Myocardium proteome remodelling after nutritional deprivation of methyl donors

Methyl donor (MD: folate, vitamin B12 and choline) deficiency causes hyperhomocysteinemia, a risk factor for cardiovascular diseases. However, the mechanisms of the association between MD deficiency, hyperhomocysteinemia, and cardiomyopathy remain unclear. Therefore, we performed a proteomic analysis of myocardium of pups from rat dams fed a MD-depleted diet to understand the impact of MD deficiency on heart at the protein level. Two-dimension gel electrophoresis and mass spectrometry-based analyses allowed us to identify 39 proteins with significantly altered abundance in MD-deficient myocardium. Ingenuity Pathway Analysis showed that 87% of them fitted to a single protein network associated with developmental disorder, cellular compromise and lipid metabolism. Concurrently increased protein carbonylation, the major oxidative post-translational protein modification, could contribute to the decreased abundance of many myocardial proteins after MD deficiency. To decipher the effect of MD deficiency on the abundance of specific proteins identified in vivo, we developed an in vitro model using the cardiomyoblast cell line H9c2. After a 4-day exposure to a MD-deprived (vs. complete) medium, cells were deficient of folate and vitamin B12, and released abnormal amounts of homocysteine. Western blot analyses of pup myocardium and H9c2 cells yielded similar findings for several proteins. Of specific interest is the result showing increased and decreased abundances of prohibitin and α-crystallin B, respectively, which underlines mitochondrial injury and endoplasmic reticulum stress within MD deficiency. The in vitro findings validate the MD-deficient H9c2 cells as a relevant model for studying mechanisms of the early metabolic changes occurring in cardiac cells after MD deprivation.     

The cell surface receptor DC-SIGN discriminates between Mycobacterium species through selective recognition of the mannose caps on lipoarabinomannan

Interactions between dendritic cells (DCs) and Mycobacterium tuberculosis, the etiological agent of tuberculosis, most likely play a key role in anti-mycobacterial immunity. We have recently shown that M. tuberculosis binds to and infects DCs through ligation of the DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and that M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) inhibits binding of the bacilli to the lectin, suggesting that ManLAM might be a key DC-SIGN ligand. In the present study, we investigated the molecular basis of DC-SIGN ligation by LAM. Contrary to what was found for slow growing mycobacteria, such as M. tuberculosis and the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin, our data demonstrate that the fast growing saprophytic species Mycobacterium smegmatis hardly binds to DC-SIGN. Consistent with the former finding, we show that M. smegmatis-derived lipoarabinomannan, which is capped by phosphoinositide residues (PILAM), exhibits a limited ability to inhibit M. tuberculosis binding to DC-SIGN. Moreover, using enzymatically demannosylated and chemically deacylated ManLAM molecules, we demonstrate that both the acyl chains on the ManLAM mannosylphosphatidylinositol anchor and the mannooligosaccharide caps play a critical role in DC-SIGN-ManLAM interaction. Finally, we report that DC-SIGN binds poorly to the PILAM and uncapped AraLAM-containing species Mycobacterium fortuitum and Mycobacterium chelonae, respectively. Interestingly, smooth colony-forming Mycobacterium avium, in which ManLAM is capped with single mannose residues, was also poorly recognized by the lectin. Altogether, our results provide molecular insight into the mechanisms of mycobacteria-DC-SIGN interaction, and suggest that DC-SIGN may act as a pattern recognition receptor and discriminate between Mycobacterium species through selective recognition of the mannose caps on LAM molecules.     

Meclizine Inhibits Mitochondrial Respiration through Direct Targeting of Cytosolic Phosphoethanolamine Metabolism

We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.     

