Preparation, characterization and in vivo conversion of new water-soluble sulfenamide prodrugs of carbamazepine

Improved synthetic methods are reported for the preparation of sulfenamide derivatives of carbamazepine (CBZ) for evaluation as prodrugs. These sulfenamide prodrugs were designed to rapidly release CBZ in vivo by cleavage of the sulfenamide bond by chemical reaction with glutathione and other sulfhydryl compounds. Physicochemical characterization and in vivo conversion of a new prodrug of CBZ was evaluated to further establish the proof of concept of the sulfenamide prodrug approach.     

Obesity-Induced Cellular Senescence Drives Anxiety and Impairs Neurogenesis

1. Download : Download high-res image (248KB)
2. Download : Download full-size image

     

Isoflurane and Sevoflurane Induce Severe Hepatic Insulin Resistance in a Canine Model

IntroductionAnesthesia induces insulin resistance, which may contribute to elevated blood glucose and adverse post-operative outcomes in critically ill patients, and impair glycemic control in surgical patients with diabetes. However, little is known about the mechanisms by which anesthesia impairs insulin sensitivity. Here we investigate the effects of anesthesia on insulin sensitivity in metabolic tissues.MethodsHyperinsulinemic-euglycemic clamps were performed in 32 lean (control diet; n = 16 conscious versus n = 16 anesthetized) and 24 fat-fed (6 weeks fat-feeding; n = 16 conscious versus n = 8 anesthetized) adult male mongrel dogs in conjunction with tracer methodology to differentiate hepatic versus peripheral insulin sensitivity. Propofol was administered as an intravenous bolus (3mg/kg) to initiate anesthesia, which was then maintained with inhaled sevoflurane or isoflurane (2–3%) for the duration of the procedure.ResultsAnesthesia reduced peripheral insulin sensitivity by approximately 50% in both lean and fat-fed animals as compared to conscious animals, and insulin action at the liver was almost completely suppressed during anesthesia such that hepatic insulin sensitivity was decreased by 75.5% and; 116.2% in lean and fat-fed groups, respectively.ConclusionInhaled anesthesia induces severe hepatic insulin resistance in a canine model. Countermeasures that preserve hepatic insulin sensitivity may represent a therapeutic target that could improve surgical outcomes in both diabetic and healthy patients.     

The C Terminus of Mouse Monocyte Chemoattractant Protein 1 (MCP1) Mediates MCP1 Dimerization while Blocking Its Chemotactic Potency

The extracellular protease plasmin cleaves mouse MCP1 (monocyte chemoattractant protein 1) at lysine 104, releasing a 50-amino acid C-terminal domain. The cleavage event increases the chemotactic activity of MCP1 and, by doing so, promotes the progression of excitotoxic injury in the central nervous system in pathological settings. The mechanism through which the cleavage event enhances MCP1-mediated chemoattraction is unknown; to investigate it, we use wild-type and mutant forms of recombinant MCP1. Full-length MCP1 (FL-MCP1) is secreted by cells as a dimer or multimer. We show that a mutant truncated at the C terminus, K104Stop-MCP1, does not dimerize, revealing that the C terminus mediates the interaction. MCP1 interacts with the monocyte/microglia receptor CCR2. The interaction is critical to the function of MCP1 because CCR2^−/− microglia do not undergo chemotaxis in response to MCP1 stimulation. We show that stimulation of microglia with FL-MCP1 or K104Stop-MCP1 triggers CCR2 internalization, whereas a mutant form unable to be cleaved at lysine 104 (K104A-MCP1) is relatively ineffective in this assay, suggesting that the C-terminal region interferes with the MCP1-CCR2 interaction. Moreover, FL-MCP1 and K104Stop-MCP1 stimulation leads to activation of Rac1, a small GTPase involved in cell migration. Conversely, MCP1-stimulated microglial migration is blocked by the Rac1 inhibitor, NSC23766, demonstrating the requirement for Rac1 effector pathways in this response. Taken together, we propose a model for MCP1 localization, activation, and function based on the initial presence and then removal of its C terminus, coupled with a requisite downstream signaling pathway from CCR2 stimulation to Rac1 activation.     

