The insulin signaling pathway controls cell growth and metabolism, thus its deregulation is associated with both cancer and diabetes. Phosphatidylinositol 3‐kinase (PI3K) contributes to the cascade ...of phosphorylation events occurring in the insulin pathway by activating the protein kinase B (PKB/AKT), which phosphorylates several substrates, including those involved in glucose uptake and storage. PI3K inactivating mutations are associated with insulin resistance while activating mutations are identified in human cancers. Here we show that RNAi‐induced depletion of the Drosophila PI3K catalytic subunit (Dp110) results in diabetic phenotypes such as hyperglycemia, body size reduction, and decreased glycogen content. Interestingly, we found that hyperglycemia produces chromosome aberrations (CABs) triggered by the accumulation of advanced glycation end‐products and reactive oxygen species. Rearing PI3KRNAi flies in a medium supplemented with pyridoxal 5′‐phosphate (PLP; the catalytically active form of vitamin B6) rescues DNA damage while, in contrast, treating PI3KRNAi larvae with the PLP inhibitor 4‐deoxypyridoxine strongly enhances CAB frequency. Interestingly, PLP supplementation rescues also diabetic phenotypes. Taken together, our results provide a strong link between impaired PI3K activity and genomic instability, a crucial relationship that needs to be monitored not only in diabetes due to impaired insulin signaling but also in cancer therapies based on PI3K inhibitors. In addition, our findings confirm the notion that vitamin B6 is a good natural remedy to counteract insulin resistance and its complications.
RNAi‐induced depletion of the Drosophila phosphatidylinositol 3‐kinase (PI3K) catalytic subunit results in diabetic phenotypes such as hyperglycemia, body size reduction, and impaired glycogen synthesis. We found that hyperglycemia produces chromosome aberrations (CABs) triggered by the accumulation of advanced glycation end‐products and reactive oxygen species. Vitamin B6 treatment rescues DNA damage and diabetic phenotypes, while its inhibitor 4‐deoxypyridoxine strongly enhances CAB frequency. Our results provide a strong link between impaired PI3K activity and genomic instability and confirm that vitamin B6 is a good remedy to counteract insulin resistance and its complications.
The Multiple Try Metropolis (MTM) algorithm is an advanced MCMC technique based on drawing and testing several candidates at each iteration of the algorithm. One of them is selected according to ...certain weights and then it is tested according to a suitable acceptance probability. Clearly, since the computational cost increases as the employed number of tries grows, one expects that the performance of an MTM scheme improves as the number of tries increases, as well. However, there are scenarios where the increase of number of tries does not produce a corresponding enhancement of the performance. In this work, we describe these scenarios and then we introduce possible solutions for solving these issues.
Pyridoxal 5'-phosphate (PLP) is a cofactor for dozens of B(6) requiring enzymes. PLP reacts with apo-B(6) enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine ...residue, thus yielding the catalytically active holo-B(6) enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4'-aldehyde moiety forms covalent adducts with other compounds and non-B(6) proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B(6) enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.
The measurement of the Si lattice parameter by X‐ray interferometry assumes the use of strain‐free crystals, which might not be true because of intrinsic stresses due to surface relaxation, ...reconstruction and oxidation. X‐ray phase‐contrast topography was used to investigate the strain sensitivity to the finishing, annealing and coating of interferometer crystals. The topography capabilities were assessed by measuring the lattice strain due to films of copper deposited on the interferometer mirror crystal. A by‐product has been the measurement of the surface stresses after complete relaxation of the coatings.
It is shown that the surface finishing has measurable effects on the bulk strain field of a 0.8 mm‐thick Si crystal, at the level of a few parts in 109. In particular, it is shown that the strain due to a Cu film a few nanometres thick is well modelled by a surface stress (equiaxial and uniform) equal to 0.29 (2) GPa × t, where t is the film thickness. This result is consistent with the stress thickness values given in the literature for similar interfaces.