Fear-like behavioral responses in mice in different odorant environments: Trigeminal versus olfactory mediation under low doses

Odors can have repulsive effects on rodents based on two complementary adaptive behaviors: the avoidance of predator odors (potentially dangerous) and the avoidance of trigeminal stimulants (potentially noxious). The present study aimed to compare the behavioral effects on mice of odors according to their trigeminal properties and ecological significance. We used three different odors: 2,4,5-trimethylthiazoline (TMT: a fox feces odor frequently used to elicit fear-induced behaviors), toluene (a strong stimulant of the trigeminal system) and phenyl ethyl alcohol (PEA: a selective stimulant of the olfactory system). First, we checked preference and avoidance behaviors in mice with and without anosmia towards these odors to ensure their olfactory/trigeminal properties. Secondly, we used a standard test (open-field and elevated plus-maze) to assess the behaviors of mice when exposed to these odors. The results show that the anosmic and control mice both avoided TMT and toluene odors. In the open-field and the elevated plus-maze, mice exhibited "anxious" behaviors when exposed to TMT. Conversely, exposure to PEA induced "anxiolytic" effects confirmed by low blood corticosterone levels resulting from completion of the elevated plus-maze. Compared with TMT exposure, toluene exposure induced moderate "anxious" effects.     

γ-Lactam-Based Antifungal Compounds against the Wheat Pathogen Zymoseptoria tritici

As new environmentally friendly and effective antifungal agents are deeply needed, efficient ecofriendly strategies were designed to access two series of compounds inspired from natural γ-lactams. Designed compounds were fully characterized and evaluated as antifungal candidates against Zymoseptoria tritici, the main pathogen on wheat crops. The targeted derivatives were prepared from natural resources using green solvents, simple procedures, and limited purification steps. These bio-inspired compounds revealed as good candidates for further development of efficient crop protection products. Indeed, the HIT compounds exhibited IC₅₀ around 1 μg/mL and were more active than the references tebuconazole and bixafen towards some multidrug-resistant strains. Two dozen of derivatives have been obtained for each series and allowed to establish early structure-activity relationships useful for the development of next generation of γ-lactam derivatives with improved efficacy.     

QTLs and candidate genes for desiccation and abscisic acid content in maize kernels

Background

Kernel moisture at harvest is an important trait since a low value is required to prevent unexpected early germination and ensure seed preservation. It is also well known that early germination occurs in viviparous mutants, which are impaired in abscisic acid (ABA) biosynthesis. To provide some insight into the genetic determinism of kernel desiccation in maize, quantitative trait loci (QTLs) were detected for traits related to kernel moisture and ABA content in both embryo and endosperm during kernel desiccation. In parallel, the expression and mapping of genes involved in kernel desiccation and ABA biosynthesis, were examined to detect candidate genes.

Results

The use of an intermated recombinant inbred line population allowed for precise QTL mapping. For 29 traits examined in an unreplicated time course trial of days after pollination, a total of 78 QTLs were detected, 43 being related to kernel desiccation, 15 to kernel weight and 20 to ABA content. Multi QTL models explained 35 to 50% of the phenotypic variation for traits related to water status, indicating a large genetic control amenable to breeding. Ten of the 20 loci controlling ABA content colocated with previously detected QTLs controlling water status and ABA content in water stressed leaves. Mapping of candidate genes associated with kernel desiccation and ABA biosynthesis revealed several colocations between genes with putative functions and QTLs. Parallel investigation via RT-PCR experiments showed that the expression patterns of the ABA-responsive Rab17 and Rab28 genes as well as the late embryogenesis abundant Emb5 and aquaporin genes were related to desiccation rate and parental allele effect. Database searches led to the identification and mapping of two zeaxanthin epoxidase (ZEP) and five novel 9-cis-epoxycarotenoid dioxygenase (NCED) related genes, both gene families being involved in ABA biosynthesis. The expression of these genes appeared independent in the embryo and endosperm and not correlated with ABA content in either tissue.

Conclusions

A high resolution QTL map for kernel desiccation and ABA content in embryo and endosperm showed several precise colocations between desiccation and ABA traits. Five new members of the maize NCED gene family and another maize ZEP gene were identified and mapped. Among all the identified candidates, aquaporins and members of the Responsive to ABA gene family appeared better candidates than NCEDs and ZEPs.     

