N, P, Si budgets for the Red River Delta (northern Vietnam): how the delta affects river nutrient delivery to the sea

The Red River Delta (RRD) (Vietnam), a region experiencing rapid population growth, industrialization, and economic development, concentrates 54% of the population of the whole Red River watershed in less than 10% of the basin area. Our study aimed at understanding and quantifying the processes by which the delta affects the nutrient fluxes coming from the upstream watershed before they reach the sea. A comprehensive budget of nitrogen (N), phosphorus (P), and silica (Si) fluxes associated with natural and anthropogenic processes in the terrestrial and hydrological system of the delta was established for five sub-basins of the delta for the period 2000–2006, based on official statistical data, available measurements, and our own sampling campaigns and enquiries. The results show that anthropogenic inputs of N and P brought into the delta area are higher than the amounts delivered by the river from the upstream watershed. However, the amounts of these two elements ultimately delivered to the coastal zone from the delta are lower than the amounts carried by the upstream river, showing extremely efficient retention of both the soils and the delta’s drainage network. For Si (taking into account both dissolved and amorphous solid forms), the retention is much lower. High retention of N and P and low retention of Si in the delta area have up to now protected the coastal zone from severe eutrophication problems.     

CCN1/Cyr61 is regulated by the canonical Wnt signal and plays an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells

Marrow mesenchymal stem cells are pluripotent progenitors that can differentiate into bone, cartilage, muscle, and fat cells. Wnt signaling has been implicated in regulating osteogenic differentiation of mesenchymal stem cells. Here, we analyzed the gene expression profile of mesenchymal stem cells that were stimulated with Wnt3A. Among the 220 genes whose expression was significantly changed by 2.5-fold, we found that three members of the CCN family, CCN1/Cyr61, CCN2/connective tissue growth factor (CTGF), and CCN5/WISP2, were among the most significantly up-regulated genes. We further investigated the role of CCN1/Cyr61 in Wnt3A-regulated osteogenic differentiation. We confirmed that CCN1/Cyr61 was up-regulated at the early stage of Wnt3A stimulation. Chromatin immunoprecipitation analysis indicates that CCN1/Cyr61 is a direct target of canonical Wnt/beta-catenin signaling. RNA interference-mediated knockdown of CCN1/Cyr61 expression diminished Wnt3A-induced osteogenic differentiation. Furthermore, exogenously expressed CCN1/Cyr61 was shown to effectively promote mesenchymal stem cell migration. These findings suggest that tightly regulated CCN1/Cyr61 expression may play an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells.     

