CFTR Protects against Mycobacterium abscessus Infection by Fine-Tuning Host Oxidative Defenses

1. Download high-res image (274KB)
2. Download full-size image

     

Immunolocalization of Non-Symbiotic Hemoglobins During Somatic Embryogenesis in Chicory

Hemoglobins are ancient O₂-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum × C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.Key Words: chicory, immunolocalization, nonsymbiotic hemoglobin, somatic embryogenesis     

Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one-pot/two-step biocatalytic whole-cell system

The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer–Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L ⁻¹·h ⁻¹) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L ⁻¹·h ⁻¹).     

G protein-dependent CCR5 signaling is not required for efficient infection of primary T lymphocytes and macrophages by R5 human immunodeficiency virus type 1 isolates

The requirement of human immunodeficiency virus (HIV)-induced CCR5 activation for infection by R5 HIV type 1 (HIV-1) strains remains controversial. Ectopic CCR5 expression in CD4(+)-transformed cells or pharmacological inhibition of G(alpha)i proteins coupled to CCR5 left unsolved whether CCR5-dependent cell activation is necessary for the HIV life cycle. In this study, we investigated the role played by HIV-induced CCR5-dependent cell signaling during infection of primary CD4-expressing leukocytes. Using lentiviral vectors, we restored CCR5 expression in T lymphocytes and macrophages from individuals carrying the homozygous 32-bp deletion of the CCR5 gene (ccr5 Delta32/Delta32). Expression of wild-type (wt) CCR5 in ccr5 Delta32/Delta32 cells permitted infection by R5 HIV isolates. We assessed the capacity of a CCR5 derivative carrying a mutated DRY motif (CCR5-R126N) in the second intracellular loop to work as an HIV-1 coreceptor. The R126N mutation is known to disable G protein coupling and agonist-induced signal transduction through CCR5 and other G protein-coupled receptors. Despite its inability to promote either intracellular calcium mobilization or cell chemotaxis, the inactive CCR5-R126N mutant provided full coreceptor function to several R5 HIV-1 isolates in primary cells as efficiently as wt CCR5. We conclude that in a primary, CCR5-reconstituted CD4(+) cell environment, G protein signaling is dispensable for R5 HIV-1 isolates to actively infect primary CD4(+) T lymphocytes or macrophages.     

Role of the chemokine stromal cell-derived factor 1 in autoantibody production and nephritis in murine lupus

In normal mice, stromal cell-derived factor 1 (SDF-1/CXCL12) promotes the migration, proliferation, and survival of peritoneal B1a (PerB1a) lymphocytes. Because these cells express a self-reactive repertoire and are expanded in New Zealand Black/New Zealand White (NZB/W) mice, we tested their response to SDF-1 in such mice. PerB1a lymphocytes from NZB/W mice were exceedingly sensitive to SDF-1. This greater sensitivity was due to the NZB genetic background, it was not observed for other B lymphocyte subpopulations, and it was modulated by IL-10. SDF-1 was produced constitutively in the peritoneal cavity and in the spleen. It was also produced by podocytes in the glomeruli of NZB/W mice with nephritis. The administration of antagonists of either SDF-1 or IL-10 early in life prevented the development of autoantibodies, nephritis, and death in NZB/W mice. Initiation of anti-SDF-1 mAb treatment later in life, in mice with established nephritis, inhibited autoantibody production, abolished proteinuria and Ig deposition, and reversed morphological changes in the kidneys. This treatment also counteracted B1a lymphocyte expansion and T lymphocyte activation. Therefore, PerB1a lymphocytes are abnormally sensitive to the combined action of SDF-1 and IL-10 in NZB/W mice, and SDF-1 is key in the development of autoimmunity in this murine model of lupus.     

