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The effect of breed [Creole (CR) vs. Large White (LW)] on performance and physiological responses during acclimation to high ambient temperature was studied in 2 experiments involving 24 (12/ breed) growing pigs each. Pigs were exposed to 24 [degrees]C for 10 d (d -10 to -1) and thereafter to a constant temperature of 31 [degrees]C for 16 d (d 1 to d 16) in Exp. 1 and for 20 d (d 1 to d 20) in Exp. 2. For both experiments, the temperature change was achieved over 4 h on d 0. The first experiment began at 105 d of age, and the average BW of CR and LW pigs was 36.6 [+ or -] 2.5 kg and 51.7 [+ or -] 3.0 kg, respectively. The second experiment was designed to compare both breeds at a similar BW (about 52 kg on d 0). Pigs were individually housed and given ad libitum access to feed. At 24 [degrees]C, ADG was lower (P < 0.01) in CR than in LW (602 vs. 913 g/d and 605 vs. 862 g/d in Exp. 1 and 2, respectively), but the ADFI was not affected by breed (190 and 221 g*[d.sup.-1] * [kg.sup.-0.60] in Exp. 1 and 2, respectively). Short-term thermoregulatory responses during the 4-h transition from 24 to 31 [degrees]C (d 0) were analyzed according to a linear plateau model to determine the break point temperature, above which rectal temperature (RT), cutaneous temperature (CT), and respiratory rate (RR) began to change. The CT increased linearly with temperature increase (0.22 [degrees]C/ [degrees]C) and was less (P < 0.05) in CR than in LW (by -0.3 [degrees]C on average). In both experiments, the break point temperature for RT was not affected by breed (27.6 [degrees]C on average), whereas for RR it was greater (P < 0.05) in CR than in LW (27.5 vs. 25.5 [degrees]C, P < 0.01). On average, ADFI declined by about 50 g*[d.sup.-1] * [kg.sup.-0.60] from d -1 to d 1 (P < 0.01), and thereafter at 31 [degrees]C, it gradually increased (23 g * [d.sup.-1] * [kg.sup.-0.60]; P < 0.05), suggesting an acclimation to high exposure. This response was not influenced by breed. After the day that marked the beginning of the acclimation response (i.e., the threshold day), RR, CT, and RT declined over the duration of exposure to 31 [degrees]C (P < 0.05) in both experiments. During this period, RT and CT were less in CR than in LW pigs (39.6 vs. 39.9 [degrees]C and 37.9 vs. 38.2 [degrees]C, respectively; P < 0.05), whereas RR was not affected by breed. The threshold day at which RT began to decline was less in CR than in LW pigs (0.18 vs. 1.17 d and 0.39 vs. 0.93 d in Exp. 1 and 2, respectively; P < 0.05). In conclusion, this study suggests that short- and long-term physiological reactions during heat acclimation differed when CR and LW pigs were compared at the same age or BW. Key words: acclimation, breed, heat stress, pig, thermoregulation

Large deletions in the first intron of the With No lysine (K) 1 (WNK1) gene are responsible for Familial Hyperkalemic Hypertension (FHHt), a rare form of human hypertension associated with hyperkalemia and hyperchloremic metabolic acidosis. We generated a mouse model of WNK1-associated FHHt to explore the consequences of this intronic deletion. WNK1(+/FHHt) mice display all clinical and biological signs of FHHt. This phenotype results from increased expression of long WNK1 (L-WNK1), the ubiquitous kinase isoform of WNK1, in the distal convoluted tubule, which in turn, stimulates the activity of the Na-Cl cotransporter. We also show that the activity of the epithelial sodium channel is not altered in FHHt mice, suggesting that other mechanisms are responsible for the hyperkalemia and acidosis in this model. Finally, we observe a decreased expression of the renal outer medullary potassium channel in the late distal convoluted tubule of WNK1(+/FHHt) mice, which could contribute to the hyperkalemia. In summary, our study provides insights into the in vivo mechanisms underlying the pathogenesis of WNK1-mediated FHHt and further corroborates the importance of WNK1 in ion homeostasis and blood pressure.