We investigate in theory and experiment the frequency dependence of magnetic losses in Grain-Oriented 0.29 mm thick high-permeability steel sheets up to 10 kHz. Such an unusually broad frequency ...range, while responding to increasing trends towards high-frequency regimes in applications, is conducive to a complex evolution of the magnetization process, as imposed by increasing frequencies to a non-linear high-permeability saturable material. We show that the concept of loss decomposition, supported by observations of the domain wall dynamics through Kerr experiments, is effective in the assessment of the broadband frequency dependence of the energy loss. By calculating, in particular, the instantaneous and time averaged macroscopic induction profiles across the sheet thickness through the Maxwell’s diffusion equation, the classical loss component Wclass, versus frequency f and peak polarization Jp, is obtained. A simplified theoretical approach is pursued in this case by identifying the normal magnetization curve with the magnetic constitutive equation of the material. While the hysteresis loss Whyst is shown to invariably increase with frequency, the excess loss Wexc, the quantity directly associated with the eddy currents circulating around the moving domain walls, tends to vanish upon increasing both frequency and induction values. The Kerr experiments actually show that, while the oscillating 180° domain walls can adjust to the depth of the induction profile by bowing at low Jp values, the magnetization reversal at high inductions and high frequencies occurs by inward motion of symmetric fronts originating at the sheet surface, according to a classical framework.
Pyridoxal-5′-phosphate oxidase (PNPO) deficiency presents as a severe neonatal encephalopathy responsive to pyridoxal-5′-phosphate (PLP) or pyridoxine. Recent studies widened the phenotype of this ...condition and detected genetic variants on PNPO gene whose pathogenic role and clinical expression remain to be established.
This paper aims to characterize the functional effects of the c.347G>A (p.Arg116Gln) mutation in the PNPO gene in order to define its pathogenicity and describe the clinical features of new patients with epilepsy carrying this mutation.
Arg116Gln protein variant was expressed as recombinant protein. The mutant protein was characterized with respect to structural and kinetic properties, thermal stability, binding constants of cofactor (FMN) and product (PLP). We also reviewed clinical data of 3 new patients carrying the mutation.
The Arg116Gln mutation does not alter the overall enzyme structure and only slightly affects its catalytic efficiency; nevertheless, this mutation affects thermal stability of PNPO, reduces its affinity for FMN and impairs transfer of PLP to PLP-dependent enzymes.
Three boys with seizure onset between 8months and 3years of age, carrying the Arg116Gln mutation, are described. These three patients exhibited different seizure types associated with interictal EEG abnormalities and slow background activity. Mild/moderate intellectual disability was observed in 2/3 patients. A dramatic therapeutic response to pyridoxine was observed in the only patient who still had active seizures when starting treatment, while in all three patients interictal EEG discharges and background activity improved after pyridoxine treatment was initiated.
The reported data support a pathogenic role of the c.347G>A (p.Arg116Gln) mutation in PNPO deficiency. The later onset of symptoms and the milder epilepsy phenotype of these expand the disease phenotype.
•A later onset of epilepsy in PNPO deficiency can be observed.•Biochemical studies evidenced pathogenic effects of c.347G>A (p.Arg116Gln).•The mutation c.347G>A (p.Arg116Gln) is associated with a milder epileptic phenotype.
Common protocols for the incorporation of noncanonical amino acids (ncAAs) into proteins require addition of the desired ncAA to the growth medium, its cellular uptake, and subsequent intracellular ...accumulation. This feeding scheme is generally suitable for small-scale proof-of-concept incorporation experiments. However, it is no general solution for orthogonal translation of ncAAs, as their chemical synthesis is generally tedious and expensive. Here, we describe a simple protocol that efficiently couples in situ semi-synthetic biosynthesis of L-azidohomoalanine and its incorporation into proteins at L-methionine (Met) positions. In our metabolically engineered Met-auxotrophic Escherichia coli strain, Aha is biosynthesized from externally added sodium azide and O-acetyl-L-homoserine as inexpensive precursors. This represents an efficient platform for expression of azide-containing proteins suitable for site-selective bioorthogonal strategies aimed at noninvasive protein modifications (Tornøe et al., J Org Chem 67:3057-3064, 2002; Kiick et al., Angew Chem Int Ed 39:2148-2152, 2000; Budisa, Angew Chem Int Ed Engl 47:6426-6463, 2004; van Hest, J Am Chem Soc 122:1282-1288, 2000).
