Improved access to 2-O-monobenzyl ethers of beta-cyclodextrin as precursors of catalysts for organophosphoryl esters hydrolysis

A comparative study of reaction conditions was performed for the synthesis of a 2-O-monobenzyl ether of cyclomaltoheptaose (beta-CD). Optimal conditions involved sodium ethoxide in Me(2)SO and benzyl bromide. The methodology was extended to the preparation of various 2(I)-O-iodobenzyl and 2(I)-O-carboxymethylbenzyl derivatives of beta-CD including a 3-carboxymethyl-4-iodobenzyl derivative of interest as precursor of an enzyme mimic to degrade the organophosphoryl ester diethyl 4-nitrophenyl phosphate (paraoxon).     

Epigenetic status of the H19 locus in human oocytes following in vitro maturation

Imprinting is an epigenetic modification that is reprogrammed in the germ line and leads to the monoallelic expression of some genes. Imprinting involves DNA methylation. Maternal imprint is reset during oocyte growth and maturation. In vitro maturation (IVM) of oocytes may, therefore, interfere with imprint acquisition and/or maintenance. To evaluate if maturing human oocytes in vitro would be hazardous at the epigenetic level, we first determined the methylation profile of the H19 differentially methylated region (DMR). The methylation status of the H19 DMR seems particularly vulnerable to in vitro culture conditions. We analyzed oocytes at different stages of maturation following IVM, germinal vesicle (GV), metaphase I (MI), and metaphase II (MII), using the bisulfite mutagenesis technique. Our results indicated that the unmethylated specific maternal profile for the H19 DMR was stably established at the GV stage. The majority of MI-arrested oocytes exhibited an altered pattern of methylation, the CTCF-binding site being methylated in half of the DNA strands analyzed. Of the 20 MII oocytes analyzed, 15 showed the normal unmethylated maternal pattern, while 5 originating from two different patients exhibited a methylated pattern. These findings highlight the need for extended analysis on MII-rescued oocytes to appreciate the epigenetic safety of the IVM procedure, before it becomes a routine and practical assisted reproductive procedure.     

Chloride salinity reduces cadmium accumulation by the Mediterranean halophyte species Atriplex halimus L.

Soil salinity usually increases bioavailability of Cd on heavy metal polluted soils but its impact on Cd absorption and accumulation by plants remains largely unknown. Plants from the halophyte species Atriplex halimus were therefore exposed for 12 and 14 days to nutrient solution containing 50 μM CdCl₂ in the presence of NaCl, KCl or NaNO₃ 50 mM. Most Cd present in solution remained as Cd–EDTA and salinity had no impact on Cd speciation. Chloride salinity (NaCl and KCl) reduced Cd accumulation in shoots and roots while NaNO₃ increased Cd accumulation in leaves. More than 30% of accumulated Cd was found at the leaf surface and accumulated in trichomes but all tested salts decreased the proportion of excreted Cd. Cadmium induced a decrease in the leaf water content. External NaCl and KCl mitigated the deleterious impact of Cd by inducing osmotic adjustment while NaNO₃ and synthesis of protecting compounds such as soluble sugars and glycinebetaine. Free polyamines (putrescine, spermidine and spermine) increased in response to Cd, Cd + NaCl and Cd + KCl while only putrescine increased in response to Cd + NaNO₃. Proline exhibited maximal concentration in the leaves of Cd + NaCl and Cd + KCl-treated plants and was correlated with osmotic adjustment. Our results suggest that chloride salinity improved the resistance of A. halimus to Cd toxicity both by decreasing the absorption of heavy metal and by improving tissular tolerance through an increase in the synthesis of osmoprotective compounds.     

Lactococcus lactis Expressing either Staphylococcus aureus Fibronectin-Binding Protein A or Listeria monocytogenes Internalin A Can Efficiently Internalize and Deliver DNA in Human Epithelial Cells

Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA⁺), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA⁺ and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA⁺) or its fibronectin binding domains C and D (L. lactis CD⁺). L. lactis FnBPA⁺ and L. lactis InlA⁺ showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD⁺ was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA⁺, L. lactis CD⁺, and L. lactis InlA⁺ were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA⁺, L. lactis CD⁺, and L. lactis InlA⁺ were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA⁺ or L. lactis InlA⁺. Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.The mucosal administration of bacterial carriers to deliver antigens and plasmid DNA constitutes a promising vaccination strategy. Pathogenic bacteria that have the capacity to invade cells, such as Listeria, Salmonella, and Shigella strains, have been used to deliver DNA constructs into mammalian cells (23). Nevertheless, the risk associated with possible reversion to a virulent phenotype of these pathogens is a major concern (5). Lactococcus lactis, the food-grade, gram-positive, noninvasive model bacterium, has been intensively used to deliver antigens and cytokines at the mucosal level (30). We previously showed (i) that native L. lactis can deliver a eukaryotic expression cassette coding for the bovine β-lactoglobulin (BLG), one of the major cow''s milk allergens, into mammalian epithelial Caco-2 cells, and (ii) that these cells were able to express and secrete BLG protein in its native conformation (10). Recently, we demonstrated the ability of native noninvasive L. lactis to deliver a fully functional plasmid to murine intestinal cells in vivo (2).The internalization of the bacterial carrier is a fundamental step to achieve efficient DNA delivery in eukaryotic cells (7). In order to increase DNA delivery by lactic acid bacteria (LAB), invasin genes were expressed in L. lactis. Due to the safety profile of LAB, recombinant lactococci expressing invasin genes from intracellular bacteria are attractive as potential DNA delivery vectors compared to the attenuated pathogens presently used.In this field, we previously demonstrated that L. lactis bacteria expressing the main Listeria monocytogenes invasin, internalin A (L. lactis InlA⁺), were able to invade eukaryotic cells and efficiently deliver a functional green fluorescent protein (GFP) expression plasmid into epithelial/endothelial cells (9). Even though attractive, the experimental use of lactococci expressing InlA in a mouse model has a major bottleneck: InlA, which binds to human E-cadherin (15), does not interact with murine E-cadherin. Consequently, in vivo experimental studies using lactococci expressing InlA as DNA delivery vehicles are limited to transgenic mice expressing human E-cadherin or to guinea pigs (13).Fibronectin binding protein A (FnBPA) of Staphylococcus aureus is another bacterial invasin that is involved in intracellular spreading of S. aureus in the host (27). It is a multifunctional adhesion protein having both fibrinogen and fibronectin binding capacities (24). Its N-terminal part, also called domain A, is responsible for fibrinogen (29) and elastin (20) binding, whereas its C-terminal part, including domains B, C, and D, binds to fibronectin (25). FnBPA is known to mediate adhesion to host tissue and bacterial uptake into nonphagocytic host cells (27). Its expression by L. lactis was previously shown to be sufficient to confer the ability to invade nonphagocytic cells in vitro and in vivo, while the expression of domains C and D confers invasivity only in vitro (19).In this study, we show that L. lactis bacteria expressing full-length FnBPA of S. aureus (L. lactis FnBPA⁺) or a truncated form encompassing only its C and D domains (L. lactis CD⁺) are internalized as efficiently as L. lactis InlA⁺ in the human intestinal cell line Caco-2. We also provide, for the first time, direct microscopic evidence of the intracellular location of the internalized lactococci, showing that the bacteria are heterogeneously distributed in the cell monolayer and that their number per cell can reach a surprisingly high level. However, we demonstrate that FbpA, a fibronectin binding protein from the commensal Lactobacillus acidophilus NCFM, does not mediate bacterial internalization: no difference in invasivity was observed between the wild-type (wt) strain and the mutant with fbpA inactivated. This result indicates that, although widely distributed among bacteria, fibronectin binding proteins are not universal mediators of bacterial internalization, even at low levels. Finally, we also demonstrate that, similarly to L. lactis InlA⁺, L. lactis FnBPA⁺ and L. lactis CD⁺ can efficiently deliver a eukaryotic GFP expression plasmid in Caco-2 cells and trigger GFP expression in these cells. Consequently, L. lactis FnBPA⁺ can be used for further DNA delivery experiments in vivo.     

Determination of the nucleotide sequence of the 23S ribosomal RNA and flanking spacers of an Enterococcus faecium strain, reveals insertion-deletion events in the ribosomal spacer 1 of enterococci

The usefulness of 16S-23S (ITS1) and 23S-5S (ITS2) ribosomal spacer nucleotide sequence determination, as a complementary approach to the biochemical tests traditionally used for enterococcal species identification, is shown by its application to the identification of a strain, E27, isolated from a natural bacteria mixture used for cheese production. Using combined approaches we showed, unambiguously, that strain E27 belongs to the Enterococcus faecium species. However, its ITS1 region has an interesting peculiarity. In our previous study of ITS1s from various enterococcal species (NAIMI et al., 1997, Microbiology 143, 823-834), the ITS1s of the two E. faecium strains studied, were found to contain an additional 115-nt long stem-loop structure as compared to the ITS1s of other enterococci, only one out of the 3 ITS1s of E. hirae ATCC 9790, was found to contain a similar 107-nt long stem-loop structure. The ITS1 of strain E27 is 100% identical to that of E. faecium ATCC 19434T, except that the 115-nt additional fragment is absent. This strongly suggests the existence of lateral DNA transfer or DNA recombination events at a hot spot position of the ITS1s from E. faecium and E. hirae. Small and large ITS1 nucleotide sequence determination for strain E27 generalized the notion of two kinds of ITSs in enterococci: one with a tRNA(Ala) gene, one without tRNA gene. To complete strain E27 characterization, its 23S rRNA sequence was established. This is the first complete 23S rRNA nucleotide sequence determined for an enterococcal species.     

Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts

Introduction  

Rheumatoid arthritis synovial fibroblasts (RASF) are key players in synovial pathophysiology and are therefore examined extensively in various experimental approaches. We evaluated, whether passaging during culture and freezing has effects on gene expression and cell proliferation.     