Glycosyltransferase Function in Core 2-Type Protein O Glycosylation

Three glycosyltransferases have been identified in mammals that can initiate core 2 protein O glycosylation. Core 2 O-glycans are abundant among glycoproteins but, to date, few functions for these structures have been identified. To investigate the biological roles of core 2 O-glycans, we produced and characterized mice deficient in one or more of the three known glycosyltransferases that generate core 2 O-glycans (C2GnT1, C2GnT2, and C2GnT3). A role for C2GnT1 in selectin ligand formation has been described. We now report that C2GnT2 deficiency impaired the mucosal barrier and increased susceptibility to colitis. C2GnT2 deficiency also reduced immunoglobulin abundance and resulted in the loss of all core 4 O-glycan biosynthetic activity. In contrast, the absence of C2GnT3 altered behavior linked to reduced thyroxine levels in circulation. Remarkably, elimination of all three C2GnTs was permissive of viability and fertility. Core 2 O-glycan structures were reduced among tissues from individual C2GnT deficiencies and completely absent from triply deficient mice. C2GnT deficiency also induced alterations in I-branching, core 1 O-glycan formation, and O mannosylation. Although the absence of C2GnT and C4GnT activities is tolerable in vivo, core 2 O glycosylation exerts a significant influence on O-glycan biosynthesis and is important in multiple physiological processes.Protein O glycosylation is a posttranslational modification implicated in a wide range of physiological processes, including cell adhesion and trafficking, T-cell apoptosis, cell signaling, endocytosis and pathogen-host interaction (1, 6, 27, 30, 54, 61, 71). Core-type protein O glycosylation is initiated in the secretory pathway by the covalent addition of a N-acetylgalactosamine (GalNAc) to the hydroxyl group of serine or threonine residues by one of multiple polypeptide GalNAc transferases (ppGalNAcTs) (20, 44, 57, 58). After linkage of the GalNAc monosaccharide to serine or threonine, other glycosyltransferases sequentially and sometimes competitively elaborate the repertoire of O-glycan structures to include different core subtypes (31, 42, 48, 49).The core 2 β1,6-N-acetylglucosaminyltransferases (C2GnTs) and the Core 2 O-glycans they generate are widely expressed among cells of mammalian species. The C2GnTs act after the core 1 β-1,3-galactosyltransferase adds a galactose in a β1,3-linkage to the GalNAc-Ser/Thr generating the initial core 1 O-glycan disaccharide structure (26). Then, one of the three C2GnTs (C2GnT1, C2GnT2, and C2GnT3) can add an N-acetylglucosamine (GlcNAc) in a β1,6-linkage to the GalNAc to initiate what is known as the core 2 O-glycan branch (Fig. ​(Fig.1a)1a) (7, 50, 51, 69). In a distinct pathway, core 3 β-1,3-N-acetylglucosaminyltransferase (C3GnT) can add a GlcNAc to the unmodified GalNAc to generate a core 3 O-glycan (24). In this case, C2GnT2 can add a GlcNAc in β1,6-linkage to the GalNAc of the core 3 O-glycan disaccharide to initiate the formation of a core 4 O-glycan (Fig. ​(Fig.1b)1b) (50, 69). In addition, both C2GnT2 and the I β-1,6-N-acetylglucosaminyltransferase (IGnT) are independently capable of forming branched polylactosamine structures (I-branches) from otherwise linear polylactosamine glycan chains (Fig. ​(Fig.1c)1c) (69).Open in a separate windowFIG. 1.Activity and expression of C2GnTs. (a to c) Monosaccharides are depicted as geometric shapes, with GalNAc as a yellow square, galactose as a yellow circle, and GlcNAc as a blue square. In addition, the vertical arrows indicate that each branch can be further elaborated by additional saccharide linkages. (a) Biantennary core 2 O-glycans are generated when any of the three C2GnTs acts on the core 1 O-glycan disaccharide. (b) C2GnT2 can generate core 4 O-glycans from core 3 O-glycans by adding a GlcNAc to the initiating GalNAc. (c) C2GnT2, in addition to IGnT, also has the ability to generate branched polylactosamine repeats from linear polylactosamine repeats. The figure depicts distal I-branching as the GlcNAc is transferred to the predistal galactose, the preferential I-branching activity of C2GnT2. However, IGnT preferentially has central I-branching activity that adds GlcNAc on the internal galactose in Galβ1→4GlcNAcβ1→3Gal-R (69). (d) RNA expression of murine Gcnt3 (left panel) and Gcnt4 (right panel), which code for C2GnT2 and C2GnT3, respectively, as determined by qPCR. The data on single animals are graphed relative to testes expression. All values are means ± the standard errors of the mean (SEM).C2GnT1-deficient mice have been shown to have an unexpected phenotype first observed as leukocytosis reflecting neutrophilia (14). This appears to be due to a severe but selective defect in selectin ligand biosynthesis among myeloid cells, leading to decreased recruitment of neutrophils that attenuates inflammation and vascular disease pathogenesis (14, 64). C2GnT1-deficient mice also exhibit a partial reduction in L-selectin ligand biosynthesis on high endothelial venules, resulting in reduced B-cell homing and colonization of peripheral lymph nodes (18, 21). Furthermore, thymic progenitors from C2GnT1-deficient mice have a reduced ability to home to the thymus due to the loss of P-selectin ligands on these cells (46). However, as of yet, C2GnT2 and C2GnT3 have not been similarly investigated, and their biological functions remain to be elucidated. To further investigate why multiple glycosyltransferases capable of core 2 O-glycan formation have been conserved, we have generated mice singly and multiply deficient in the three known C2GnTs and characterized the resulting physiology and alterations to the glycome.     