Polyamine depletion switches the form of 2-deoxy-D-ribose-induced cell death from apoptosis to necrosis in HL-60 cells

Our previous studies demonstrated that intracellular polyamine depletion blocked HL-60 cell apoptosis triggered by exposure to 2-deoxy-d-ribose (dRib). Here, we have characterized the intracellular events underlying the apoptotic effects of dRib and the involvement of polyamines in these effects. Treatment of HL-60 cells with dRib induces loss of mitochondrial transmembrane potential, radical oxygen species production, intracellular glutathione depletion and translocation of Bax from cytosol to membranes. These effects are followed by cell death. However, the mode of cell death caused by dRib depends on intracellular levels of polyamines. d-Rib-treated cells with normal polyamine levels, progressing through the G(1) into the S and G(2)/M phases, undergo apoptosis, while in polyamine-depleted cells, being blocked at the G(1) phase, cell death mechanisms are switched to necrosis. The present study points to a relationship between the cell cycle distribution and the mode of cell death, and suggests that the level of intracellular spermidine, essential to cell cycle progression, may determine whether a cell dies by apoptosis or necrosis in response to a death stimulus.     

Structural features of linear (alphaMe)Val-based peptides in solution by photophysical and theoretical conformational studies

In continuation of our studies on the determination of the structural features of functionalized peptides in solution by combining time-resolved fluorescence data and molecular mechanics results, the conformational features of a series of linear, L-(alphaMe)Val-based peptides have been investigated in methanol. These foldamers have the general formula F[(alphaMe)Val](r)-T-[(alphaMe)Val](2)NHtBu, where (alphaMe)Val = C(alpha)-methylvaline and r = 0-3, while F [= fluoren-9-ylmethoxycarbonyl (Fmoc)] and T [= 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-carboxylic (Toac)] are a fluorophoric N(alpha)-protecting group and a nitroxide-based alpha-amino acid quencher, respectively. According to ir and CD spectra, the longest term of the series (r = 3) attains a 3(10)-helical structure, while the other peptides populate an intramolecularly H-bonded, 3(10)-helix-like conformation affected by dynamic helical distortions, which are enhanced by the shortness of the backbone chain. Such distortions are reflected in both the energy of the stretching mode and the molar extinction coefficient of the H-bonded N-H groups, the former being higher and the latter smaller than those of a stable 3(10)-helix. Steady-state and time-resolved fluorescence measurements in methanol show a strong quenching of Fmoc by the Toac residue, located at different helix positions, depending on the r value. Comparison of quenching efficiencies and lifetime preexponents with those theoretically obtained from the deepest energy minimum conformers, assuming a F?rster mechanism, is satisfactory. The computed structures exhibit a rather compact arrangement, which accounts for the few sterically favored conformations for each peptide, in full agreement with the time-resolved fluorescence data. Orientational effects between the probes must be taken into account for a correct interpretation of the fluorescence decay results, implying that interconversion among conformational substates involving the probes is slower than the energy transfer rate.     

Antifungal Resistance and Virulence Among <Emphasis Type="Italic">Candida</Emphasis> spp. from Captive <Emphasis Type="Italic">Amazonian manatees</Emphasis> and West Indian Manatees: Potential Impacts on Animal and Environmental Health

This work aimed at evaluating the antifungal susceptibility and production of virulence factors by Candida spp. isolated from sirenians in Brazil. The isolates (n = 105) were recovered from the natural cavities of Amazonian and West Indian manatees and were tested for the susceptibility to amphotericin B, itraconazole, and fluconazole and for the production of phospholipases, proteases, and biofilm. The minimum inhibitory concentrations (MICs) for amphotericin B ranged from 0.03 to 1 µg/mL, and no resistant isolates were detected. Itraconazole and fluconazole MICs ranged from 0.03 to 16 µg/mL and from 0.125 to 64 µg/mL, respectively, and 35.2% (37/105) of the isolates were resistant to at least one of these azole drugs. Concerning the production of virulence factors, phospholipase activity was observed in 67.6% (71/105) of the isolates, while protease activity and biofilm production were detected in 50.5% (53/105) and 32.4% (34/105) of the isolates, respectively. Since the natural cavities of manatees are colonized by resistant and virulent strains of Candida spp., these animals can act as sources of resistance and virulence genes for the environment, conspecifics and other animal species, demonstrating the potential environmental impacts associated with their release back into their natural habitat.     

Best practices for the visualization,mapping, and manipulation of R‐loops

R‐loops represent an abundant class of large non‐B DNA structures in genomes. Even though they form transiently and at modest frequencies, interfering with R‐loop formation or dissolution has significant impacts on genome stability. Addressing the mechanism(s) of R‐loop‐mediated genome destabilization requires a precise characterization of their distribution in genomes. A number of independent methods have been developed to visualize and map R‐loops, but their results are at times discordant, leading to confusion. Here, we review the main existing methodologies for R‐loop mapping and assess their limitations as well as the robustness of existing datasets. We offer a set of best practices to improve the reproducibility of maps, hoping that such guidelines could be useful for authors and referees alike. Finally, we propose a possible resolution for the apparent contradictions in R‐loop mapping outcomes between antibody‐based and RNase H1‐based mapping approaches.