 63), whereas the cytoplasmic dynein DHC1b is responsible for the retrograde IFT (41, 43, 53). Importantly, in the well-studied amphid cilia of C. elegans, two distinct kinesin-2 complexes are involved in the anterograde IFT and differ in movement velocity: the “slow” heterotrimeric kinesin-II and the “fast” homodimeric OSM-3 kinesin (54). While kinesin-II and OSM-3 work redundantly to assemble the middle segment, OSM-3 alone functions in the assembly of the distal segment (39, 56).In C. elegans, DYF-1 is specifically required for assembly of the distal segment (39). In the DYF-1 mutant, the rate of IFT in the remaining middle segment is reduced to the level of the slow kinesin-II, suggesting that the Osm3 complex is nonfunctional and that kinesin-II functions alone in the middle segment. Thus, DYF-1 could either activate OSM-3 kinesin or dock OSM-3 to IFT particles (14, 39).However, a recent study of zebrafish has led to a different model for DYF-1 function. Zebrafish embryos that are homozygous for a loss of function of fleer, an ortholog of DYF-1, have shortened olfactory and pronephric cilia and ultrastructural defects in the axonemes. In the middle segment, the fleer axonemes have B tubules that are disconnected from the A tubule, indicating that DYF-1 functions in the middle segment and could play a role in the stability of doublet microtubules (40). Earlier, a similar mutant phenotype was reported in Tetrahymena for a mutation in the C-terminal tail domain of β-tubulin, at the glutamic acid residues that are used by posttranslational polymodifications (glycylation and glutamylation) (47). Glycylation (46) and glutamylation (12) are conserved polymeric posttranslational modifications that affect tubulin and are highly enriched on microtubules of axonemes and centrioles (reviewed in reference 20). Other studies have indicated that tubulin glutamylation contributes to the assembly and stability of axonemes and centrioles (4, 8). The fleer mutant zebrafish cilia have reduced levels of glutamylated tubulin (40). Pathak and colleagues proposed that the primary role of DYF-1/fleer is to serve as an IFT cargo adapter for a tubulin glutamic acid ligase (25) and that the effects of lack of function of DYF-1/fleer could be caused by deficiency in tubulin glutamylation in the axoneme (40). As an alternative hypothesis, the same authors proposed that DYF-1 is a structural component that stabilizes the doublet microtubules in the axoneme (40).Here, we evaluate the significance of a DYF-1 ortholog, Dyf1p, in Tetrahymena thermophila. Unexpectedly, we found that Tetrahymena cells lacking Dyf1p either fail to assemble an axoneme or can assemble an axoneme remnant. While our observations revealed major differences in the significance of DYF-1 for segmental differentiation in diverse models, it is clear that DYF-1 is a conserved and critical component that is required for assembly of the axoneme.     

Thyroid receptor ligands. Part 5: novel bicyclic agonist ligands selective for the thyroid hormone receptor beta

Based on the examination of the crystal structure of rat TRbeta complexed with 3,5,3'-triiodo-l-thyronine (2) a novel TRbeta-selective indole derivative 6b was prepared and tested in vitro. This compound was found to be 14 times selective for TRbeta over TRalpha in binding and its beta-selectivity could be rationalized through the comparison of the X-ray crystallographic structures of 6b complexed with TRalpha and TRbeta.     

Mutations in Traf3ip1 reveal defects in ciliogenesis, embryonic development, and altered cell size regulation

Tumor necrosis factor alpha receptor 3 interacting protein 1 (Traf3ip1), also known as MIPT3, was initially characterized through its interactions with tubulin, actin, TNFR-associated factor-3 (Traf3), IL-13R1, and DISC1. It functions as an inhibitor of IL-13-mediated phosphorylation of Stat6 and in sequestration of Traf3 and DISC1 to the cytoskeleton. Studies of the Traf3ip1 homologs in C. elegans (DYF-11), Zebrafish (elipsa), and Chlamydomonas (IFT54) revealed that the protein localizes to the cilium and is required for ciliogenesis. Similar localization data has now been reported for mammalian Traf3ip1. This raises the possibility that Traf3ip1 has an evolutionarily conserved role in mammalian ciliogenesis in addition to its previously indicated functions. To evaluate this possibility, a Traf3ip1 mutant mouse line was generated. Traf3ip1 mutant cells are unable to form cilia. Homozygous Traf3ip1 mutant mice are not viable and have both neural developmental defects and polydactyly, phenotypes typical of mouse mutants with ciliary assembly defects. Furthermore, in Traf3ip1 mutants the hedgehog pathway is disrupted, as evidenced by abnormal dorsal–ventral neural tube patterning and diminished expression of a hedgehog reporter. Analysis of the canonical Wnt pathway indicates that it was largely unaffected; however, specific domains in the pharyngeal arches have elevated levels of reporter activity. Interestingly, Traf3ip1 mutant embryos and cells failed to show alterations in IL-13 signaling, one of the pathways associated with its initial discovery. Novel phenotypes observed in Traf3ip1 mutant cells include elevated cytosolic levels of acetylated microtubules and a marked increase in cell size in culture. The enlarged Traf3ip1 mutant cell size was associated with elevated basal mTor pathway activity. Taken together, these data demonstrate that Traf3ip1 function is highly conserved in ciliogenesis and is important for proper regulation of a number of essential developmental and cellular pathways. The Traf3ip1 mutant mouse and cell lines will provide valuable resources to assess cilia function in mammalian development and also serve as a tool to explore the potential connections between cilia and cytoskeletal dynamics, mTor regulation, and cell volume control.     

FXYD proteins stabilize Na,K-ATPase: amplification of specific phosphatidylserine-protein interactions

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared with the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C₁₂E₈), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E₁ ↔ E₂ conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.