We present the first data on the life history traits and growth patterns of Boana riojana from 35 individuals belonging to two populations in central Argentina. We determined age using ...skeletochronology and analyzed morphometric variables in relation to these ages. The mean snout-vent length of sexually mature males was 53.7 mm, with an average age of 2.4 years. The species had a maximum longevity of four years, and the minimum age at sexual maturity was two years, which was consistent across both populations. Body size and age showed a positive correlation. The von Bertalanffy growth equation revealed an asymptotic snout-vent length of 59.7 mm and a growth coefficient of 0.87. Additionally, a decline in growth rate was observed from the second to the third year of age. We found no significant differences in the variables between both populations. In conclusion, determining the individual age of different populations of B. riojana has, for the first time, allowed us to establish the life history traits of the species.
The disturbed metabolism of vitamins B1 or B6, which are essential for neurotransmitters homeostasis, may cause seizures. Our study aims at revealing therapeutic potential of vitamins B1 and B6 by ...estimating the short- and long-term effects of their combined administration with the seizure inductor pentylenetetrazole (PTZ). The PTZ dose dependence of a seizure and its parameters according to modified Racine’s scale, along with delayed physiological and biochemical consequences the next day after the seizure are assessed regarding sexual dimorphism in epilepsy. PTZ sensitivity is stronger in the female than the male rats. The next day after a seizure, sex differences in behavior and brain biochemistry arise. The induced sex differences in anxiety and locomotor activity correspond to the disappearance of sex differences in the brain aspartate and alanine, with appearance of those in glutamate and glutamine. PTZ decreases the brain malate dehydrogenase activity and urea in the males and the phenylalanine in the females. The administration of vitamins B1 and B6 24 h before PTZ delays a seizure in female rats only. This desensitization is not observed at short intervals (0.5–2 h) between the administration of the vitamins and PTZ. With the increasing interval, the pyridoxal kinase (PLK) activity in the female brain decreases, suggesting that the PLK downregulation by vitamins contributes to the desensitization. The delayed effects of vitamins and/or PTZ are mostly sex-specific and interacting. Our findings on the sex differences in sensitivity to epileptogenic factors, action of vitamins B1/B6 and associated biochemical events have medical implications.
The diploid Odontophrynus cordobae and its autopolyploid counterpart O. americanus (4n) co-occur in a small-sized contact zone in Central Argentina, together with numerous specimens of a cryptic ...triploid taxon. Additionally, we monitor another five localities inhabited by taxa of this species complex within a radius of 76 km north and south of the contact zone to look for possible co-occurrence of taxa. In this study, we analyze the reproductive interactions in this breeding assemblage covering three levels of ploidy. We focus on the advertisement call structure as a powerful mechanism of premating isolation and on the mating preferences of males and females in the natural habitat. Advertisement calls of triploids were indistinguishable from those of tetraploids, and both differed significantly in pulse rate from diploids. Analyses of 21 amplexi demonstrated that heterospecific matings dominated breeding: one between a diploid female and tetraploid male, and ten between triploids and tetraploids. At three localities with syntopic diploids and tetraploids, premating isolation was almost perfect, preventing the formation of triploids. Therefore, we question the putative origin of triploids from hybridization and discuss alternatives. This unique system, including three bisexual taxa of distinct ploidy, which interact reproductively, remains to be investigated in more detail to fully understand the mechanism stabilizing its persistence.
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