Evolutionary history of the alpha2,8-sialyltransferase (ST8Sia) gene family: Tandem duplications in early deuterostomes explain most of the diversity found in the vertebrate ST8Sia genes

<p>Abstract</p> <p>Background</p> <p>The animal sialyltransferases, which catalyze the transfer of sialic acid to the glycan moiety of glycoconjugates, are subdivided into four families: ST3Gal, ST6Gal, ST6GalNAc and ST8Sia, based on acceptor sugar specificity and glycosidic linkage formed. Despite low overall sequence identity between each sialyltransferase family, all sialyltransferases share four conserved peptide motifs (L, S, III and VS) that serve as hallmarks for the identification of the sialyltransferases. Currently, twenty subfamilies have been described in mammals and birds. Examples of the four sialyltransferase families have also been found in invertebrates. Focusing on the ST8Sia family, we investigated the origin of the three groups of α2,8-sialyltransferases demonstrated in vertebrates to carry out poly-, oligo- and mono-α2,8-sialylation.</p> <p>Results</p> <p>We identified in the genome of invertebrate deuterostomes, orthologs to the common ancestor for each of the three vertebrate ST8Sia groups and a set of novel genes named ST8Sia EX, not found in vertebrates. All these ST8Sia sequences share a new conserved family-motif, named "C-term" that is involved in protein folding, via an intramolecular disulfide bridge. Interestingly, sequences from <it>Branchiostoma floridae </it>orthologous to the common ancestor of polysialyltransferases possess a polysialyltransferase domain (PSTD) and those orthologous to the common ancestor of oligosialyltransferases possess a new ST8Sia III-specific motif similar to the PSTD. In osteichthyans, we have identified two new subfamilies. In addition, we describe the expression profile of ST8Sia genes in <it>Danio rerio</it>.</p> <p>Conclusion</p> <p>Polysialylation appeared early in the deuterostome lineage. The recent release of several deuterostome genome databases and paralogons combined with synteny analysis allowed us to obtain insight into events at the gene level that led to the diversification of the ST8Sia genes, with their corresponding enzymatic activities, in both invertebrates and vertebrates. The initial expansion and subsequent divergence of the ST8Sia genes resulted as a consequence of a series of ancient duplications and translocations in the invertebrate genome long before the emergence of vertebrates. A second subset of ST8sia genes in the vertebrate genome arose from whole genome duplication (WGD) R1 and R2. Subsequent selective ST8Sia gene loss is responsible for the characteristic ST8Sia gene expression pattern observed today in individual species.</p

Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0

<p>Abstract</p> <p>Background</p> <p>The rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of <it>fut8 </it>gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat <it>fut8 </it>gene.</p> <p>Results</p> <p>The cDNAs encoding the rat α1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a α1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat <it>fut8 </it>gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of <it>fut8 </it>in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 <it>fut8 </it>gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells.</p> <p>Conclusion</p> <p>Altogether, these data contribute to a better knowledge of <it>fut8 </it>expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.</p

Analysis of sequence variability in the CART gene in relation to obesity in a Caucasian population

BACKGROUND: Cocaine and amphetamine regulated transcript (CART) is an anorectic neuropeptide located principally in hypothalamus. CART has been shown to be involved in control of feeding behavior, but a direct relationship with obesity has not been established. The aim of this study was to evaluate the effect of polymorphisms within the CART gene with regards to a possible association with obesity in a Caucasian population. RESULTS: Screening of the entire gene as well as a 3.7 kb region of 5' upstream sequence revealed 31 SNPs and 3 rare variants ; 14 of which were subsequently genotyped in 292 French morbidly obese subjects and 368 controls. Haplotype analysis suggested an association with obesity which was found to be mainly due to SNP-3608T>C (rs7379701) (p = 0.009). Genotyping additional cases and controls also of European Caucasian origin supported further this possible association between the CART SNP -3608T>C T allele and obesity (global p-value = 0.0005). Functional studies also suggested that the SNP -3608T>C could modulate nuclear protein binding. CONCLUSION: CART SNP -3608T>C may possibly contribute to the genetic risk for obesity in the Caucasian population. However confirmation of the importance of the role of the CART gene in energy homeostasis and obesity will require investigation and replication in further populations

Comparison of SMOS and SMAP Soil Moisture Retrieval Approaches Using Tower-based Radiometer Data over a Vineyard Field