Wnt antagonist SFRP3 inhibits the differentiation of mouse hepatic progenitor cells

Wnt/β‐catenin pathway plays an important role in regulating embryonic development. Hepatocytes differentiate from endoderm during development. Hepatic progenitor cells (HPCs) have been isolated from fetal liver and extrahepatic tissues. Most current studies in liver development and hepatic differentiation have been focused on Wnts, β‐catenin, and their receptors. Here, we sought to determine the role of Wnt antagonists in regulating hepatic differentiation of fetal liver‐derived HPCs. Using mouse liver tissues derived from embryonic day E12.5 to postnatal day (PD) 28, we found that 13 of the 19 Wnt genes and almost all of Wnt receptors/co‐receptors were expressed in most stages. However, Wnt antagonists SFRP2, SFRP3, and Dkk2 were only detected in the early stages. We established and characterized the reversible stable HPCs derived from E14.5 mouse fetal liver (HP14.5). HP14.5 cells were shown to express high levels of early liver progenitor cell markers, but low levels or none of late liver markers. HP14.5 cells were shown to differentiate into mature hepatocytes upon dexamethasone (Dex) stimulation. Dex‐induced late marker expression and albumin promoter activity in HP14.5 cells were inhibited by exogenous expression of SFRP3. Furthermore, Dex‐induced glycogen synthesis of PAS‐positive HP14.5 cells was significantly inhibited by SFRP3. Therefore, our results have demonstrated that the expression of Wnt antagonists decreases as hepatic differentiation progresses, suggesting that a balanced Wnt signaling may be critical during mouse liver development and hepatic differentiation. J. Cell. Biochem. 108: 295–303, 2009. © 2009 Wiley‐Liss, Inc.     

Analysis of 17β-hydroxysteroid dehydrogenase types 5, 7, and 12 genetic sequence variants in breast cancer cases from French Canadian Families with high risk of breast and ovarian cancer

A family history and estrogen exposure are well-known risk factors for breast cancer. Members of the 17β-hydroxysteroid dehydrogenase family are responsible for important steps in the metabolism of androgens and estrogens in peripheral tissues, including the mammary gland. The crucial biological function of 17β-HSDs renders these genes good candidates for being involved in breast cancer etiology. This study screened for mutations in HSD17B7 and HSD17B12 genes, which encode enzymes involved in estradiol biosynthesis and in AKR1C3, which codes for 17β-HSD type 5 enzyme involved in androgen and progesterone metabolism, to assess whether high penetrance allelic variants in these genes could be involved in breast cancer susceptibility. Mutation screening of 50 breast cancer cases from non-BRCA1/2 high-risk French Canadian families failed to identify germline likely high-risk mutations in HSD17B7, HSD17B12 and AKR1C3 genes. However, 107 sequence variants were identified, including seven missense variants. Assessment of the impact of missense variants on enzymatic activity of the corresponding enzymes revealed no difference in catalytic properties between variants of 17β-HSD types 7 and 12 and wild-type enzymes, while variants p.Glu77Gly and p.Lys183Arg in 17β-HSD type 5 showed a slightly decreased activity. Finally, a haplotype-based approach was used to determine tagging SNPs providing valuable information for studies investigating associations of common variants in these genes with breast cancer risk.     

