Antioxidant effects of an aqueous extract of Ilex paraguariensis

In this work we investigate the antioxidant properties of an aqueous extract prepared from an infusion of Ilex paraguariensis (Aquifoliaceae) using free radical-generating systems. The extract inhibited the enzymatic and nonenzymatic lipid peroxidation in rat liver microsomes in a concentration-dependent fashion, with IC(50) values of 18 microg/ml and 28 microg/ml, respectively. The extract also inhibited the H(2)O(2)-induced peroxidation of red blood cell membranes with an IC(50) of 100 microg/ml and exhibited radical scavenging properties toward superoxide anion (IC(50) = 15 microg/ml) and 2,2-diphenyl-1-picrylhydrazyl radical. In the range of concentrations used, the extract was not a scavenger of the hydroxyl radical. Our results suggest that ingestion of extracts of Ilex paraguariensis could contribute to increase the antioxidant defense of an organism against free radicals attack.     

Plasma membrane coating with cationic silica particles and osmotic shock alters the morphology of bovine aortic endothelial cells

We have used a published method of membrane preparation based on the precoating of the apical membrane of aortic endothelial cells with cationic silica microbeads (with or without polyacrylic acid) in combination with an osmotic shock and mechanical shearing to isolate the apical from the basal plasma membranes of these cells, in vitro. After labeling of the plasma membrane of adherent endothelial cells with a fluorescent derivative of phosphatidylcholine and by using laser confocal fluorescence scanning microscopy, we found that this method of membrane isolation rapidly induced invaginations of the basal plasma membrane to an extent which makes this method unsuitable for further membrane lipid analysis. Morphological analysis of the cells and fluorescence recovery after photobleaching experiments on the plasma membranes were performed at each step of the purification procedure and showed that only hypotonic shock and mechanical shearing of the cells enabled the basal plasma membranes to be purified without significant morphological changes.     

Function and Expression of CD44 during Spreading, Migration, and Invasion of Murine Carcinoma Cells

The cell surface glycoprotein CD44 is proposed as a main participant in cell adhesion and migration. We studied the function, expression, and distribution of CD44 in the invasive and metastatic F3II murine carcinoma cell line during adhesion, spreading, migration, and invasion. A mAb anti-CD44 (KM 201) dramatically blocked F3II cell adhesion on both plastic and hyaluronic acid coatings, as well as spreading on uncoated plastic surfaces (P< 0.01). KM201 mAb significantly inhibited F3II cell migration and invasion in Transwell chambers. Immunocytochemistry of spreading cells revealed that CD44 distributed in bands on the cell surface, particularly in the tip of leading edges and in the perinuclear zones of the cell membrane. CD44 antigen was never detected in filopodia or lamellipodia nor in focal adhesion-like structures, but was also detectable as strong interlamellar bands. Fully spread cells showed a decreased CD44 signal compared to cells in early stages of spreading. This decrease correlated with a reduced expression of CD44 as detected by Western blot. We also investigated the signals that may regulate CD44 expression in F3II cells. Treatment of F3II cells, with phorbol myristate acetate (PMA) or phosphatidic acid (PA, the product of PLD-dependent hydrolysis of phosphatidylcholine), significantly enhanced CD44 expression. Conversely, the treatment of F3II cells with H7, a specific PKC inhibitor, or propranolol, which blocks PA conversion to DAG, significantly decreased CD44 expression levels. These results suggest the involvement of PKC and PLD pathways in CD44 expression. These results demonstrate that CD44 plays an important role during F3II cells adhesion, spreading, migration, and invasion. In addition we provide information linking the PLD- and PKC-dependent pathways with the regulation of CD44 expression.     

Cytoskeletal Polarization of T Cells Is Regulated by an Immunoreceptor Tyrosine-based Activation Motif–dependent Mechanism

Abstract. Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell–cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex–dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-ζ and CD3ε, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-ζ and once in CD3ε, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling requirements.     

