The aqueous accessibility in the external half of transmembrane domain I of the GABA transporter GAT-1 Is modulated by its ligands

The sodium- and chloride-dependent gamma-aminobutyric acid (GABA) transporter GAT-1 is the first identified member of a family of transporters, which maintain low synaptic neurotransmitter levels and thereby enable efficient synaptic transmission. To obtain evidence for the idea that the highly conserved transmembrane domain I (TMD I) participates in the permeation pathway, we have determined the impact of impermeant methanethiosulfonate (MTS) reagents on cysteine residues engineered into this domain. As a background the essentially insensitive but fully active C74A mutant has been used. Transport activity of mutants with a cysteine introduced cytoplasmic to glycine 63 is largely unaffected and is resistant to the impermeant MTS reagents. Conversely, transport activity in mutants extracellular to glycine 63 is strongly impacted. Nevertheless, transport activity could be measured in all but three mutants: G65C, N66C, and R69C. In each of the six active cysteine mutants the activity is highly sensitive to the impermeant MTS reagents. This sensitivity is potentiated by sodium in L64C, F70C, and Y72C, but is protected in V67C and P71C. GABA protects in L64C, W68C, F70C, and P71C. The non-transportable GABA analogue SKF100330A also protects in L64C, W68C, and P71C as well as V67C, but strikingly potentiates inhibition in F70C. Although cysteine substitution in this region may have perturbed the native structure of GAT-1, our observations, taken together with the recently published accessibility study on the related serotonin transporter (Henry, L. K., Adkins, E. M., Han, Q., and Blakely, R. D. (2003) J. Biol. Chem. 278, 37052-37063), suggest that the extracellular part of TMD I is conformationally sensitive, lines the permeation pathway, and forms a more extended structure than expected from a membrane-embedded alpha-helix.     

Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors

In the embryonic mouse retina, retinoic acid (RA) is unevenly distributed along the dorsoventral axis: RA-rich zones in dorsal and ventral retina are separated by a horizontal RA-poor stripe that contains the RA-inactivating enzyme CYP26A1. To explore the developmental role of this arrangement, we studied formation of the retina and its projections in Cyp26a1 null-mutant mice. Expression of several dorsoventral markers was not affected, indicating that CYP26A1 is not required for establishing the dorsoventral retina axis. Analysis of the mutation on a RA-reporter mouse background confirmed, as expected, that the RA-poor stripe was missing in the retina and its projections at the time when the optic axons first grow over the diencephalon. A day later, however, a gap appeared both in retina and retinofugal projections. As explanation, we found that CYP26C1, another RA-degrading enzyme, had emerged centrally in a narrower domain within the RA-poor stripe. While RA applications increased retinal Cyp26a1 expression, they slightly reduced Cyp26c1. These observations indicate that the two enzymes function independently. The safeguard of the RA-poor stripe by two distinct enzymes during later development points to a role in maturation of a significant functional feature like an area of higher visual acuity that develops at its location.     

Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (K _i) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride K _i by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn²⁺ ions, in particular the αS583C mutant showing a K _i for Zn²⁺of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride K _i, the later mutation generating a Ca²⁺blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na⁺ions at an external Na⁺binding site preventing ion permeation through the channel pore.     

Bovine hemoglobin: an attractive source of antibacterial peptides

A peptic hemoglobin hydrolysate was fractioned by a semi-preparative reversed-phase HPLC and some fractions have an antibacterial activity against four bacteria strains: Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis. These fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize the peptides in these fractions. Each fraction contains at least three peptides and some fractions contain five peptides. All these fractions were purified several times by HPLC to obtain pure peptides. Thirty antibacterial peptides were identified. From the isolated antibacterial peptides, 24 peptides were derived from the chains of hemoglobin and 6 peptides were derived from the β chains of hemoglobin. The lowest concentration of these peptides (minimum inhibitory concentration (MIC)) necessary to completely inhibit the growth of four bacteria strain was determined. The cell population of all of the tested bacteria species decreased by at least 97% after a 24-h incubation with any of the peptides at the minimum inhibitory concentration.     

Accumulation of metals in GOLD4 COPD lungs is associated with decreased CFTR levels

Background

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that primarily resides in airway epithelial cells. Decreased CFTR expression and/or function lead to impaired airway surface liquid (ASL) volume homeostasis, resulting in accumulation of mucus, reduced clearance of bacteria, and chronic infection and inflammation.

Methods

Expression of CFTR and the cigarette smoke metal content were assessed in lung samples of controls and COPD patients with established GOLD stage 4. CFTR protein and mRNA were quantified by immunohistochemistry and quantitative RT-PCR, respectively. Metals present in lung samples were quantified by ICP-AES. The effect of cigarette smoke on down-regulation of CFTR expression and function was assessed using primary human airway epithelial cells. The role of leading metal(s) found in lung samples of GOLD 4 COPD patients involved in the alteration of CFTR was confirmed by exposing human bronchial epithelial cells 16HBE14o- to metal-depleted cigarette smoke extracts.

