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.     

Imidazo[1,2-α]pyridines possess adenosine A1 receptor affinity for the potential treatment of cognition in neurological disorders

Previous research has shown that bicyclic 6:5-fused heteroaromatic compounds with two N-atoms have variable degrees of adenosine A₁ receptor antagonistic activity. Prompted by this imidazo[1,2-α]pyridine analogues were synthesized and evaluated for their adenosine A₁ and A_2A receptor affinity via radioligand binding studies and subjected to a GTP shift assay to determine its adenosine A₁ receptor agonistic or antagonistic functionality. Imidazo[1,2-α]pyridine, the parent scaffold, was found devoid of affinity for the adenosine A₁ and A_2A receptors. The influence of substitution on position C2 showed no improvement for either adenosine A₁ or A_2A receptor affinity. The addition of an amino or a cyclohexylamino group to position C3 also showed no improvement of adenosine A₁ or A_2A receptor affinity. Surprisingly para-substitution on the phenyl ring at position C2 in combination with a cyclohexylamino group at position C3 led to adenosine A₁ receptor affinity in the low micromolar range with compound 4d showing: (1) the highest affinity for the adenosine A₁ receptor with a K_i value of 2.06 µM and (2) adenosine A₁ receptor antagonistic properties. This pilot study concludes that para-substituted 3-cyclohexylamino-2-phenyl-imidazo[1,2-α]pyridine analogues represent an interesting scaffold to investigate further structure-activity relationships in the design of novel imidazo[1,2-α]pyridine-based adenosine A₁ receptor antagonists for the treatment of neurodegenerative disorders.     

Immobilization of <Emphasis Type="BoldItalic">Irpex lacteus</Emphasis> to liquid-core alginate beads and their application to degradation of pollutants

White rot fungi (WRF) are applicable to biodegradation of recalcitrant pollutants. However, excessive biomass growth typical for WRF cultivation can hinder their large scale applications. Therefore, immobilization of Irpex lacteus to liquid-core alginate beads restricting excessive mycelium growth and simultaneously keeping high degradation rate of pollutants was tested. Effective diffusivities of dyes to the beads varied from (2.98 ± 0.69) × 10^?10 to (10.27 ± 2.60) × 10^?10 m²/s. Remazol Brilliant Blue R (RBBR), Reactive Orange 16 (RO16), and Naphthol Blue Black (NBB) were used as model dyes. The immobilized fungus decolorized model dyes when applied both in microwell plates and in fluidized bed reactors. Using the microwell plates, the apparent reaction rate constants ranged from (2.06 ± 0.11) × 10^?2 to (11.06 ± 0.27) × 10^?2 1/h, depending on the dye used and its initial concentration. High initial concentrations negatively affected the dye decolorization rate. No fungal growth outside the beads was observed in fluidized bed reactors and thus no operational problems linked to an excessive biomass growth occurred. When RBBR was decolorized in subsequent batches in the fluidized bed reactor, the apparent reaction rate constant increased from (11.63 ± 0.35) × 10^?2 to (29.26 ± 7.19) × 10^?2 1/h.     

Stoichiometry and absolute quantification of proteins with mass spectrometry using fluorescent and isotope-labeled concatenated peptide standards

We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent protein (GFP) with a synthetic fusion protein (GAB-GFP) in Escherichia coli to function as a spectroscopic standard for the quantification of an analogous stable isotope-labeled, non-fluorescent fusion protein (GAB*) and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB* contain concatenated sequences of specific proteotypic peptides that are derived from the alpha, beta, and gamma protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB* and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits alpha, beta, and gamma was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with a relative standard deviation of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic, or antibody-based technologies.     

