Christian Richard

Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide

Mycobacterium tuberculosis, the causative agent of human tuberculosis, and Mycobacteriumbovis each express two genes, glbN and glbO, encoding distantly related truncated hemoglobins (trHbs), trHbN and trHbO, respectively. Here we report that disruption of M. bovis bacillus Calmette–Guérin glbN caused a dramatic reduction in the NO-consuming activity of stationary phase cells, and that activity could be restored fully by complementing knockout cells with glbN. Aerobic respiration of knockout cells was inhibited markedly by NO in comparison to that of wild-type cells, indicating a protective function for trHbN. TyrB10, which is highly conserved in trHbs and interacts with the bound oxygen, was found essential for NO consumption. Titration of oxygenated trHbN (trHbN⋅O2) with NO resulted in stoichiometric oxidation of the protein with nitrate as the major product of the reaction. The second-order rate constant for the reaction between trHbN⋅O2 and NO at 23°C was 745 μM−1⋅s−1, demonstrating that trHbN detoxifies NO 20-fold more rapidly than myoglobin. These results establish a role for a trHb and demonstrate an NO-metabolizing activity in M. tuberculosis or M. bovis. trHbN thus might play an important role in persistence of mycobacterial infection by virtue of trHbN′s ability to detoxify NO.

Regional differences in adipose tissue metabolism in women : Minor effect of obesity and body fat distribution

Studies comparing adipose tissue metabolism in central versus peripheral fat depots have generated equivocal data. We examined whether regional differences in abdominal subcutaneous and omental adipose tissue metabolism in women exist and whether they persist across the spectrum of body fatness and abdominal adiposity values. We measured adipocyte size; lipoprotein lipase (LPL) activity; and basal, isoproterenol-, forskolin-, and dibutyryl cAMP–stimulated lipolysis in adipose tissue or mature adipocytes isolated from the omental and subcutaneous fat depots in a sample of 55 healthy women undergoing elective gynecological surgery. Measures of body fat mass and body fat distribution were also obtained by dual-energy X-ray absorptiometry and computed tomography. Subcutaneous adipocytes were significantly larger than omental adipocytes (P < 0.0001). LPL activity expressed as a function of cell number was significantly higher in subcutaneous versus omental adipose tissue (P < 0.0001). Basal, isoproterenol-stimulated, dibutyryl cAMP–stimulated (10−3 mol/l) and forskolin-stimulated (10−5 mol/l) lipolysis (expressed as a function of cell number) were all significantly higher in subcutaneous versus omental adipocytes (P < 0.05 to P < 0.0001). However, the response of omental adipocytes to lipolytic stimuli tested (fold increase over basal level) was significantly greater in magnitude compared with subcutaneous adipocytes (P < 0.01). These differences were relatively constant across total body fat mass and visceral adipose tissue area tertiles. In conclusion, compared with adipocytes from the omental fat compartment, subcutaneous adipocytes are larger, have higher LPL activity, and are more lipolytic on an absolute basis, which may reflect a higher fat storage capacity in this depot in women. In contrast, omental adipocytes display greater relative responsiveness to both adrenergic receptor–and postreceptor-acting agents compared with subcutaneous adipocytes. Overall and visceral obesity have only minor effects on regional differences in adipose tissue metabolism.

The Chlamydomonas reinhardtii LI818 gene represents a distant relative of the cabI/II genes that is regulated during the cell cycle and in response to illumination

In the green unicellular alga Chlamydomonas reinhardtii, as in higher plants, the expression of the genes encoding the chlorophyll a/b-binding (CAB) polypeptides associated with photosystem I (PSI) and photosystem II (PSII) is regulated by endogenous (circadian clock) and exogenous signals (light and temperature). The circadian clock ensures that the oscillation in the levels of the different cab mRNAs is continuously kept in phase with light/dark (LD) cycles and is maximal by the middle of the day. On the other hand, light controls the amplitude of the oscillations. We report here the cloning and characterization of the C. reinhardtii LI818 gene, which identifies a CAB-related polypeptide and whose expression is regulated quite differently from the cabI/II genes. We show: (1) that in LD synchronized Chlamydomonas cells LI818 mRNA accumulation is subject to dual regulation that involves separable regulation by light and an endogenous oscillator; (2) that LI818 mRNA is fully expressed several hours before the cab I/II mRNAs and that the latter accumulate concomitantly; (3) that blocking the electron flow through PSII using DCMU prevents cells from accumulating cab I/II mRNAs but not LI818 mRNA and (4) that the accumulation of LI818 mRNA is abolished by blocking cytoplasmic protein synthesis, suggesting that these regulatory mechanisms are mediated by labile proteins.

Characterization of the LI818 polypeptide from the green unicellular alga Chlamydomonas reinhardtii.

The LI818 gene from Chlamydomonas encodes a polypeptide that is related to the chlorophyll a/b-binding proteins (CAB) of higher plants and green algae. However, despite this relatedness, LI818 gene expression is not coordinated with that of cab genes and is regulated differently by light, suggesting a different role for LI818 polypeptide. We show here that, in contrast to CAB polypeptides, LI818 polypeptide is not tightly embedded into the thylakoid membranes and is localized in stroma-exposed regions. Moreover, during chloroplast development, LI818 polypeptide accumulates before CAB polypeptides. We also show that the LI818 polypeptide forms with certain chlorophyll a/c-binding proteins (CAC) from the haptophyte Isochrysis galbana and the diatom Cyclotella cryptica a natural group that is distinct from those constituted by CAB, CAC and the chlorophyll a/a-binding proteins (CAA). Such an association suggests a very ancient origin for this group of polypeptides, which predates the division of the early photosynthetic eukaryotes into green (chlorophyte), red (rhodophyte) and brown (chromophyte) algae. Possible roles for the LI818 polypeptide are discussed.