The objective of this study was to compare several approaches to soil moisture (SM) retrieval using L-band microwave radiometry. The comparison was based on a brightness temperature (TB) data set acquired since 2010 by the L-band radiometer ELBARA-II over a vineyard field at the Valencia Anchor Station (VAS) site. ELBARA-II, provided by the European Space Agency (ESA) within the scientific program of the SMOS (Soil Moisture and Ocean Salinity) mission, measures multiangular TB data at horizontal and vertical polarization for a range of incidence angles (30-60). Based on a three year data set (2010-2012), several SM retrieval approaches developed for spaceborne missions including AMSR-E (Advanced Microwave Scanning Radiometer for EOS), SMAP (Soil Moisture Active Passive) and SMOS were compared. The approaches include: the Single Channel Algorithm (SCA) for horizontal (SCA-H) and vertical (SCA-V) polarizations, the Dual Channel Algorithm (DCA), the Land Parameter Retrieval Model (LPRM) and two simplified approaches based on statistical regressions (referred to as 'Mattar' and 'Saleh'). Time series of vegetation indices required for three of the algorithms (SCA-H, SCA-V and Mattar) were obtained from MODIS observations. The SM retrievals were evaluated against reference SM values estimated from a multiangular 2-Parameter inversion approach. The results obtained with the current base line algorithms developed for SMAP (SCA-H and -V) are in very good agreement with the reference SM data set derived from the multi-angular observations (R2 around 0.90, RMSE varying between 0.035 and 0.056 m3m3 for several retrieval configurations). This result showed that, provided the relationship between vegetation optical depth and a remotely-sensed vegetation index can be calibrated, the SCA algorithms can provide results very close to those obtained from multi-angular observations in this study area. The approaches based on statistical regressions provided similar results and the best accuracy was obtained with the Saleh methods based on either bi-angular or bipolarization observations (R2 around 0.93, RMSE around 0.035 m3m3). The LPRM and DCA algorithms were found to be slightly less successful in retrieving the 'reference' SM time series (R2 around 0.75, RMSE around 0.055 m3m3). However, the two above approaches have the great advantage of not requiring any model calibrations previous to the SM retrievals

GAD2 on chromosome 10p12 is a candidate gene for human obesity

The gene GAD2 encoding the glutamic acid decarboxylase enzyme (GAD65) is a positional candidate gene for obesity on Chromosome 10p11&ndash;12, a susceptibility locus for morbid obesity in four independent ethnic populations. GAD65 catalyzes the formation of &gamma;-aminobutyric acid (GABA), which interacts with neuropeptide Y in the paraventricular nucleus to contribute to stimulate food intake. A case-control study (575 morbidly obese and 646 control subjects) analyzing GAD2 variants identified both a protective haplotype, including the most frequent alleles of single nucleotide polymorphisms (SNPs) +61450 C&gt;A and +83897 T&gt;A (OR = 0.81, 95% CI [0.681&ndash;0.972], p = 0.0049) and an at-risk SNP (&minus;243 A&gt;G) for morbid obesity (OR = 1.3, 95% CI [1.053&ndash;1.585], p = 0.014). Furthermore, familial-based analyses confirmed the association with the obesity of SNP +61450 C&gt;A and +83897 T&gt;A haplotype (&chi;2 = 7.637, p = 0.02). In the murine insulinoma cell line &beta;TC3, the G at-risk allele of SNP &minus;243 A&gt;G increased six times GAD2 promoter activity (p &lt; 0.0001) and induced a 6-fold higher affinity for nuclear extracts. The &minus;243 A&gt;G SNP was associated with higher hunger scores (p = 0.007) and disinhibition scores (p = 0.028), as assessed by the Stunkard Three-Factor Eating Questionnaire. As GAD2 is highly expressed in pancreatic &beta; cells, we analyzed GAD65 antibody level as a marker of &beta;-cell activity and of insulin secretion. In the control group, &minus;243 A&gt;G, +61450 C&gt;A, and +83897 T&gt;A SNPs were associated with lower GAD65 autoantibody levels (p values of 0.003, 0.047, and 0.006, respectively). SNP +83897 T&gt;A was associated with lower fasting insulin and insulin secretion, as assessed by the HOMA-B% homeostasis model of &beta;-cell function (p = 0.009 and 0.01, respectively). These data support the hypothesis of the orexigenic effect of GABA in humans and of a contribution of genes involved in GABA metabolism in the modulation of food intake and in the development of morbid obesity.<br /

Tumour-associated carbohydrate antigens in breast cancer

Glycosylation changes that occur in cancer often lead to the expression of tumour-associated carbohydrate antigens. In breast cancer, these antigens are usually associated with a poor prognosis and a reduced overall survival. Cellular models have shown the implication of these antigens in cell adhesion, migration, proliferation and tumour growth. The present review summarizes our current knowledge of glycosylation changes (structures, biosynthesis and occurrence) in breast cancer cell lines and primary tumours, and the consequences on disease progression and aggressiveness. The therapeutic strategies attempted to target tumour-associated carbohydrate antigens in breast cancer are also discussed