Dynamics of glucosamine-6-phosphate synthase catalysis

Glucosamine-6P synthase, which catalyzes glucosamine-6P synthesis from fructose-6P and glutamine, channels ammonia over 18 Å between its glutaminase and synthase active sites. The crystal structures of the full-length Escherichia coli enzyme have been determined alone, in complex with the first bound substrate, fructose-6P, in the presence of fructose-6P and a glutamine analog, and in the presence of the glucosamine-6P product. These structures represent snapshots of reaction intermediates, and their comparison sheds light on the dynamics of catalysis. Upon fructose-6P binding, the C-terminal loop and the glutaminase domains get ordered, leading to the closure of the synthase site, the opening of the sugar ring and the formation of a “closed” ammonia channel. Then, glutamine binding leads to the closure of the Q-loop to protect the glutaminase site, the activation of the catalytic residues involved in glutamine hydrolysis, the swing of the side chain of Trp74, which allows the communication between the two active sites through an “open” channel, and the rotation of the glutaminase domains relative to the synthase domains dimer. Therefore, binding of the substrates at the appropriate reaction time is responsible for the formation and opening of the ammonia channel and for the activation of the enzyme glutaminase function.     

Integrins in mammary development

Integrins are ubiquitously expressed major cell surface receptors for extracellular matrix. Integrin interaction with their extracellular ligands triggers activation of the intracellular signaling pathways that control cell shape, motility, proliferation, survival, cell-type-specific gene expression. In this review, we summarize recent studies analyzing contribution of integrins to the control of the mammary morphogenesis and differentiation, function and maintenance of mammary stem and progenitor cells and resume the data from mouse models revealing the contribution of the integrin-mediated signaling to mammary tumorigenesis.     

Relationships between Categorical Perception of Phonemes,Phoneme Awareness,and Visual Attention Span in Developmental Dyslexia

We tested the hypothesis that the categorical perception deficit of speech sounds in developmental dyslexia is related to phoneme awareness skills, whereas a visual attention (VA) span deficit constitutes an independent deficit. Phoneme awareness tasks, VA span tasks and categorical perception tasks of phoneme identification and discrimination using a d/t voicing continuum were administered to 63 dyslexic children and 63 control children matched on chronological age. Results showed significant differences in categorical perception between the dyslexic and control children. Significant correlations were found between categorical perception skills, phoneme awareness and reading. Although VA span correlated with reading, no significant correlations were found between either categorical perception or phoneme awareness and VA span. Mediation analyses performed on the whole dyslexic sample suggested that the effect of categorical perception on reading might be mediated by phoneme awareness. This relationship was independent of the participants’ VA span abilities. Two groups of dyslexic children with a single phoneme awareness or a single VA span deficit were then identified. The phonologically impaired group showed lower categorical perception skills than the control group but categorical perception was similar in the VA span impaired dyslexic and control children. The overall findings suggest that the link between categorical perception, phoneme awareness and reading is independent from VA span skills. These findings provide new insights on the heterogeneity of developmental dyslexia. They suggest that phonological processes and VA span independently affect reading acquisition.     

Determination of the biological diatom index (IBD NF T 90–354): results of an intercomparison exercise

A diatom intercomparison exercise on sampling, slide preparation, countsand identification has been carried out in September 1999 with 24French-speaking diatomists on the River Loup (Alpes Maritimes, France). Fewoperators strictly respected the standard, especially for the number ofindividuals to be counted. It has however been demonstrated this had a very lowimpact on the index value and that old counts can be used for indexcalculation,if there are at least 300 frustules or valves. Counts higher than 400 did notlead to a better precision. The total variability largely depended on theoperator and was distributed unevenly between sampling, which appears to be acritical stage, slide preparation and counting. For those who strictlyrespectedthe standard procedure, the natural variability can be estimated to about oneunit value of the Biological Diatom Index. This intra-operator variability canbe considered as very low, as the IBD scale is from 0 to 20, and notsignificantin assessing water quality. Despite this, the inter-operator variability isgreater. When comparing operators' results, after excluding those who did notrespect the standard requirements, the main source of variability is due tomisidentifications, especially for small Achnanthes(A. minutissima and A. biasolettiana)and Cocconeis (C. placentula var.placentula, var. euglypta and var.lineata). Intercomparison exercises, internet exchangesforchecking and reference material collections should be encouraged both tosatisfythe internal and external quality controls and to acquaint biologists with theimportance of field and laboratory protocols, as well as to allow progress indiatom identification.     

Beta1 integrin deletion from the basal compartment of the mammary epithelium affects stem cells

The mammary gland epithelium comprises two major cell types: basal and luminal. Basal cells interact directly with the extracellular matrix (ECM) and express higher levels of the ECM receptors, integrins, than luminal cells. We show that deletion of beta1 integrin from basal cells abolishes the regenerative potential of the mammary epithelium and affects mammary gland development. The mutant epithelium was characterized by an abnormal ductal branching pattern and aberrant morphogenesis in pregnancy, although at the end of gestation, the secretory alveoli developed from beta1 integrin-positive progenitors. Lack of beta1 integrin altered the orientation of the basal-cell division axis and in mutant epithelium, in contrast to control tissue, the progeny of beta1 integrin-null basal cells, identified by a genetic marker, was found in the luminal compartment. These results reveal, for the first time, the essential role of the basal mammary epithelial cell-ECM interactions mediated by beta1 integrins in the maintenance of a functional stem cell population, mammary morphogenesis and segregation of the two major mammary cell lineages.     