PPARγ Ligands Switched High Fat Diet-Induced Macrophage M2b Polarization toward M2a Thereby Improving Intestinal Candida Elimination

Obesity is associated with a chronic low-grade inflammation that predisposes to insulin resistance and the development of type 2 diabetes. In this metabolic context, gastrointestinal (GI) candidiasis is common. We recently demonstrated that the PPARγ ligand rosiglitazone promotes the clearance of Candida albicans through the activation of alternative M2 macrophage polarization. Here, we evaluated the impact of high fat diet (HFD)-induced obesity and the effect of rosiglitazone (PPARγ ligand) or WY14643 (PPARα ligand) both on the phenotypic M1/M2 polarization of peritoneal and cecal tissue macrophages and on the outcome of GI candidiasis. We demonstrated that the peritoneal macrophages and the cell types present in the cecal tissue from HF fed mice present a M2b polarization (TNF-α^high, IL-10^high, MR, Dectin-1). Interestingly, rosiglitazone induces a phenotypic M2b-to-M2a (TNF-α^low, IL-10^low, MR^high, Dectin-1^high) switch of peritoneal macrophages and of the cells present in the cecal tissue. The incapacity of WY14643 to switch this polarization toward M2a state, strongly suggests the specific involvement of PPARγ in this mechanism. We showed that in insulin resistant mice, M2b polarization of macrophages present on the site of infection is associated with an increased susceptibility to GI candidiasis, whereas M2a polarization after rosiglitazone treatment favours the GI fungal elimination independently of reduced blood glucose. In conclusion, our data demonstrate a dual benefit of PPARγ ligands because they promote mucosal defence mechanisms against GI candidiasis through M2a macrophage polarization while regulating blood glucose level.     

Antigen receptor engagement selectively induces macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta chemokine production in human B cells

We show herein that B cell Ag receptor (BCR) triggering, but not stimulation by CD40 mAb and/or IL-4, rapidly induced the coordinated expression of two closely related T cell chemoattractants, macrophage inflammatory protein-1 beta (MIP-1 beta) and MIP-1 alpha, by human B cells. Naive, memory, and germinal center B cells all produced MIP-1 alpha/beta in response to BCR triggering. In contrast to MIP-1 alpha/beta, IL-8, which is spontaneously produced by germinal center B cells but not by naive and memory B cells, was not regulated by BCR triggering. Culturing follicular dendritic cell-like HK cells with activated B cells did not regulate MIP-1 alpha/beta production, but it did induce production of IL-8 by HK cells. Microchemotaxis assays showed that CD4+CD45RO+ T cells of the effector/helper phenotype actively migrated along a chemotactic gradient formed by BCR-stimulated B cells. This effect was partially blocked by anti-MIP-1 beta and anti-CC chemokine receptor 5 Ab, but not by anti-MIP-1 alpha Ab suggesting that MIP-1 beta plays a major role in this chemoattraction. Since maturation of the B cell response to a peptide Ag is mostly dependent on the availability of T cell help, the ability of Ag-stimulated B cells to recruit T cells via MIP-1 alpha/beta, may represent one possible mechanism enabling cognate interactions between rare in vivo Ag-specific T and B cells.     

Age and gonadotropins control Ca2+-spike acquisition in mouse oocytes isolated from early preantral follicles

The action of gonadotropins upon the oocyte is known to be crucial at later stages of follicular development in mammals. However, its influence on oocytes at early preantral stages is still a matter of debate. In the present study we evaluated the onset of mouse oocyte's capacity to exhibit calcium spikes during preantral stages of follicular development, prior to meiotic competence acquisition. In particular, through the use of confocal microscopy, we probed for the specific effects of age and gonadotropin stimulation upon the calcium dynamics of preantral follicle oocytes. We found that important developmental changes on the Ca2+ signalling mechanisms take place early during follicular development. Specifically we demonstrate that both age and gonadotropin stimulation increase the capacity of oocytes recovered from preantral follicles to exhibit calcium spikes. We propose that a strictly morphological staging of follicular development is insufficient to predict oocyte behaviour and must take in consideration animal age and gonadotropin environment.     

A burial cave in the western Aleutian Islands, Alaska

During the 1998 field season, the Western Aleutians Archaeological and Paleobiological Project (WAAPP) team located a cave in the Near Islands, Alaska. Near the entrance of the cave, the team identified work areas and sleeping/sitting areas surrounded by cultural debris and animal bones. Human burials were found in the cave interior. In 2000, with permission from The Aleut Corporation, archaeologists revisited the site. Current research suggests three distinct occupations or uses for this cave. Aleuts buried their dead in shallow graves at the rear of the cave circa 1,200 to 800 years ago. Aleuts used the front of the cave as a temporary hunting camp as early as 390 years ago. Finally, Japanese and American military debris and graffiti reveal that the cave was visited during and after World War II. Russian trappers may have also taken shelter there 150 to 200 years ago. This is the first report of Aleut cave burials west of the Delarof Islands in the central Aleutians.