Reproductive isolation among two interfertile Rhododendron species: low frequency of post-F1 hybrid genotypes in alpine hybrid zones

Hybrids between the acid-loving species Rhododendron ferrugineum and the basic soil species Rhododendron hirsutum occur on soils of intermediate pH in the European Alps. Material from two hybrid zones ~500 m apart, and also nearby populations of each parent species, was surveyed for presence/absence of 31 random amplified polymorphic DNA markers that distinguish parents. Based on morphological assessment, the material comprised 51 putative hybrids, 18 putative R. ferrugineum individuals and 26 putative R. hirsutum plants. RAPD data were analysed using a Bayesian approach implemented by the program newhybrids , and also by principal coordinates analysis. The identity of all R. ferrugineum plants examined was confirmed; however, of the putative R. hirsutum individuals examined, two were certainly and 11 possibly hybrid derivatives. Among all hybrid derivatives examined, about half were designated as F₁s or a similar class, otherwise backcrosses to R. hirsutum appeared to be common whereas other hybrid classes were rare and backcrosses to R. ferrugineum possibly absent. Despite this, artificially generated seed of F₂ class and backcrosses in each direction showed greater viability than one parent ( R. hirsutum ). Introgression from R. ferrugineum was also detected in a population that from morphology appeared to contain only R. hirsutum . Hence, the direction of backcrossing might be highly asymmetric within hybrid zones, causing unidirectional gene flow from R. ferrugineum into R. hirsutum . Conversely, the rarity of backcrosses to R. ferrugineum , F₂s and later hybrid generations, which might be due to phenology effects and habitat-mediated selection, could play a part in restricting gene flow towards R. ferrugineum .     

Extended polypeptide linkers establish the spatial architecture of a pyruvate dehydrogenase multienzyme complex

Icosahedral pyruvate dehydrogenase (PDH) enzyme complexes are molecular machines consisting of a central E2 core decorated by a shell of peripheral enzymes (E1 and E3) found localized at a distance of approximately 75-90 A from the core. Using a combination of biochemical, biophysical, and cryo-electron microscopic techniques, we show here that the gap between the E2 core and the shell of peripheral enzymes is maintained by the flexible but extended conformation adopted by 60 linker polypeptides that radiate outwards from the inner E2 core, irrespective of the E1 or E3 occupancy. The constancy of the gap is thus not due to protein-protein interactions in the outer protein shell. The extended nature of the E2 inner-linker regions thereby creates the restricted annular space in which the lipoyl domains of E2 that carry catalytic intermediates shuttle between E1, E2, and E3 active sites, while their conformational flexibility facilitates productive encounters.     

Three-dimensional imaging of the highly bent architecture of Bdellovibrio bacteriovorus by using cryo-electron tomography

Bdellovibrio bacteriovorus cells are small deltaproteobacterial cells that feed on other gram-negative bacteria, including human pathogens. Using cryo-electron tomography, we demonstrated that B. bacteriovorus cells are capable of substantial flexibility and local deformation of the outer and inner membranes without loss of cell integrity. These shape changes can occur in less than 2 min, and analysis of the internal architecture of highly bent cells showed that the overall distribution of molecular machines and the nucleoid is similar to that in moderately bent cells. B. bacteriovorus cells appear to contain an extensive internal network of short and long filamentous structures. We propose that rearrangements of these structures, in combination with the unique properties of the cell envelope, may underlie the remarkable ability of B. bacteriovorus cells to find and enter bacterial prey.     

Nature meets nurture: molecular genetics of gastric cancer

The immensity of genes and molecules implicated in gastric carcinogenesis is overwhelming and the relevant importance of some of these molecules is too often unclear. This review serves to bring us up-to-date with the latest findings as well as to look at the larger picture in terms of how to tackle the problem of solving this multi-piece puzzle. In this review, the environmental nurturing of intestinal cancer is discussed, beginning with epidemiology (known causative factors for inducing molecular change), an update of H. pylori research, including the role of inflammation and stem cells in premalignant lesions. The role of E-cadherin in the nature (genotype) of diffuse gastric cancer is highlighted, and finally the ever growing discipline of SNP analysis (including IL1B) is discussed.