From NMR chemical shifts to amino acid types: Investigation of the predictive power carried by nuclei

An approach to automatic prediction of the amino acid type from NMR chemical shift values of its nuclei is presented here, in the frame of a model to calculate the probability of an amino acid type given the set of chemical shifts. The method relies on systematic use of all chemical shift values contained in the BioMagResBank (BMRB). Two programs were designed, one (BMRB stats) for extracting statistical chemical shift parameters from the BMRB and another one (RESCUE2) for computing the probabilities of each amino acid type, given a set of chemical shifts. The Bayesian prediction scheme presented here is compared to other methods already proposed: PROTYP RESCUE and PLATON and is found to be more sensitive and more specific. Using this scheme, we tested various sets of nuclei. The two nuclei carrying the most information are C(beta) and H(beta), in agreement with observations made in Grzesiek and Bax, 1993. Based on four nuclei: H(beta), C(beta), C(alpha) and C', it is possible to increase correct predictions to a rate of more than 75%. Taking into account the correlations between the nuclei chemical shifts has only a slight impact on the percentage of correct predictions: indeed, the largest correlation coefficients display similar features on all amino acids.     

N-3 long chain polyunsaturated fatty acids: a nutritional tool to prevent insulin resistance associated to type 2 diabetes and obesity?

n-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), mainly eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3), are present in mammal tissues both from endogenous synthesis from desaturation and elongation of 18:3 n-3 and/or from dietary origin (marine products and fish oils). In rodents in vivo, n-3 LC-PUFA have a protective effect against high fat diet induced insulin resistance. Such an effect is explained at the molecular level by the prevention of many alterations of insulin signaling induced by a high fat diet. Indeed, the protective effect of n-3 LC-PUFA results from the following: (a) the prevention of the decrease of phosphatidyl inositol 3' kinase (PI3 kinase) activity and of the depletion of the glucose transporter protein GLUT4 in the muscle; (b) the prevention of the decreased expression of GLUT4 in adipose tissue. In addition, n-3 LC-PUFA inhibit both the activity and expression of liver glucose-6-phosphatase which could explain the protective effect with respect to the excessive hepatic glucose output induced by a high fat diet. n-3 LC-PUFA also decrease muscle intramyofibrillar triglycerides and liver steatosis. This last effect results on the one hand, from a decreased expression of lipogenesis enzymes and of delta 9 desaturase (via a depleting effect on sterol response element binding protein 1c (SREBP-1c). On the other hand, n-3 LC-PUFA stimulate fatty acid oxidation in the liver (via the activation of peroxisome proliferator activated receptor alpha (PPAR-alpha)). In patients with type 2 diabetes, fish oil dietary supplementation fails to reverse insulin resistance for unclear reasons, but systematically decreases plasma triglycerides. Conversely, in healthy humans, fish oil has many physiological effects. Indeed, fish oil reduces insulin response to oral glucose without altering the glycaemic response, abolishes extraggression at times of mental stress, decreases the activation of sympathetic activity during mental stress and also decreases plasma triglycerides. These effects are encouraging in the perspective of prevention of insulin resistance but further clinical and basic studies must be designed to confirm and complete our knowledge in this field.     

Discovery of superoxide reductase: an historical perspective

For more than 30 years, the only enzymatic system known to catalyze the elimination of superoxide was superoxide dismutase, SOD. SOD has been found in almost all organisms living in the presence of oxygen, including some anaerobic bacteria, supporting the notion that superoxide is a key and general component of oxidative stress. Recently, a new concept in the field of the mechanisms of cellular defense against superoxide has emerged. It was discovered that elimination of superoxide in some anaerobic and microaerophilic bacteria could occur by reduction, a reaction catalyzed by a small metalloenzyme thus named superoxide reductase, SOR. Having played a major role in this discovery, we describe here how the concept of superoxide reduction emerged and how it was experimentally substantiated independently in our laboratory.Abbreviations Dfx desulfoferrodoxin - SOD superoxide dismutase - SOR superoxide reductase     