Two different primate species express an identical functional MHC class I allele

 The products of the highly polymorphic and variable major histocompatibility complex (MHC) class I loci play a crucial role in host defenses against infectious disease. While similar alleles have been found in closely related species, sharing of a functional MHC class I allele between two species has never been reported. Here we show that an identical functional MHC class I molecule is present in two different primate species with an approximate divergence time of 0.7 million years. Lymphocytes from the red-crested tamarin (Saguinus geoffroyi) expressed an MHC class I allele (Sage-G ^* 01) that was identical in coding sequence to an MHC class I allele (Saoe-G ^* 08) found in the cotton-top tamarin (Saguinus oedipus). Furthermore, influenza virus-specific cytotoxic T lymphocytes (CTLs) generated in the cotton-top tamarin killed lymphocytes expressing the influenza virus nucleoprotein (NP) from the red-crested tamarin. Since the influenza virus NP epitope is bound by Saoe-G^*08 in the cotton-top tamarin, it is likely that this molecule is functional in both species. These data provide the first evidence that functional MHC class I molecules can be maintained entirely intact in two separate species. Received: 6 June 1997 / Revised: 21 July 1997     

Identification of a novel gene involved in pilin glycosylation in Neisseria meningitidis

The pili of Neisseria meningitidis are a key virulence factor, being major adhesins of this capsulate organism that contribute to specificity for the human host. Recently it has been reported that meningococcal pili are post-translationally modified by the addition of an O-linked trisaccharide, Gal (β1–4) Gal (α1–3) 2,4-diacetimido-2,4,6-trideoxyhexose. Using a set of random genomic sequences from N. meningitidis strain MC58, we have identified a novel gene homologous to a family of glycosyltransferases. A plasmid clone containing the gene was isolated from a genomic library of N. meningitidis strain MC58 and its nucleotide sequence determined. The clone contained a complete copy of the gene, here designated pglA (pilin glycosylation). Insertional mutations were constructed in pglA in a range of meningococcal strains with well-defined lipopolysaccharide (LPS) or pilin-linked glycan structures to determine whether pglA had a role in the biosynthesis of these molecules. There was no alteration in the phenotype of LPS from pglA mutant strains as judged by gel migration and the binding of monoclonal antibodies. In contrast, decreased gel migration of the pilin subunit molecules of pglA mutants was observed, which was similar to the migration of pilins of galE mutants of same strains, supporting the notion that pglA is a glycosyltransferase involved in the biosynthesis of the pilin-linked trisaccharide structure. The pglA mutation, like the galE mutation reported previously, had no effect on pilus-mediated adhesion to human epithelial or endothelial cells. Pilin from pglA mutants were unable to bind to monospecific antisera recognizing the Gal (β1–4) Gal structure, suggesting that PglA is a glycosyltransferase involved in the addition of galactose of the trisaccharide substituent of pilin.     

Circulating Insulin Concentrations,Smoking, and Alcohol Intake Are Important Independent Predictors of Leptin in Young Healthy Men

Objective : Leptin, an adipocyte-secreted hormone, has been shown to signal the status of energy stores to the brain, regulate energy homeostasis, and mediate the neuroendocrine response to food deprivation. Obesity is associated with increased leptin levels, and several hormones, including insulin and glucocorticoids, have been associated with leptin levels and expression in rodents. Although obesity has been strongly associated with increased leptin in humans, a significant percentage of leptin's variability remains unexplained. The role of endogenous hormones, demographic factors, or certain life-style factors in explaining the residual variability of leptin levels has not yet been clarified. We performed this cross-sectional study to document the relative importance of obesity, lifestyle factor, and endogenous hormones in determining serum leptin levels. Research Methods and Procedures : We measured serum concentrations of insulin, Cortisol, testosterone, growth hormone, and dehydroepiandrosterone sulfate; ascertained anthropometric, demographic, and lifestyle characteristics; and studied these variables in relationship to serum leptin concentrations in a sample of young healthy men. Results : Obesity and alcohol intake were independently and positively associated with circulating leptin concentrations. Additionally, cigarette smoking was negatively and independently associated with leptin concentrations. Finally, serum insulin concentration was an independent hormonal determinant of circulating leptin concentrations, whereas serum testosterone was negatively associated with leptin only by bivariate analysis. Discussion : We conclude that, in addition to obesity, cigarette smoking, alcohol intake, and serum insulin levels are associated with leptin levels in a population of healthy young men.     

