An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Complex regulation of nucleoside transporter expression in epithelial and immune system cells

Nucleoside transporters have a variety of functions in the cell, such as the provision of substrates for nucleic acid synthesis and the modulation of purine receptors by determining agonist availability. They also transport a wide range of nucleoside-derived antiviral and anticancer drugs. Most mammalian cells co-express several nucleoside transporter isoforms at the plasma membrane, which are differentially regulated. This paper reviews studies on nucleoside transporter regulation, which has been extensively characterized in the laboratory in several model systems: the hepatocyte, an epithelial cell type, and immune system cells, in particular B cells, which are non-polarized and highly specialized. The hepatocyte co-expresses at least two Na+-dependent nucleoside transporters, CNT1 and CNT2, which are up-regulated during cell proliferation but may undergo selective loss in certain experimental models of hepatocarcinomas. This feature is consistent with evidence that CNT expression also depends on the differentiation status of the hepatocyte. Moreover, substrate availability also modulates CNT expression in epithelial cells, as reported for hepatocytes and jejunum epithelia from rats fed nucleotide-deprived diets. In human B cell lines, CNT and ENT transporters are co-expressed but differentially regulated after B cell activation triggered by cytokines or phorbol esters, as described for murine bone marrow macrophages induced either to activate or to proliferate. The complex regulation of the expression and activity of nucleoside transporters hints at their relevance in cell physiology.     

Tumor necrosis factor-alpha stimulates focal adhesion kinase activity required for mitogen-activated kinase-associated interleukin 6 expression

Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that promotes cell migration, survival, and gene expression. Here we show that FAK signaling is important for tumor necrosis factor-alpha (TNFalpha)-induced interleukin 6 (IL-6) mRNA and protein expression in breast (4T1), lung (A549), prostate (PC-3), and neural (NB-8) tumor cells by FAK short hairpin RNA knockdown and by comparisons of FAK-null (FAK(-/-)) and FAK(+/+) mouse embryo fibroblasts. FAK promoted TNFalpha-stimulated MAPK activation needed for maximal IL-6 production. FAK was not required for TNFalpha-mediated nuclear factor-kappaB or c-Jun N-terminal kinase activation. TNFalpha-stimulated FAK catalytic activation and IL-6 production were inhibited by FAK N-terminal but not FAK C-terminal domain overexpression. Analysis of FAK(-/-) fibroblasts stably reconstituted with wild type or various FAK point mutants showed that FAK catalytic activity, Tyr-397 phosphorylation, and the Pro-712/713 proline-rich region of FAK were required for TNFalpha-stimulated MAPK activation and IL-6 production. Constitutively activated MAPK kinase-1 (MEK1) expression in FAK(-/-) and A549 FAK short hairpin RNA-expressing cells rescued TNFalpha-stimulated IL-6 production. Inhibition of Src protein-tyrosine kinase activity or mutation of Src phosphorylation sites on FAK (Tyr-861 or Tyr-925) did not affect TNFalpha-stimulated IL-6 expression. Moreover, analyses of Src(-/-), Yes(-/-), and Fyn(-/-) fibroblasts showed that Src expression was inhibitory to TNFalpha-stimulated IL-6 production. These studies provide evidence for a novel Src-independent FAK to MAPK signaling pathway regulating IL-6 expression with potential importance to inflammation and tumor progression.     

Complex regulation of nucleoside transporter expression in epithelial and immune system cells

Nucleoside transporters have a variety of functions in the cell, such as the provision of substrates for nucleic acid synthesis and the modulation of purine receptors by determining agonist availability. They also transport a wide range of nucleoside-derived antiviral and anticancer drugs. Most mammalian cells coexpress several nucleoside transporter isoforms at the plasma membrane, which are differentially regulated. This paper reviews studies on nucleoside transporter regulation, which has been extensively characterized in the laboratory in several model systems: the hepatocyte, an epithelial cell type, and immune system cells, in particular B cells, which are non-polarized and highly specialized. The hepatocyte co-expresses at least two Na+-dependent nucleoside transporters, CNT1 and CNT2, which are up-regulated during cell proliferation but may undergo selective loss in certain experimental models of hepatocarcinomas. This feature is consistent with evidence that CNT expression also depends on the differentiation status of the hepatocyte. Moreover, substrate availability also modulates CNT expression in epithelial cells, as reported for hepatocytes and jejunum epithelia from rats fed nucleotide-deprived diets. In human B cell lines, CNT and ENT transporters are co-expressed but differentially regulated after B cell activation triggered by cytokines or phorbol esters, as described for murine bone marrow macrophages induced either to activate or to proliferate. The complex regulation of the expression and activity of nucleoside transporters hints at their relevance in cell physiology.     