Conjugated linoleic acid isomers in mitochondria: evidence for an alteration of fatty acid oxidation

The beneficial effects exerted by low amounts of conjugated linoleic acids (CLA) suggest that CLA are maximally conserved and raise the question about their mitochondrial oxidizability. Cis-9,trans-11-C(18:2) (CLA1) and trans-10,cis-12-C(18:2) (CLA2) were compared to cis-9,cis-12-C(18:2) (linoleic acid; LA) and cis-9-C(16:1) (palmitoleic acid; PA), as substrates for total fatty acid (FA) oxidation and for the enzymatic steps required for the entry of FA into rat liver mitochondria. Oxygen consumption rate was lowest when CLA1 was used as a substrate with that on CLA2 being intermediate between it and the respiration on LA and PA. The order of the radiolabeled FA oxidation rate was PA > LA > CLA2 > CLA1. Transesterification to acylcarnitines of the octadecadienoic acids were similar, while uptake across inner membranes of CLA1 and, to a lesser extent, of CLA2 was greater than that of LA or PA. Prior oxidation of CLA1 or CLA2 made re-isolated mitochondria much less capable of oxidising PA or LA under carnitine-dependent conditions, but without altering the carnitine-independent oxidation of octanoic acid. Therefore, the CLA studied appeared to be both poorly oxidizable and capable of interfering with the oxidation of usual FA at a step close to the beginning of the beta-oxidative cycle.     

Interactions between heme d and heme b595 in quinol oxidase bd from Escherichia coli: a photoselection study using femtosecond spectroscopy

Femtosecond spectroscopy was performed on CO-liganded (fully reduced and mixed-valence states) and O(2)-liganded quinol oxidase bd from Escherichia coli. Substantial polarization effects, unprecedented for optical studies of heme proteins, were observed in the CO photodissociation spectra, implying interactions between heme d (the chlorin ligand binding site) and the close-lying heme b(595) on the picosecond time scale; this general result is fully consistent with previous work [Vos, M. H., Borisov, V. B., Liebl, U., Martin, J.-L., and Konstantinov, A. A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1554-1559]. Analysis of the data obtained under isotropic and anisotropic polarization conditions and additional flash photolysis nanosecond experiments on a mutant of cytochrome bd mostly lacking heme b(595) allow to attribute the features in the well-known but unusual CO dissociation spectrum of cytochrome bd to individual heme d and heme b(595) transitions. This renders it possible to compare the spectra of CO dissociation from reduced and mixed-valence cytochrome bd under static conditions and on a picosecond time scale in much more detail than previously possible. CO binding/dissociation from heme d is shown to perturb ferrous heme b(595), causing induction/loss of an absorption band centered at 435 nm. In addition, the CO photodissociation-induced absorption changes at 50 ps reveal a bathochromic shift of ferrous heme b(595) relative to the static spectrum. No evidence for transient binding of CO to heme b(595) after dissociation from heme d is found in the picosecond time range. The yield of CO photodissociation from heme d on a time scale of < 15 ps is found to be diminished more than 3-fold when heme b(595) is oxidized rather than reduced. In contrast to other known heme proteins, molecular oxygen cannot be photodissociated from the mixed-valence cytochrome bd at all, indicating a unique structural and electronic configuration of the diheme active site in the enzyme.     

Bacterial mesosomes: Method dependent artifacts

The occurrence of mesosomes was investigated during septum formation of vegetative and sporulating cells of Bacillus cereus. It has been demonstrated that bacterial mesosomes which are considered by numerous microbiologists as an integrated constituent of Gram positive bacteria, are in reality artifacts arising during the preparation for electron microscopy. The conventional fixation methods allowed enough time for the cytoplasmic membrane to react to the changed conditions and to form the typical pocket-like membrane invaginations. With cryofixation followed by freeze-substitution it was shown in ultrathin sections that mesosomes do not occur. The extremely rapid freezing and the substitution of the ice by an organic solvent containing the fixative prevented the formation of membraneous artifacts.Non-standard abbreviations OsO₄ osmium tetroxide - UO₂Ac uranylacetate - PHB poly--hydroxy-butyric acid - M mesosome - CW cell wall - CM cytoplasmic membrane - PF plasmatic fracture of the cytoplasmic membrane     

Structure de la paroi de Aremoricanium rigaudae Deunff, 1955 (Acritarche,Ordovicien)

The wall of Aremoricanium rigaudaeDeunff, 1955, type-species by original designation of the genus AremoricaniumDeunff, 1955, is composed of two layers: the internal one has a circular opening while the second is the only one which develops the processes and a tubular expansion.