U-richness is a defining feature of plant introns and may function as an intron recognition signal in maize

Using a large set of plant gene sequences we compared individual introns to their flanking exons. Both Zea mays and Arabidopsis thaliana introns are U-rich but display no apparent bias for A. We identified fifteen 11-mer U-rich motifs as frequent elements of maize introns, and these are virtually absent from exons. By mutagenesis, we show that the single U-rich motif in the Bronze2 intron of maize plays a key role in intron processing in vivo.     

Spatial patterns in phage-Rhizobium coevolutionary interactions across regions of common bean domestication

Bacteriophages play significant roles in the composition, diversity, and evolution of bacterial communities. Despite their importance, it remains unclear how phage diversity and phage-host interactions are spatially structured. Local adaptation may play a key role. Nitrogen-fixing symbiotic bacteria, known as rhizobia, have been shown to locally adapt to domesticated common bean at its Mesoamerican and Andean sites of origin. This may affect phage-rhizobium interactions. However, knowledge about the diversity and coevolution of phages with their respective Rhizobium populations is lacking. Here, through the study of four phage-Rhizobium communities in Mexico and Argentina, we show that both phage and host diversity is spatially structured. Cross-infection experiments demonstrated that phage infection rates were higher overall in sympatric rhizobia than in allopatric rhizobia except for one Argentinean community, indicating phage local adaptation and host maladaptation. Phage-host interactions were shaped by the genetic identity and geographic origin of both the phage and the host. The phages ranged from specialists to generalists, revealing a nested network of interactions. Our results suggest a key role of local adaptation to resident host bacterial communities in shaping the phage genetic and phenotypic composition, following a similar spatial pattern of diversity and coevolution to that in the host.Subject terms: Microbial ecology, Bacteriophages, Microbial ecology, Biogeography, Microbial communities     

Metazoans of redoxcline sediments in Mediterranean deep-sea hypersaline anoxic basins

Background

The deep-sea hypersaline anoxic basins (DHABs) of the Mediterranean (water depth ~3500 m) are some of the most extreme oceanic habitats known. Brines of DHABs are nearly saturated with salt, leading many to suspect they are uninhabitable for eukaryotes. While diverse bacterial and protistan communities are reported from some DHAB haloclines and brines, loriciferans are the only metazoan reported to inhabit the anoxic DHAB brines. Our goal was to further investigate metazoan communities in DHAB haloclines and brines.

Results

We report observations from sediments of three DHAB (Urania, Discovery, L’Atalante) haloclines, comparing these to observations from sediments underlying normoxic waters of typical Mediterranean salinity. Due to technical difficulties, sampling of the brines was not possible. Morphotype analysis indicates nematodes are the most abundant taxon; crustaceans, loriciferans and bryozoans were also noted. Among nematodes, Daptonema was the most abundant genus; three morphotypes were noted with a degree of endemicity. The majority of rRNA sequences were from planktonic taxa, suggesting that at least some individual metazoans were preserved and inactive. Nematode abundance data, in some cases determined from direct counts of sediments incubated in situ with CellTracker^TM Green, was patchy but generally indicates the highest abundances in either normoxic control samples or in upper halocline samples; nematodes were absent or very rare in lower halocline samples. Ultrastructural analysis indicates the nematodes in L’Atalante normoxic control sediments were fit, while specimens from L’Atalante upper halocline were healthy or had only recently died and those from the lower halocline had no identifiable organelles. Loriciferans, which were only rarely encountered, were found in both normoxic control samples as well as in Discovery and L’Atalante haloclines. It is not clear how a metazoan taxon could remain viable under this wide range of conditions.

Conclusions

We document a community of living nematodes in normoxic, normal saline deep-sea Mediterranean sediments and in the upper halocline portions of the DHABs. Occurrences of nematodes in mid-halocline and lower halocline samples did not provide compelling evidence of a living community in those zones. The possibility of a viable metazoan community in brines of DHABs is not supported by our data at this time.