Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.     

Sensational placodes: Neurogenesis in the otic and olfactory systems

For both the intricate morphogenetic layout of the sensory cells in the ear and the elegantly radial arrangement of the sensory neurons in the nose, numerous signaling molecules and genetic determinants are required in concert to generate these specialized neuronal populations that help connect us to our environment. In this review, we outline many of the proteins and pathways that play essential roles in the differentiation of otic and olfactory neurons and their integration into their non-neuronal support structures. In both cases, well-known signaling pathways together with region-specific factors transform thickened ectodermal placodes into complex sense organs containing numerous, diverse neuronal subtypes. Olfactory and otic placodes, in combination with migratory neural crest stem cells, generate highly specialized subtypes of neuronal cells that sense sound, position and movement in space, odors and pheromones throughout our lives.     

Intraspecific diversity in the fungal speciesChaunopycnis alba: Implications for microbial screening programs

Intraspecific variation among 84 isolates of the anamorphic fungusChaunopycnis alba from 26 different geographical locations was analyzed by investigating optimal growth temperatures, differences in the production of secondary metabolites and presence or absence of the cyclosporin synthetase gene. The genetic diversity was assessed using random amplified polymorphic DNA (RAPD). Analysis of these data showed high genetic, metabolic and physiological diversity within this species. Isolates from the Antarctic represented the most homogeneous group withinC. alba and together with isolates from the Arctic these polar strains differed from alpine, temperate and tropical strains by low optimal growth temperatures and by low production of secondary metabolites. Isolates from tropical climes were characterized by high optimal growth temperatures and by the production of comparatively diverse metabolite spectra. Most of the isolates that were similar in the combination of their physiological and metabolic characters were also genetically related. Isolates from different geographical origins did not show many similarities, with the exception of the cyclosporin A-producing isolates, and large diversity could be observed even within a single habitat. This leads us to the suggestion that for pharmaceutical screening programs samples should be collected from a diversity of different geographical and climatic locations. For the selection of strains for screening the RAPD assay seems to be the most powerful tool. It reflected the highest intraspecific diversity and the results corresponded well with the other characteristics.     

Interaction of morphine and naloxone with mixed monolayers of lecithin and gangliosides

Monomolecular films of lecithin, gangliosides or lecithin/gangliosides mixtures were studied on a Langmuir through in order to examine the interactions between these lipids and opioid agonists or antagonists. Lecithin alone did not interact in a monolayer structure with opioids. However, gangliosides and lecithin/gangliosides mixtures were expanded by both morphine and naloxone. The expansion of ganglioside-containing monolayers was greater with morphine than with the antagonist, naloxone.     

Polymyxin B-lipid interactions in Langmuir-Blodgett monolayers of Escherichia coli lipids: a thermodynamic and atomic force microscopy study

The dramatically increased frequency of antibiotic resistance has led to intensive efforts towards developing new families of antibiotics. Membrane-active antibiotic peptides such as polymyxin B (PxB) hold promise as the next generation of antibiotics, since they rarely spur the evolution of resistance. At low concentrations in the membrane, PxB forms vesicle-vesicle contacts and induces lipid exchange without leakage or fusion, a phenomenon that can explain its specificity towards gram-negative bacteria by contact formation between the two phospholipids interfaces in the periplasmatic space. In this work, the interaction of PxB and the nonantibiotic derivative polymyxin B nonapeptide (PxB-NP) with monolayers of Escherichia coli membrane lipids (ECL) has been studied by thermodynamic and structural methods. PxB inserts itself into ECL monolayers as a conformation that forms intermembrane contacts with vesicles injected underneath, and induces lipid exchange when the monolayer surface pressure is set at 32 mN/m (membrane equivalence pressure) or net transfer vesicle-to-monolayer at lower surface pressures. Thermodynamic analysis of the compression isotherms of mixed monolayers indicates that PxB inserts into the monolayer with an expansion of the mean molecular area, implying that peptide and lipids form nonideal mixtures. At low concentrations, corresponding to the membrane-membrane contact form of PxB, the mixed monolayers present positive excess energy values (deltaGm(Ex)), and atomic force microscopy (AFM) imaging reveals structures of approximately 120-nm diameter that protrude from the lipid surface approximately 0.7 nm. At concentrations of PxB above 4 mol %, thermodynamic analysis gives a very high deltaGm(Ex), corresponding to nonfavorable interactions, and AFM images show round structures of 20-30 nm diameter. PxB-NP behaves in a totally different way, in agreement with its inability to form vesicle-vesicle contacts and its lack of antibiotic effect. These results are discussed in the light of the mechanism of action of PxB on the membrane of gram-negative bacteria.     