Structural Determinants in the Group III Truncated Hemoglobin from Campylobacter jejuni

Truncated hemoglobins (trHbs) constitute a distinct lineage in the globin superfamily, distantly related in size and fold to myoglobin and monomeric hemoglobins. Their phylogenetic analyses revealed that three groups (I, II, and III) compose the trHb family. Group I and II trHbs adopt a simplified globin fold, essentially composed of a 2-on-2 α-helical sandwich, wrapped around the heme group. So far no structural data have been reported for group III trHbs. Here we report the three-dimensional structure of the group III trHbP from the eubacterium Campylobacter jejuni. The 2.15-Å resolution crystal structure of C. jejuni trHbP (cyano-met form) shows that the 2-on-2 trHb fold is substantially conserved in the trHb group III, despite the absence of the Gly-based sequence motifs that were considered necessary for the attainment of the trHb specific fold. The heme crevice presents important structural modifications in the C-E region and in the FG helical hinge, with novel surface clefts at the proximal heme site. Contrary to what has been observed for group I and II trHbs, no protein matrix tunnel/cavity system is evident in C. jejuni trHbP. A gating movement of His(E7) side chain (found in two alternate conformations in the crystal structure) may be instrumental for ligand entry to the heme distal site. Sequence conservation allows extrapolating part of the structural results here reported to the whole trHb group III.

Effect of the Mediterranean diet with and without weight loss on cardiovascular risk factors in men with the metabolic syndrome

BACKGROUND AND AIMS No study has yet examined how weight loss modifies the impact of the Mediterranean diet (MedDiet) on cardiovascular risk factors in men with the metabolic syndrome (MetS). The objective of the study was to assess the efficacy of MedDiet, with and without weight loss, to modify the cardiometabolic risk profile of male patients with MetS. METHODS AND RESULTS Twenty-six men aged between 24 and 62 years with the MetS consumed a North American control diet for 5 weeks followed by a 5-week MedDiet, both under weight-maintaining conditions. Participants then underwent a 20-week weight loss period, after which they consumed the MedDiet for five weeks under weight stable conditions. Body weight was reduced by 10.2% ± 2.9% after the weight loss period (p < 0.001). All foods were provided to participants during the weight stable phases of the study. The MedDiet in the absence of weight loss decreased total plasma cholesterol (C) (-7.1%), LDL-C (-9.3%) and the total/HDL-C ratio (-6.5%) compared to the control diet (all p < 0.04). The MedDiet combined with weight loss led to reductions in systolic blood pressure (-4.7%), diastolic blood pressure (-7.7%), triglycerides (-18.2%), ApoB (-10.7%), fasting glucose (-4.2%) and insulin (-29.9%) compared to the control diet (all p < 0.001). CONCLUSION The MedDiet in the absence of weight loss leads to significant changes in plasma cholesterol concentrations but has little effects on other cardiometabolic risk factors associated with the MetS in men.

Impact of buttermilk consumption on plasma lipids and surrogate markers of cholesterol homeostasis in men and women

BACKGROUND AND AIMS Sphingolipids (SL) are important components of the milk fat globule membrane (MFGM) found in buttermilk. While studies in animal models suggest that dietary SL may have cholesterol-lowering properties, data in human are lacking. The aim of this study was to investigate the impact of buttermilk consumption on plasma lipids and surrogate markers of cholesterol (C) homeostasis in humans. METHODS AND RESULTS Men and women (n = 34) with serum LDL-C <5.0 mmol/L at screening (mean LDL-C = 3.8 mmol/L) were recruited in this double-blinded randomized crossover placebo controlled study. Their diets were supplemented with 45 g/d of buttermilk and with 45 g/d of a macro/micronutrient matched placebo (4 weeks each in random order). Serum lipid concentrations and surrogate markers of cholesterol homeostasis were measured post diet and compared using mixed models for repeated measures. Consumption of buttermilk led to reduction in serum cholesterol (-3.1%, P = 0.019), LDL-C (-3.1%, P = 0.057) and triacylglycerol (-10.7%, P = 0.007). Buttermilk consumption increased plasma lathosterol concentrations (+12.1%, P = 0.001), but multiple regression analysis indicated that variations in β-sitosterol concentrations (P = 0.002) were the only significant predictor of the LDL-C response to buttermilk consumption. CONCLUSION Buttermilk consumption may be associated with reduced cholesterol concentrations in men and women, primarily through inhibition of intestinal absorption of cholesterol. REGISTRATION NUMBER This trial is registered at clinicaltrials.gov as NCT01248026.

Structural characterization of a group II 2/2 hemoglobin from the plant pathogen Agrobacterium tumefaciens.

Within the 2/2 hemoglobin sub-family, no group II 2/2Hbs from proteobacteria have been so far studied. Here we present the first structural characterization of a group II 2/2Hb from the soil and phytopathogenic bacterium Agrobacterium tumefaciens (At-2/2HbO). The crystal structure of ferric At-2/2HbO (reported at 2.1Å resolution) shows the location of specific/unique heme distal site residues (e.g., His(42)CD1, a residue distinctive of proteobacteria group II 2/2Hbs) that surround a heme-liganded water molecule. A highly intertwined hydrogen-bonded network, involving residues Tyr(26)B10, His(42)CD1, Ser(49)E7, Trp(93)G8, and three distal site water molecules, stabilizes the heme-bound ligand. Such a structural organization suggests a path for diatomic ligand diffusion to/from the heme. Neither a similar distal site structuring effect nor the presence of distal site water molecules has been so far observed in group I and group III 2/2Hbs, thus adding new distinctive information to the complex picture of currently available 2/2Hb structural and functional data. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.