Progression through meiosis I and meiosis II in Arabidopsis anthers is regulated by an A-type cyclin predominately expressed in prophase I

Meiosis is often described as a special case of cell division since it differs from mitosis in having two nuclear divisions without an intervening S-phase. It will be of great interest to uncover what molecular mechanisms underlie these special features of meiosis. We previously reported that the tardy asynchronous meiosis (tam) mutant of Arabidopsis (Arabidopsis thaliana) is slower in cell cycle progression in male meiosis. Here we report that TAM encodes the A-type cyclin, CYCA1;2. The point mutation in tam replaced a conserved threonine with an isoleucine in the linker region between the alpha4 and alpha5 helices of the first cyclin fold. By studying the dynamics of a CYCA1;2-green fluorescent protein fusion protein under the control of the CYCA1;2 promoter, we found that the fusion protein was most abundant at pachytene, but was undetectable from late prophase I until telophase II. Nonetheless, cell cycle progression in tam was delayed in both pachytene and meiosis II. We conclude either that the CYCA1;2 produced in prophase I indirectly regulates meiosis II progression, or that a very low level of CYCA1;2 directly regulates meiosis II progression. Either of these scenarios is a deviation from the typical mode of action of mitotic cyclins in mitosis and meiosis I, in which each nuclear division is coupled with a peak of expression of mitotic cyclins.     

The uptake of apoptotic cells drives Coxiella burnetii replication and macrophage polarization: a model for Q fever endocarditis

Patients with valvulopathy have the highest risk to develop infective endocarditis (IE), although the relationship between valvulopathy and IE is not clearly understood. Q fever endocarditis, an IE due to Coxiella burnetii, is accompanied by immune impairment. Patients with valvulopathy exhibited increased levels of circulating apoptotic leukocytes, as determined by the measurement of active caspases and nucleosome determination. The binding of apoptotic cells to monocytes and macrophages, the hosts of C. burnetii, may be responsible for the immune impairment observed in Q fever endocarditis. Apoptotic lymphocytes (AL) increased C. burnetii replication in monocytes and monocyte-derived macrophages in a cell-contact dependent manner, as determined by quantitative PCR and immunofluorescence. AL binding induced a M2 program in monocytes and macrophages stimulated with C. burnetii as determined by a cDNA chip containing 440 arrayed sequences and functional tests, but this program was in part different in monocytes and macrophages. While monocytes that had bound AL released high levels of IL-10 and IL-6, low levels of TNF and increased CD14 expression, macrophages that had bound AL released high levels of TGF-beta1 and expressed mannose receptor. The neutralization of IL-10 and TGF-beta1 prevented the replication of C. burnetii due to the binding of AL, suggesting that they were critically involved in bacterial replication. In contrast, the binding of necrotic cells to monocytes and macrophages led to C. burnetii killing and typical M1 polarization. Finally, interferon-gamma corrected the immune deactivation induced by apoptotic cells: it prevented the replication of C. burnetii and re-directed monocytes and macrophages toward a M1 program, which was deleterious for C. burnetii. We suggest that leukocyte apoptosis associated with valvulopathy may be critical for the pathogenesis of Q fever endocarditis by deactivating immune cells and creating a favorable environment for bacterial persistence.     

Pentosan polysulfate,a potent anti HIV and anti tumor agent,inhibits protein serine/threonine and tyrosine kinases

The effect of nicotinamide-adenine dinucleotides (NAD⁺ and NADP⁺) on Ca²⁺ transport in rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺ uptake was dependent on adenosine triphosphate (ATP; 2mM). The presence of NAD⁺ (2mM) or NADP⁺ (1 and 2mM) caused a significant inhibition of Ca²⁺ uptake following addition of 2mM ATP. Ca²⁺, which accumulated in the nuclei during 6 min after ATP addition, was significantly released by the addition of NAD⁺ (0.5–2mM) or NADP⁺ (0.1–2mM). However, the effect of NADH (2mM) or NADPH (2mM) on Ca²⁺ uptake and release clearly weakened in comparison with the effects of NAD⁺ and NADP⁺. Meanwhile, ryanodine (10M), thapsigargin (10M) or oxalate (0.5mM) had no effect on Ca²⁺ uptake and release in rat liver nuclei. These reagents did not significantly alter the effects of 2mM NAD⁺ on Ca²⁺ uptake and release. Thus, NAD⁺ and NADP⁺ had a potent effect on Ca²⁺ transport in rat liver nuclei. The present findings suggest that the liver cytosolic NAD⁺ (NADP⁺) is a factor in the regulation of the nuclear Ca²⁺ concentration. (Mol Cell Biochem121: 127–133, 1993)