Mycolic acids, very long-chain α-alkyl, β-hydroxylated fatty acids, occur in the members of the order Corynebacteriales where their chain lengths (C(26)-C(88)) and structural features (oxygen functions, cis or trans double bonds, cyclopropane rings and methyl branches) are genus- and species-specific. The molecular composition and structures of the mycolic acids of two species belonging to the genus Segniliparus were determined by a combination of modern analytical chemical techniques, which include MS and NMR. They consist of mono-ethylenic C(62-)C(64) (α'), di-ethylenic C(77)-C(79) (α) and extremely long-chain mycolic acids (α(+)) ranging from 92 to 98 carbon atoms and containing three unsaturations, cis and/or trans double bonds and/or cyclopropanes. The double bonds in each class of mycolic acids were positioned by oxidative cleavage and exhibit locations similar to those of α- and α'-mycolic acids of mycobacteria. For the ultralong chain α-mycolic acids, the three double bonds were located at equally spaced carbon intervals (C(13)-C(16)), with the methyl branches adjacent to the proximal and distal trans double bonds. Examination of the Segniliparus rotundus genome compared with those of other members of the Corynebacteriales indicated two obvious differences in genes encoding the elongation fatty acid (FAS-II) enzymes involved in the biosynthesis of mycolic acids: the organization of 3-ketoacyl-ACP synthases (KasA and KasB) and (3R)-hydroxyacyl-ACP dehydratases (HadAB/BC), on one hand, and the presence of two copies of the hadB gene encoding the catalytic domain of the latter enzyme type, on the other. This observation is discussed in light of the most recent data accumulated on the biosynthesis of this hallmark of Corynebacteriales.

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The cell wall of these bacteria is composed of a heteropolymer of peptidoglycan (PG) linked to arabinogalactan (AG), which in turn is covalently associated with an atypical outer membrane, here called mycomembrane (M). The latter structure has been visualized by cryo-electron microscopy of vitreous sections, but its biochemical composition is still poorly defined, thereby hampering the elucidation of its physiological function. In this report, we show for the first time that the mycomembrane-linked heteropolymer of PG and AG (M-AG-PG) of C. glutamicum can be physically separated from the inner membrane on a flotation density gradient. Analysis of purified M-AG-PG showed that the lipids that composed the mycomembrane consisted almost exclusively of mycolic acid derivatives, with only a tiny amount, if any, of phospholipids and lipomannans, which were found with the characteristic lipoarabinomannans in the plasma membrane. Proteins associated with or inserted in the mycomembrane were extracted from M-AG-PG with lauryl-dimethylamine-oxide (LDAO), loaded on an SDS-PAGE gel, and analyzed by tandem mass spectrometry or by Western blotting. Sixty-eight different proteins were identified, 19 of which were also found in mycomembrane fragments released by the terminal-arabinosyl-transferase-defective ΔAftB strain. Almost all of them are predicted to contain a signal sequence and to adopt the characteristic β-barrel structure of Gram-negative outer membrane proteins. These presumed mycomembrane proteins include the already-known pore-forming proteins (PorA and PorB), 5 mycoloyltransferases (cMytA, cMytB, cMytC, cMytD, and cMytF), several lipoproteins, and unknown proteins typified by a putative C-terminal hydrophobic anchor.

The mechanisms permitting nonpolymorphic CD1 molecules to present lipid antigens that differ considerably in polar head and aliphatic tails remain elusive. It is also unclear why hydrophobic motifs in the aliphatic tails of some antigens, which presumably embed inside CD1 pockets, contribute to determinants for T-cell recognition. The 1.9-Å crystal structure of an active complex of CD1b and a mycobacterial diacylsulfoglycolipid presented here provides some clues. Upon antigen binding, endogenous spacers of CD1b, which consist of a mixture of diradylglycerols, moved considerably within the lipid-binding groove. Spacer displacement was accompanied by F' pocket closure and an extensive rearrangement of residues exposed to T-cell receptors. Such structural reorganization resulted in reduction of the A' pocket capacity and led to incomplete embedding of the methyl-ramified portion of the phthioceranoyl chain of the antigen, explaining why such hydrophobic motifs are critical for T-cell receptor recognition. Mutagenesis experiments supported the functional importance of the observed structural alterations for T-cell stimulation. Overall, our data delineate a complex molecular mechanism combining spacer repositioning and ligand-induced conformational changes that, together with pocket intricacy, endows CD1b with the required molecular plasticity to present a broad range of structurally diverse antigens.

O-acylation of proteins was known only in a few eukaryotic proteins but never in bacteria. We demonstrate, using a combination of protein chemistry and mass spectrometry, the occurrence of three O-acylated polypeptides in Corynebacterium glutamicum, PorA, PorH, and an unknown small protein. The three polypeptides are O-substituted by mycolic acids, long chain alpha-alkyl and beta-hydroxy fatty acids specifically produced by members of the Corynebacterineae suborder. To date these acids were described only as esterifying trehalose and arabinogalactan, and less frequently glycerol, important components of the highly impermeable outer barrier of Corynebacterineae. We show that the post-translational mycoloylation of PorA occurs at Ser-15 and is necessary for the pore-forming activity of C. glutamicum.