The COUP-TF nuclear receptors regulate cell migration in the mammalian basal forebrain

Cells migrate via diverse pathways and in different modes to reach their final destinations during development. Tangential migration has been shown to contribute significantly to the generation of neuronal diversity in the mammalian telencephalon. GABAergic interneurons are the best-characterized neurons that migrate tangentially, from the ventral telencephalon, dorsally into the cortex. However, the molecular mechanisms and nature of these migratory pathways are only just beginning to be unravelled. In this study we have first identified a novel dorsal-to-ventral migratory route, in which cells migrate from the interganglionic sulcus, located in the basal telencephalon between the lateral and medial ganglionic eminences, towards the pre-optic area and anterior hypothalamus in the diencephalon. Next, with the help of transplantations and gain-of-function studies in organotypic cultures, we have shown that COUP-TFI and COUP-TFII are expressed in distinct and non-overlapping migratory routes. Ectopic expression of COUP-TFs induces an increased rate of cell migration and cell dispersal, suggesting roles in cellular adhesion and migration processes. Moreover, cells follow a distinct migratory path, dorsal versus ventral, which is dependent on the expression of COUP-TFI or COUP-TFII, suggesting an intrinsic role of COUP-TFs in guiding migrating neurons towards their target regions. Therefore, we propose that COUP-TFs are directly involved in tangential cell migration in the developing brain, through the regulation of short- and long-range guidance cues.     

Differential roles of multiple signal peptidases in the virulence of Listeria monocytogenes

Most bacteria contain one type I signal peptidase (Spase I) for cleavage of signal peptides from exported and secreted proteins. Here, we identified a locus encoding three contiguous Spase I genes in the genome of Listeria monocytogenes. The deduced Sip proteins (denoted SipX, SipY and SipZ) are significantly similar to SipS and SipT, the major SPase I proteins of Bacillus subtilis (38% to 44% peptidic identity). We studied the role of these multiple signal peptidases in bacterial pathogenicity by constructing a series of single- and double-chromosomal knock-out mutants. Inactivation of sipX did not affect intracellular multiplication of L. monocytogenes but significantly reduced bacterial virulence (approximately 100-fold). Inactivation of sipZ impaired the secretion of phospholipase C (PC-PLC) and listeriolysin O (LLO), restricted intracellular multiplication and almost abolished virulence (LD(50) of 10(8.3)), inactivation of sipY had no detectable effects. Most importantly, a mutant expressing only SipX was impaired in intracellular survival and strongly attenuated in the mouse (LD(50) of 10(7.2)), whereas, a mutant expressing only SipZ behaved like wild-type EGD in all the assays performed. The data establish that SipX and SipZ perform distinct functions in bacterial pathogenicity and that SipZ is the major Spase I of L. monocytogenes. This work constitutes the first report on the differential role of multiple Spases I in a pathogenic bacterium and suggests a possible post-translational control mechanism of virulence factors expression.     

HPLC-PAD-APCI/MS assay of phenylpropanoids in cereals

A new, rapid HPLC-PAD-APCI/MS assay has been developed in order to measure accurately the amount of p-coumaric, E- and Z-ferulic acid and the dehydrodimers of ferulic acid in cereal grain. In the positive ionisation mode, MS patterns gave additional information for the identification of the dimers. The time required and the quantities of solvents employed in the developed analytical method are much lower than those involved in previously available assays of these compounds, thus making the method suitable for the screening of cereal genotypes. Application of the method to accessions of maize, wheat and sorghum showed that E-ferulic was the most abundant phenylpropanoid, whilst the major dimer was 8-O-4' dehydrodimer of ferulic acid followed by the 5-5' and then the 8-5' forms. Maize grains, especially of the Mexican landraces, contained the highest levels of these dimers.     