Results

We found that CFTR expression is reduced in the lungs of GOLD 4 COPD patients, especially in bronchial epithelial cells. Assessment of metals present in lung samples revealed that cadmium and manganese were significantly higher in GOLD 4 COPD patients when compared to control smokers (GOLD 0). Primary human airway epithelial cells exposed to cigarette smoke resulted in decreased expression of CFTR protein and reduced airway surface liquid height. 16HBE14o-cells exposed to cigarette smoke also exhibited reduced levels of CFTR protein and mRNA. Removal and/or addition of metals to cigarette smoke extracts before exposure established their role in decrease of CFTR in airway epithelial cells.

Conclusions

CFTR expression is reduced in the lungs of patients with severe COPD. This effect is associated with the accumulation of cadmium and manganese suggesting a role for these metals in the pathogenesis of COPD.     

Cytokinin treated microcalli of Phelipanche ramosa: an efficient model for studying haustorium formation in holoparasitic plants

The ability of Orobanchaceae to establish a parasitic relationship is based on the development of a specific organ called haustorium. Previous studies in Phelipanche aegyptiaca and P. ramosa have underlined the interest of producing calli from germinated seeds as an efficient genetic transformation system, but they also pointed the need to improve the attachment rate of infectious calli to the host plant as well as the seed yield of the regenerated plants following attachment. It was previously shown that haustorium formation in P. ramosa is triggered by cytokinins. The present work demonstrates that one month-old microcalli produced from germinated seeds displayed a response to c/tZ similar to germinated seeds, as shown by the temporal expression profile of markers genes of cytokinin-related haustorium initiation, including PrRR5, PrCKX2, PrCKX4, PrTRN2 and PrZFP6. In addition, a 48 h treatment of microcalli with c/tZ (10^?7 M) before infestation triggered a 4-fold increase in the attachment rate of microcalli to tomato roots after 18 days in minirhizotrons when compared to untreated microcalli. Finally, it also outlines that increasing the aggressiveness of microcalli with a c/tZ treatment followed by the transfer of parasitized tomato plants into pots significantly improved seed yield of regenerated P. ramosa plants. Indeed, high amounts of viable seeds which germinated at more than 90% in response to GR24 were harvested after a 10 weeks-co-cultivation period. According to these results, cytokinin treated P. ramosa microcalli thus appear to be a good tool for further functional studies in holoparasitic plants, especially on haustorium formation.

     

Resin duct size and density as ecophysiological traits in fire scars of Pseudotsuga menziesii and Larix occidentalis

Background and Aims

Resin ducts (RDs) are features present in most conifer species as defence structures against pests and pathogens; however, little is known about RD expression in trees following fire injury. This study investigates changes in RD size and density in fire scars of Douglas fir (Pseudotsuga menziesii) and western larch (Larix occidentalis) as a means to evaluate the ecophysiological significance of traumatic resinosis for tree defence and survival.

Methods

Transverse and tangential microsections were prepared for light microscopy and image analysis in order to analyse axial and radial RDs, respectively. Epithelial cells of RDs and fusiform rays associated with radial RDs were also examined. RDs were compared between normal xylem and wound xylem at four different section heights along the fire-injured stem.

Key Results

Following fire injury, P. menziesii axial RDs narrowed by 38–43 % in the first year after injury, and the magnitude of this change increased with stem height. Larix occidentalis axial RDs widened by 46–50 % in the second year after injury. Radial RDs were of equivalent size in P. menziesii, but widened by 162–214 % in L. occidentalis. Fusiform rays were larger following fire injury, by 4–14 % in P. menziesii and by 23–38 % in L. occidentalis. Furthermore, axial RD density increased in both species due to the formation of tangential rows of traumatic RDs, especially in the first and second years after injury. However, radial RD density did not change significantly.

Conclusions

These results highlight traumatic resinosis as a species-specific response. Pseudotsuga menziesii produce RDs of equivalent or reduced size, whereas L. occidentalis produce wider RDs in both the axial and radial duct system, thereby increasing resin biosynthesis and accumulation within the whole tree. Larix occidentalis thus appears to allocate more energy to defence than P. menziesii.     

The genome of alpha-proteobacteria : complexity, reduction, diversity and fluidity

The alpha-proteobacteria displayed diverse and often unconventional life-styles. In particular, they keep close relationships with the eucaryotic cell. Their genomic organization is often atypical. Indeed, complex genomes, with two or more chromosomes that could be linear and sometimes associated with plasmids larger than one megabase, have been described. Moreover, polymorphism in genome size and topology as well as in replicon number was observed among very related bacteria, even in a same species. Alpha-proteobacteria provide a good model to study the reductive evolution, the role and origin of multiple chromosomes, and the genomic fluidity. The amount of new data harvested in the last decade should lead us to better understand emergence of bacterial life-styles and to build the conceptual basis to improve the definition of the bacterial species.