Monoclonal anti-MAGE-3 CTL responses in melanoma patients displaying tumor regression after vaccination with a recombinant canarypox virus

We have analyzed the T cell responses of HLA-A1 metastatic melanoma patients with detectable disease, following vaccination with a recombinant ALVAC virus, which bears short MAGE-1 and MAGE-3 sequences coding for antigenic peptides presented by HLA-A1. To evaluate the anti-MAGE CTL responses, we resorted to antigenic stimulation of blood lymphocytes under limiting dilution conditions, followed by tetramer analysis and cloning of the tetramer-positive cells. The clones were tested for their specific lytic ability and their TCR sequences were obtained. Four patients who showed tumor regression were analyzed, and an anti-MAGE-3.A1 CTL response was observed in three of these patients. Postvaccination frequencies of anti-MAGE-3.A1 CTL were 3 x 10(-6), 3 x 10(-3), and 3 x 10(-7) of the blood CD8 T cells, respectively. These three responses were monoclonal. No anti-MAGE-1.A1 CTL response was observed. These results indicate that, like peptide immunization, ALVAC immunization produces monoclonal responses. They also suggest that low-level antivaccine CTL responses can initiate a tumor regression process. Taken together, our analysis of anti-MAGE-3.A1 T cell responses following peptide or ALVAC vaccination shows a degree of correlation between CTL response and tumor regression, but firm conclusions will require larger numbers.     

An enzyme-coupled assay for amidotransferase activity of glucosamine-6-phosphate synthase

An assay for glucosamine-6-phosphate synthase using a yeast glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) as coupling enzyme was developed. GNA1 transfers the acetyl moiety from acetyl-coenzyme A (CoA) to glucosamine-6-phosphate, releasing coenzyme A. The assay measures the production of glucosamine-6-phosphate by either following the consumption of acetyl-CoA spectrophotometrically at 230nm or quantifying the free thiol with 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent) in a discontinuous manner. This method is simple to perform and can be adapted to a 96-well microtiter plate format, which will facilitate high-throughput inhibitor screening and mechanistic studies using purified GlmS.     

Study of banana and coconut fibers Botanical composition, thermal degradation and textural observations

Four fibers from banana-trees (leaf, trunk) and coconut-tree (husk, fabric) were examined before their incorporation in cementitious matrices, in order to prepare insulating material for construction. Their botanical compositions have been determined following a method described previously. Thermal degradation of these fibers were studied between 200 and 700 degrees C under nitrogen gas flow. Temperature of pyrolysis was the experimental parameter investigated. The solid residues obtained were analyzed by classical elemental analysis, Fourier Transform Infra Red (FTIR) spectroscopy and were observed by Scanning Electron Microscopy (SEM). This study has shown (1) the relation between botanical, chemical composition with both localization of fibers in the tree and type of tree; (2) the rapid and preferential decomposition of banana fibers with increasing temperature of pyrolysis and (3) the rough samples are made of hollow fibers.     

Discovering new inhibitors of bacterial glucosamine-6P synthase (GlmS) by docking simulations

Results of an in silico screening of a freely accessible database encompassing 50,000 commercial compounds on bacterial glucosamine-6P synthase (Glms) are described. Each product was docked with the GOLD software in a region of 20A surrounding the sugar binding site and ranked according to its score. Among the 14 best-scored molecules, three molecules exhibited good experimental inhibition properties (IC(50)=70 microM) giving a high hit rate (H.R.: 0.23). Interestingly, these molecules are predicted to interact with a protein region that forms a pocket at the interface between the two enzyme monomers, opening the route to dimerization inhibitors.     

Identification of a novel serine phosphorylation site in human glutamine:fructose-6-phosphate amidotransferase isoform 1

Glutamine:fructose-6-phosphate amidotransferase (Gfat) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway. The increasing amount of evidence that links excess hexosamine biosynthesis with pathogenic complications of type II diabetes highlights the need to understand the regulation of Gfat. Previous studies showed that eukaryotic Gfat is subjected to feedback inhibition by UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) and to phosphorylation by cAMP-activated protein kinase A (PKA). In this study, overexpression of human Gfat isoform 1 (hGfat1) in insect cells revealed that hGfat1 is phosphorylated in vivo. Using matrix-assisted laser desorption/ionization and electrospray tandem mass spectrometry, we have identified Ser243 as a novel phosphorylation site. Biochemical properties of the wild type and the Ser243Glu mutant of hGfat1 overexpressed in Escherichia coli were compared. Our results provide evidence that phosphorylation at Ser243 stimulates glucosamine 6-phosphate-synthesizing activity, lowers amidohydrolyzing activity in the absence of fructose 6-phosphate (F6P) (glutaminase activity), and lowers Km(F6P) 2-fold, but has no effect on UDP-GlcNAc inhibition. On the basis of the sequence consensus, AMP-activated protein kinase and calcium/calmodulin-dependent kinase II were identified to phosphorylate specifically Ser243 in vitro. Phosphorylation by these two kinases results in an increase of enzymatic activity by 1.4-fold. These findings suggest for the first time that hGfat1 may be regulated by kinases other than PKA.