Widespread Distribution of Poribacteria in Demospongiae

Poribacteria were found in nine sponge species belonging to six orders of Porifera from three oceans. Phylogenetic analysis revealed four distinct poribacterial clades, which contained organisms obtained from several different geographic regions, indicating that the distribution of poribacteria is cosmopolitan. Members of divergent poribacterial clades were also found in the same sponge species in three different sponge genera.Recently, a novel bacterial phylum, termed “Poribacteria,” was discovered, and members of this phylum have been found exclusively in sponges (2). Phylogenetic analyses of 16S rRNA genes indicated that poribacteria are evolutionarily deeply branching organisms and related to a superphylum composed of Planctomycetes, Verrucomicrobia, and Chlamydia (11). Poribacterial 16S rRNA genes contain 13 of 15 planctomycete signature nucleotides, but a level of sequence divergence of more than 25% compared to any other bacterial phylum, including the Planctomycetes, justifies the status of this taxon as an independent phylum. A consistent treeing pattern is difficult to resolve in comparative phylogenetic sequence analyses, making the poribacteria an unusual line of phylogenetic descent. In addition to their divergent status as a separate phylum on the basis of the 16S rRNA sequence, poribacteria are also divergent because they may have a compartmentalized cell structure, a cell plan they share only with members of the phyla Planctomycetes and Verrucomicrobia (2). They are also of interest for understanding the potential contribution of obligate sponge-associated bacteria to the sponges harboring them and as an example of a yet-to-be-cultured group of bacteria associated with invertebrate tissue apparently exclusively but for unknown reasons. This study aimed to further explore the presence and diversity of poribacteria in different marine demosponge genera using samples from around the world.The Mediterranean sponges were collected by scuba divers offshore at Banyuls sur Mer, France (42°29′N, 03°08′E). The Caribbean sponges were collected offshore at Little San Salvador Island, Bahamas (24°32′N, 75°55′W). The eastern Pacific sponge Aplysina fistularis was collected offshore at San Diego, CA (32°51′N, 117°15′W). The western Pacific sponge Theonella swinhoei was collected offshore at Palau (07°23′N, 134°38′E). All non-Great Barrier Reef (non-GBR) sponges were collected between May and July 2000, and once individual sponge specimens were brought to the surface, they were frozen in liquid nitrogen on board ship and stored at −80°C until microbiological processing (9). The GBR marine sponges were collected off Heron Island Research Station (23°27′S, 151°5′E) in April 2002 (5). Pseudoceratina clavata was collected by scuba divers at a depth of 14 m, and Rhabdastrella globostellata was collected at a depth of ca. 0.5 m after a reef walk consisting of a few hundred meters. The samples were immediately placed in plastic bags and brought to Heron Island Research Station, where they were stored at −80°C until processing. Sponge DNA was extracted as described previously (2, 5).Total sponge-derived genomic DNA was screened by PCR for the presence of poribacteria using a 16S rRNA gene primer set. Poribacterial 16S rRNA genes were amplified by employing a pair of Poribacteria-specific primers, POR389f (5′-ACG ATG CGA CGC CGC GTG-3′) and POR1130r (5′-GGC TCG TCA CCA GCG GTC-3′) (2). The poribacterial PCR products that were ca. 740 bp long derived from one sponge individual were cloned into the pGEM-T Easy vector (Promega, Madison, WI). Clone inserts were digested with restriction endonucleases MspI and HaeIII (New England Biolabs, Inc., United States), characterized to obtain restriction profiles and unique profiles, and sequenced. The compiled partial 16S rRNA gene sequences were then analyzed using BLASTN to select the most closely related poribacterial reference sequences.The sequences exhibiting levels of similarity of less than 97% were used for further analysis. Poribacterial 16S rRNA gene sequences were aligned using the ARB software package (7). The resulting alignment was imported into PAUP (10) and analyzed by using distance, maximum parsimony, and maximum likelihood algorithms together with bootstrap resamplings (3,000, 3,000, and 200 resamplings, respectively), and the resulting bootstrap values were applied to nodes on the ARB neighbor-joining tree. Signature sequences were detected using the ARB software package. A signature sequence is defined here as a short sequence that is present in a group of poribacterial sequences in a phylogenetic clade but is not found in any other clade in the poribacterial tree.Analysis of the 16S rRNA gene clone library sequences generated from sponge tissues revealed the presence of poribacteria in sponge individuals belonging to the orders Verongida, Astrophorida, Dictyoceratida, Haplosclerida, Lithistida, and Homosclerophorida, while poribacteria could not be detected in sponges belonging to the orders Hadromerida and Agelasida. In the order Halichondrida, poribacteria were detected in Xestospongia muta but not in Haliclona sp. Altogether, nine sponge species were added to the list of Poribacteria-containing sponges (Table ​(Table1).1). Three distinct clades were observed that were clearly supported by bootstrap values greater than 75 with every tree-building algorithm applied (Fig. ​(Fig.1),1), and one clade (clade I) was supported by bootstrap values of 64, 98, and 71 in distance, maximum parsimony, and maximum likelihood trees, respectively. Similarity calculations using approximately 740-bp amplified poribacterial 16S rRNA gene fragments and other poribacterial sequences from the NCBI database showed that the dissimilarity between clades was consistent with their separation in phylogenetic trees. For example, the levels of dissimilarity between members of clade I and clade II were 3 to 8%, while the levels of dissimilarity between members of clades I and III and between members of clades I and IV were 10 to 14% and 11 to 15%, respectively.Open in a separate windowFIG. 1.Neighbor-joining phylogenetic tree for poribacterial clones based on Poribacteria-specific PCR products (740 bp) of the 16S rRNA gene, showing relationships of poribacterial clones from different global regions. The poribacterial clones on the right are additional clones belonging to the same clades as strains in the tree at the same level. Bootstrap confidence values of >75% for distance, maximum parsimony, and maximum likelihood algorithm analyses are indicated by filled circles at nodes, and open circles indicate unsupported nodes. Prefixes for clones: A, Aplysina aerophoba; C, Aplysina cavernicola; F, Aplysina fistularis; L, Aplysina lacunosa; S, Ircinia sp.; P, Plakortis sp.; PC, Pseudoceratina clavata; RG, Rhabdastrella globostellata; T, Theonella swinhoei; X, Xestospongia muta. Scale bar = 0.1 nucleotide substitution per site.