Corynebacterineae are gram-positive bacteria that possess a true outer membrane composed of mycolic acids and other lipids. Little is known concerning the modulation of mycolic acid composition and content in response to changes in the bacterial environment, especially temperature variations. To address this question, we investigated the function of the Rv3802c gene, a gene conserved in Corynebacterineae and located within a gene cluster involved in mycolic acid biosynthesis. We showed that the Rv3802 ortholog is essential in Mycobacterium smegmatis, while its Corynebacterium glutamicum ortholog, NCgl2775, is not. We provided evidence that the NCgl2775 gene is transcriptionally induced under heat stress conditions, and while the corresponding protein has no detectable activity under normal growth conditions, the increase in its expression triggers an increase in mycolic acid biosynthesis concomitant with a decrease in phospholipid content. We demonstrated that these lipid modifications are part of a larger outer membrane remodeling that occurs in response to exposure to a moderately elevated temperature (42 degrees C). In addition to showing an increase in the ratio of saturated corynomycolates to unsaturated corynomycolates, our results strongly suggested that the balance between mycolic acids and phospholipids is modified inside the outer membrane following a heat challenge. Furthermore, we showed that these lipid modifications help the bacteria to protect against heat damage. The NCgl2775 protein and its orthologs thus appear to be a protein family that plays a role in the regulation of the outer membrane lipid composition of Corynebacterineae under stress conditions. We therefore propose to name this protein family the envelope lipids regulation factor (ElrF) family.

The major cell wall carbohydrate of Corynebacterineae is arabinogalactan (AG), a branched polysaccharide that is essential for the physiology of these bacteria. Decaprenylphosphoryl-D-arabinose (DPA), the lipid donor of D-arabinofuranosyl residues of AG, is synthesized through a series of unique biosynthetic steps, the last one being the epimerization of decaprenylphosphoryl-beta-D-ribose (DPR) into DPA, which is believed to proceed via a sequential oxidation-reduction mechanism. Two proteins from Mycobacterium tuberculosis (Rv3790 and Rv3791) have been shown to catalyse this epimerization in an in vitro system. The present study addressed the exact function of these proteins through the inactivation of the corresponding orthologues in Corynebacterium glutamicum (NCgl0187 and NCgl0186, respectively) and the analysis of their in vivo effects on AG biosynthesis. We showed that NCgl0187 is essential, whereas NCgl0186 is not. Deletion of NCgl0186 led to a mutant possessing an AG that contained half the arabinose and rhamnose, and less corynomycolates linked to AG but more trehalose mycolates, compared with the parental strain. A candidate gene that may encode a protein functionally similar to NCgl0186 was identified in both C. glutamicum (NCgl1429) and M. tuberculosis (Rv2073c). While the deletion of NCgl1429 had no effect on AG biosynthesis of the mutant, the gene could complement the mycolate defect of the AG of the NCgl0186 mutant, strongly supporting the concept that the two proteins play a similar function in vivo. Consistent with this, the NCgl1429 gene appeared to be essential in the NCgl0186-inactivated mutant. A detailed bioinformatics analysis showed that NCgl1429, NCgl0186, Rv3791 and Rv2073c could constitute, with 52 other proteins belonging to the actinomycetales, a group of closely related short-chain reductases/dehydrogenases (SDRs) with atypical motifs. We propose that the epimerization of DPR to DPA involves three enzymes that catalyse two distinct steps, each being essential for the viability of the bacterial cells.

The absorbed fraction phi to the cell nucleus for low energy electrons (0.5 keV-50 keV) was evaluated. Distributions of radioactivity within either the nucleus (nu) and the cytoplasm (cy), or on the cell membrane (mem) were considered. phi was computed as a function of the cell sizes and of the electron energy E. For a strictly intranuclear distribution, phi(nu) is close to 1 for very low energy values (E < 4 keV), i.e. the energy is totally absorbed in the cell nucleus itself. The absorbed fraction decreases when the energy increases and phi(nu) becomes less than 0.1 for E > or = 40 keV. For a cell membrane distribution, the absorbed fraction remains always less than 0.2. For very low energy electrons (E < 6 keV), phi(mem) = 0, due to the fact that the electron falls short to the nucleus target. The absorbed fraction is maximum for E ranging from 12 keV to 20 keV. For higher values of E, phi(mem) decreases when E increases. When considering a cytoplasmic distribution, the maximum absorbed fraction phi(cy) is obtained for E values ranging from 10 keV to 25 keV (phi(cy max) = 0.27). Dosimetric computations at the cellular level show that the absorbed fraction to the cell nucleus may have values ranging from 0 to 1, depending on the dimensions of the cell, the energy of the emitted electron and on the intracellular localization of the Auger emitter.