Comparative effects of 7 beta-hydroxycholesterol towards murine lymphomas, lymphoblasts and lymphocytes: selective cytotoxicity and blastogenesis inhibition

The effects of 7 beta-hydroxycholesterol on lymphoma cells in culture, on lymphocytes entering blastic transformation and on quiescent murine spleen lymphocytes have been investigated. The early events of blastogenesis as well as YAC-1, RDM-4 and EL-4 cells were shown to be very sensitive to this sterol at microM concentration, whereas constituted lymphoblasts and normal lymphocytes remained insensitive at 50 times higher concentration. The effect of some classical antitumor drugs (Adriamycin, Mitomycin-C, Methotrexate) on these lymphoma cells were of the same order of magnitude. However, the activity of 7 beta-hydroxycholesterol was closely related to the composition of the culture medium. Indeed, the cytotoxic effects of this compound were less in medium supplemented with foetal calf serum than in lipoprotein poor, Ultroser-G supplemented medium. The possible impairment of the same, or closely related, events occurring in blastic transformation and in rapid proliferation of cells is pointed out. Our results raise the question of the possible use of these compounds for an antitumor strategy.     

5-hydroxylation of benzimidazole by micromycetes II. Optimisation of production with Absidia spinosa

Summary The bioconversion of benzimidazole to 5-hydroxybenzimidazole was investigated as a function of the physiological state of the fungus and culture conditions. Using an inoculum composed only of spores, the addition of benzimidazole at different times of development led to a better definition of optimal physiological conditions for obtaining a good hydroxylation yield. The relationship between the degree of hydroxylation and that of culture medium aeration, was shown by the use of four different conditions: flowing medium on an inert support, in static, agitated or highly aerated medium.     

Adult ovaries of Locusta migratoria contain the sequence of biosynthetic intermediates for ecdysone

Ovaries of adult $\text{Locusta migratoria}$ have recently been shown to produce impressive amounts of ecdysone together with low polarity ecdysteroids, some of which cross-react with ecdysone in our RIA. A gas chromatographic-mass spectrometric analysis of extracts from ovaries of $\text{Locusta}$ has shown the presence of following compounds (less polar than ecdysone): 2-deoxy-ecdysone, 2,22-bis-deoxy-ecdysone, 2, 22, 2 5-tri-deoxy-ecdysone, 2, 14, 22, 2 5-tetra-deoxy-ecdysone. No other related ecdysteroids were present in our extracts. Cholesterol is used by $\text{Locusta}$ ovaries as a precursor for ecdysone biosynthesis, as our previous studies with labelled products have shown, and we propose that the compounds detected in the present work represent biosynthetic intermediates between cholesterol and ecdysone in $\text{Locusta}$ ovaries.     

Full length cDNA structure and deduced amino acid sequence of human 3 beta-hydroxy-5-ene steroid dehydrogenase

Polyclonal antibodies raised against 3 beta-hydroxysteroid dehydrogenase isolated from human placenta were used to screen a lambda gt11 expression cDNA library from the same tissue. The protein deduced from cDNA sequences contains 372 amino acids with a calculated mol wt of 42,216. Since 3 beta-hydroxysteroid dehydrogenase is the enzyme catalyzing the formation of all classes of hormonal steroids, the availability of the cDNA encoding this enzyme opens new possibilities for a detailed investigation of the factors regulating the expression and activity of this crucial enzyme in adrenal, gonadal as well as peripheral tissues.     

A protocol for rapid generation of recombinant adenoviruses using the AdEasy system

Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We have developed an approach that simplifies the generation and production of such viruses called the AdEasy system. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, employing homologous recombination in bacteria rather than in eukaryotic cells. After transfection of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of GFP encoded by a gene incorporated into the viral backbone. This system has expedited the process of generating and testing recombinant adenoviruses for a variety of purposes. In this protocol, we describe the practical aspects of using the AdEasy system for generating recombinant adenoviruses. The full protocol usually takes 4-5 weeks to complete.     

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.