TABLE 1.

Distribution of poribacteria in different demosponge orders

TGFβ/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-induced Osteogenic Signaling in Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs.     

A sensitive and specific liquid chromatography–tandem mass spectrometric method for determination of belinostat in plasma from liver cancer patients

A novel, sensitive and reliable liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the determination of belinostat (PXD101) in human plasma. Oxamflatin was used as the internal standard. Liquid–liquid extraction of the plasma sample was performed using tert-butyl methyl ether as the organic solvent. Chromatographic separation was achieved on a BDS Hypersil C18 column (2.1 mm × 100 mm, 5 μm) using gradient elution mode using 0.05% formic acid in water and 0.05% formic acid in acetonitrile as solvents A and B, respectively, 60/40. The run time was 6 min. The mass spectrometer was operated under a positive electrospray ionization condition and a multiple reaction monitoring mode. An excellent linear calibration was achieved in the range of 0.5–1000 ng/mL. An average recovery of belinostat for four quality controls was 72.6% and the recovery of the internal standard at 1000 ng/mL was 67.8%. The intra-day and inter-day precisions for belinostat were ≤8.0 and ≤10.3%, respectively, and their accuracy ranged from 100.2 to 106.7%. No significant matrix effect was identified. In analysis of patient samples, belinostat glucuronide was identified and baseline separated from belinostat. This well-validated assay has been applied for quantification of belinostat in plasma samples within 24 h after the start of infusion for Asian hepatocellular carcinoma patients in a dose escalation study.