Natriuretic peptides modify <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> cytotoxicity by regulating cyclic nucleotides and modifying LPS structure

Background  

Nervous tissues express various communication molecules including natriuretic peptides, i.e. Brain Natriuretic Peptide (BNP) and C-type Natriuretic Peptide (CNP). These molecules share structural similarities with cyclic antibacterial peptides. CNP and to a lesser extent BNP can modify the cytotoxicity of the opportunistic pathogen Pseudomonas aeruginosa. The psychrotrophic environmental species Pseudomonas fluorescens also binds to and kills neurons and glial cells, cell types that both produce natriuretic peptides. In the present study, we investigated the sensitivity of Pseudomonas fluorescens to natriuretic peptides and evaluated the distribution and variability of putative natriuretic peptide-dependent sensor systems in the Pseudomonas genus.     

Involvement of a phospholipase C in the hemolytic activity of a clinical strain of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Background  

Pseudomonas fluorescens is a ubiquitous Gram-negative bacterium frequently encountered in hospitals as a contaminant of injectable material and surfaces. This psychrotrophic bacterium, commonly described as unable to grow at temperatures above 32°C, is now considered non pathogenic. We studied a recently identified clinical strain of P. fluorescens biovar I, MFN1032, which is considered to cause human lung infection and can grow at 37°C in laboratory conditions.     

Design,synthesis, and evaluation of substituted nicotinamide adenine dinucleotide (NAD+) synthetase inhibitors as potential antitubercular agents

Nicotinamide adenine dinucleotide (NAD⁺) synthetase catalyzes the last step in NAD⁺ biosynthesis. Depletion of NAD⁺ is bactericidal for both active and dormant Mycobacterium tuberculosis (Mtb). By inhibiting NAD⁺ synthetase (NadE) from Mtb, we expect to eliminate NAD⁺ production which will result in cell death in both growing and nonreplicating Mtb. NadE inhibitors have been investigated against various pathogens, but few have been tested against Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied to understand the structure–activity-relationships of substituents on this position. Compounds were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent compound displayed very weak inhibition against Mtb NadE (IC₅₀ = 1000 µM), we observed up to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of the series with an IC₅₀ value of 90 µM against Mtb NadE. Our modeling results show that these urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is supported by data showing that, even when treated with potent inhibitors, NadE catalysis is restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb cell growth with MIC values of 19–100 µg/mL. These results improve our understanding of the SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a potential antitubercular drug target.     

Structural studies of N-terminal mutants of Connexin 32 using (1)H NMR spectroscopy

Microtubules (MTs) control cell replication, material transport and motion in eukaryotic cells, but MT role in several pathologies is still unknown. These functions are related to the MT physico-chemical properties and MT formation mode starting from tubulin molecules. This study describes a new method, based on the computer aided analysis of the electron paramagnetic resonance (EPR) spectra of selected spin probes to obtain structural and dynamical information on tubulins and MTs and the kinetics of MTs formation promoted by guanosine-5'-triphosphate (GTP). It was found that tubulin and MTs avoid radical quenching caused by ethylene glycol tetraacetic acid (EGTA). MT formation showed different kinetics as a function of tubulin concentration. At 5 mg/mL of tubulin, MTs were formed in 8 min. These results are also useful for getting information on MT-drug interactions.     

Roles of GPR41 and GPR43 in leptin secretory responses of murine adipocytes to short chain fatty acids

GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)-stimulated leptin secretion by activating Gα_i. Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Gα_i signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild-type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild-type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Gα_i signalling mediated by GPR43 in SCFA-stimulated leptin secretion.     

31P NMR and saturation transfer studies of the effect of Pb2+ on cultured osteoblastic bone cells

The mechanism of lead toxicity at the cellular level remains unknown, although an effect of lead on intracellular Ca2+ has been described. Since bone is a major target for lead, we have investigated the effect of lead on bioenergetic rates and on the intracellular free Mg2+ concentration in cultured osteoblastic bone cells. Using 31P NMR and the saturation transfer technique we have detected a sizable (18%) transfer of saturation from gamma ATP to Pi in a perfused osteoblastic osteosarcoma bone cell line, Ros 17/2.8, and have found a large (greater than 82%) reduction in the Pi----ATP rate upon treatment with 10 microM Pb2+. The NMR-measured unidirectional rate was much greater than the net rate of ATP synthesis through glycolysis and oxidative phosphorylation. By using iodoacetate we investigated the mechanism of the saturation transfer and found that it is catalyzed by the glycolytic enzyme couple glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. The net rate of glycolysis as measured by lactate production and that of oxidative phosphorylation as measured by O2 consumption were found to be significantly decreased by 18 and 74%, respectively, with lead treatment. In addition, from the chemical shifts of intracellular ATP resonances, we found a significant reduction of 21% in the intracellular free Mg2+ concentration upon Pb2+ treatment. The observed lead-induced reduction in ATP synthesis/utilization and the decrease in intracellular free Mg2+ may contribute to the impairment of bone formation during lead intoxication.     

Maize lateral root developmental plasticity induced by mild water stress. II: Genotype-specific spatio-temporal effects on determinate development

Maize lateral roots exhibit determinate growth, whereby the meristem is genetically programmed to stop producing new cells. To explore whether lateral root determinacy is modified under water deficits, we studied two maize genotypes (B73 and FR697) with divergent responses of lateral root growth to mild water stress using an experimental system that provided near-stable water potential environments throughout lateral root development. First-order laterals of the primary root system of FR697 exhibited delayed determinacy when grown at a water potential of −0.28 MPa, resulting in longer and wider roots than in well-watered (WW) controls. In B73, in contrast, neither the length nor width of lateral roots was affected by water deficit. In water-stressed FR697, root elongation continued at or above the maximum rate in WW roots for 3 days longer, and was still 45% of maximum when WW roots approached their determinate length. Maintenance of root elongation was associated with sustained rates of cell production. In addition, kinematic analyses showed that reductions in tissue expansion rates with aging were delayed in the longitudinal, radial and tangential planes throughout the root growth zone. Thus, this study reveals large genotypic differences in the interaction of water stress with developmental determinacy of maize lateral roots.