High-throughput comparison of gene fitness among related bacteria

ABSTRACT: BACKGROUND: The contribution of a gene to the fitness of a bacterium can be assayed by whether and to what degree the bacterium tolerates transposon insertions in that gene. We use this fact to compare the fitness of syntenic homologous genes among related Salmonella strains to reveal differences not apparent at the gene sequence level. RESULTS: A transposon Tn5 derivative was used to construct mutants in Salmonella Typhimurium ATCC14028 (STM1) and Salmonella Typhi Ty2 (STY1), which were then grown in rich media. The locations of 234,152 and 53,556 integration sites, respectively, were mapped by sequencing. These data were compared to similar data available for a different Ty2 strain (STY2) and essential genes identified in E. coli K-12 (ECO). Of 277 genes considered essential in ECO, all had syntenic homologs in STM1, STY1, and STY2, and all but nine genes were either devoid of Tn insertions or had very few. For three of these nine genes, part of the annotated gene lacked Tn integrations (yejM, ftsN and murB). At least one of the other six genes, trpS, had a potentially functionally redundant gene encoded elsewhere in Salmonella but not in ECO. An additional 165 genes were almost entirely devoid of transposon integrations in all three Salmonella strains examined, including many genes associated with protein and DNA synthesis. Four of these genes (STM14_1498.L, STM14_2872, STM14_3360.RJ, and STM14_5442) are not found in E. coli. Notable differences in the extent of gene selection were also observed among the three different Salmonella isolates. Mutations in hns, for example, were selected against in STM1 but not in the two STY strains, which have a defect in rpoS rendering hns nonessential. CONCLUSIONS: Comparisons among transposon integration profiles from different members of a species and among related species, all grown in similar conditions, identify differences in gene fitness among syntenic homologous genes. Further differences in fitness profiles among shared genes can be expected in other selective environments, with potential relevance for comparative systems biology.     

Nitrite oxidation in the Namibian oxygen minimum zone

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in ¹⁵N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (⩽372 nM NO₂⁻ d⁻¹) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO₃⁻ was re-oxidized back to NO₃⁻ via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.     

Bone marrow stroma confers resistance to Apo2 ligand/TRAIL in multiple myeloma in part by regulating c-FLIP

Apo2 ligand (Apo2L)/TRAIL induces apoptosis of cancer cells that express the specific receptors while sparing normal cells. Because the tumor microenvironment protects myeloma from chemotherapy, we investigated whether hemopoietic stroma induces resistance to Apo2L/TRAIL apoptosis in this disease. Apo2L/TRAIL-induced death was diminished in myeloma cell lines (RPMI 8226, U266, and MM1s) directly adhered to a human immortalized HS5 stroma cell line but not adhered to fibronectin. In a Transwell assay, with myeloma in the upper well and HS5 cells in the lower well, Apo2L/TRAIL apoptosis was reduced when compared with cells exposed to medium in the lower well. Using HS5 and myeloma patients' stroma-conditioned medium, we determined that soluble factor(s) produced by stroma-myeloma interactions are responsible for a reversible Apo2/TRAIL apoptosis resistance. Soluble factor(s) attenuated procaspase-8, procaspase-3, and poly(ADP-ribose) polymerase cleavage and diminished mitochondrial membrane potential changes without affecting Bcl-2 family proteins and/or Apo2L/TRAIL receptors. Soluble factor(s) increased the baseline levels of the anti-apoptotic protein c-FLIP in all cell lines tested. Inhibition of c-FLIP by means of RNA interference increased Apo2/TRAIL sensitivity in RPMI 8226 cells. Unlike direct adhesion to fibronectin, soluble factor(s) have no impact on c-FLIP redistribution within cellular compartments. Cyclohexamide restored Apo2L/TRAIL sensitivity in association with down-regulation of c-FLIP, suggesting that c-FLIP synthesis, not intracellular traffic, is essential for soluble factor(s) to regulate c-FLIP. Additionally, IL-6 conferred resistance to Apo2L/TRAIL-mediated apoptosis in association with increased c-FLIP levels. In conclusion, the immune cytotoxic effect of Apo2L/TRAIL can be restored at least in part by c-FLIP pathway inhibitors.     

Spatial and temporal segregation of auditory and vestibular neurons in the otic placode

The otic placode generates the auditory and vestibular sense organs and their afferent neurons; however, how auditory and vestibular fates are specified is unknown. We have generated a fate map of the otic placode and show that precursors for vestibular and auditory cells are regionally segregated in the otic epithelium. The anterior-lateral portion of the otic placode generates vestibular neurons, whereas the posterior-medial region gives rise to auditory neurons. Precursors for vestibular and auditory sense organs show the same distribution. Thus, different regions of the otic placode correspond to particular sense organs and their innervating neurons. Neurons from contiguous domains rarely intermingle suggesting that the regional organisation of the otic placode dictates positional cues to otic neurons. But, in addition, vestibular and cochlear neurogenesis also follows a stereotyped temporal pattern. Precursors from the anterior-lateral otic placode delaminate earlier than those from its medial-posterior portion. The expression of the proneural genes NeuroM and NeuroD reflects the sequence of neuroblast formation and differentiation. Both genes are transiently expressed in vestibular and then in cochlear neuroblasts, while differentiated neurons express Islet1, Tuj1 and TrkC, but not NeuroM or NeuroD. Together, our results indicate that the position of precursors within the otic placode confers identity to sensory organs and to the corresponding otic neurons. In addition, positional information is integrated with temporal cues that coordinate neurogenesis and sensory differentiation.