PTH-1R responses to PTHrP and regulation by vitamin D in keratinocytes and adjacent fibroblasts

Vitamin D and PTHrP are essential for the differentiation of keratinocytes and epidermal development. The action of PTHrP on skin is mediated via its PTH-1R receptors present in both epidermal and dermal cells. This suggests that PTHrP may have a paracrine/autocrine role, and its receptors may act in association or in negative cooperativity. We compared the intracellular signaling pathways in response to PTHrP (1-34) and to various PTHrP peptides, the N-terminal (1-34), Mid region (67-89), and C-terminal (107-139) fragments, and the possible modulation of PTHrP and its receptor mRNA expressions by vitamin D. Adjacent dermal fibroblasts as freshly isolated keratinocytes expressed both PTHrP and PTH-1R mRNAs, and responded to the various PTHrP fragments. bPTH and PTHrP(1-34) increased both cellular cAMP and [Ca(2+)]i in keratinocytes and fibroblasts. In contrast, PTHrP (107-139) increased [Ca(2+)]i but not cAMP in the two cell types. PTHrP (67-89) had no effect in keratinocytes, and only increased [Ca(2+)]i in fibroblasts. Vitamin D deficiency in weaned rats increased the expression of PTHrP mRNA in keratinocytes, and decreased it in fibroblasts and kidneys. Vitamin D deficiency increased PTH-1R mRNA expression in keratinocytes and kidneys, but not in fibroblasts. Although keratinocytes and skin fibroblasts are target cells for PTHrP and express PTH-1R, the two adjacent cell types differ as regards their intracellular signaling in response to PTHrP peptides. Moreover vitamin D regulates PTHrP and PTH-1R in a cell-specific manner.     

In silico screening of a saturated mutation library of tomato

A comprehensive mutant population is a basic resource for exploring gene function. We developed an isogenic tomato 'mutation library' in the genetic background of the inbred variety M82. A total of 13 000 M(2) families, derived from EMS (ethyl methane sulfonate) and fast-neutron mutagenesis, were visually phenotyped in the field and categorized into a morphological catalog that includes 15 primary and 48 secondary categories. Currently, 3417 mutations have been cataloged; among them are most of the previously described phenotypes from the monogenic mutant collection of The Tomato Genetics Resource Center, and over a thousand new mutants, with multiple alleles per locus. The phenotypic database indicates that most mutations fall into more than a single category (pleiotropic), with some organs such as leaves more prone to alterations than others. All data and images can be searched and accessed in the Solanaceae Genome Network (SGN) on a site called 'The Genes That Make Tomatoes' (http://zamir.sgn.cornell.edu/mutants/).     

Selective deficits in the circadian light response in mice lacking PACAP

Previous studies indicate that light information reaches the suprachiasmatic nucleus through a subpopulation of retinal ganglion cells that contain both glutamate and pituitary adenylyl cyclase-activating peptide (PACAP). Although the role of glutamate in this pathway has been well studied, the involvement of PACAP and its receptors is only beginning to be understood. To investigate the functions of PACAP in vivo, we developed a mouse model in which the gene coding for PACAP was disrupted by targeted homologous recombination. RIA was used to confirm a lack of detectable PACAP protein in these mice. PACAP-deficient mice exhibited significant impairment in the magnitude of the response to brief light exposures with both light-induced phase delays and advances of the circadian system impacted. This mutation equally impacted phase shifts induced by bright and dim light exposure. Despite these effects on phase shifting, the loss of PACAP had only limited effects on the generation of circadian oscillations, as measured by rhythms in wheel-running activity. Unlike melanopsin-deficient mice, the mice lacking PACAP exhibited no loss of function in the direct light-induced inhibition of locomotor activity, i.e., masking. Finally, the PACAP-deficient mice exhibited normal phase shifts in response to exposure to discrete dark treatments. The results reported here show that the loss of PACAP produced selective deficits in the light response of the circadian system.