Calorie restriction reduces biomarkers of cellular senescence in humans

Calorie restriction (CR) with adequate nutrient intake is a potential geroprotective intervention. To advance this concept in humans, we tested the hypothesis that moderate CR in healthy young-to-middle-aged individuals would reduce circulating biomarkers of cellular senescence, a fundamental mechanism of aging and aging-related conditions. Using plasma specimens from the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE™) phase 2 study, we found that CR significantly reduced the concentrations of several senescence biomarkers at 12 and 24 months compared to an ad libitum diet. Using machine learning, changes in biomarker concentrations emerged as important predictors of the change in HOMA-IR and insulin sensitivity index at 12 and 24 months, and the change in resting metabolic rate residual at 12 months. Finally, using adipose tissue RNA-sequencing data from a subset of participants, we observed a significant reduction in a senescence-focused gene set in response to CR at both 12 and 24 months compared to baseline. Our results advance the understanding of the effects of CR in humans and further support a link between cellular senescence and metabolic health.     

Arsenic Exposure Increases Monocyte Adhesion to the Vascular Endothelium,a Pro-Atherogenic Mechanism

Epidemiological studies have shown that arsenic exposure increases atherosclerosis, but the mechanisms underlying this relationship are unknown. Monocytes, macrophages and platelets play an important role in the initiation of atherosclerosis. Circulating monocytes and macrophages bind to the activated vascular endothelium and migrate into the sub-endothelium, where they become lipid-laden foam cells. This process can be facilitated by platelets, which favour monocyte recruitment to the lesion. Thus, we assessed the effects of low-to-moderate arsenic exposure on monocyte adhesion to endothelial cells, platelet activation and platelet-monocyte interactions. We observed that arsenic induces human monocyte adhesion to endothelial cells in vitro. These findings were confirmed ex vivo using a murine organ culture system at concentrations as low as 10 ppb. We found that both cell types need to be exposed to arsenic to maximize monocyte adhesion to the endothelium. This adhesion process is specific to monocyte/endothelium interactions. Hence, no effect of arsenic on platelet activation or platelet/leukocyte interaction was observed. We found that arsenic increases adhesion of mononuclear cells via increased CD29 binding to VCAM-1, an adhesion molecule found on activated endothelial cells. Similar results were observed in vivo, where arsenic-exposed mice exhibit increased VCAM-1 expression on endothelial cells and increased CD29 on circulating monocytes. Interestingly, expression of adhesion molecules and increased binding can be inhibited by antioxidants in vitro and in vivo. Together, these data suggest that arsenic might enhance atherosclerosis by increasing monocyte adhesion to endothelial cells, a process that is inhibited by antioxidants.     

Kinetics of Anti-Phlebotomus perniciosus Saliva Antibodies in Experimentally Bitten Mice and Rabbits

Background

Sand flies are hematophagous arthropods that act as vectors of Leishmania parasites. When hosts are bitten they develop cellular and humoral responses against sand fly saliva. A positive correlation has been observed between the number of bites and antibody levels indicating that anti-saliva antibody response can be used as marker of exposure to sand flies. Little is known about kinetics of antibodies against Phlebotomus perniciosus salivary gland homogenate (SGH) or recombinant salivary proteins (rSP). This work focused on the study of anti-P. perniciosus saliva antibodies in sera of mice and rabbits that were experimentally exposed to the bites of uninfected sand flies.

Methodology/Principal Findings

Anti-saliva antibodies were evaluated by ELISA and Western blot. In addition, antibody levels against two P. perniciosus rSP, apyrase rSP01B and D7 related protein rSP04 were determined in mice sera. Anti-saliva antibody levels increased along the immunizations and correlated with the number of sand fly bites. Anti-SGH antibody levels were detected in sera of mice five weeks after exposure, and persisted for at least three months. Anti-apyrase rSP01B antibodies followed similar kinetic responses than anti-SGH antibodies while rSP04 showed a delayed response and exhibited a greater variability among sera of immunized mice. In rabbits, anti-saliva antibodies appeared after the second week of exposure and IgG antibodies persisted at high levels, even 7 months post-exposure.

Conclusions/Significance

Our results contributed to increase the knowledge on the type of immune response P. perniciosus saliva and individual proteins elicited highlighting the use of rSP01B as an epidemiological marker of exposure. Anti-saliva kinetics in sera of experimentally bitten rabbits were studied for the first time. Results with rabbit model provided useful information for a better understanding of the anti-saliva antibody levels found in wild leporids in the human leishmaniasis focus in the Madrid region, Spain.     

The cylindrical or tubiliform glands of Nephila clavipes

The cylindrical or tubiliform glands of the spider Nephila clavipes have been studied and compared to the large ampullates on which we have previously reported. The three pairs of cylindrical or tubiliform glands secrete the fibroin for the organism's egg case. Their solubilized luminar contents migrate as a homogeneous band in Sodium dodecyl sulfate polyacrylamide gel electrophoresis and turn out to be a larger protein than that produced by the large ampullates. The excised cylindrical glands remain metabolically active for several hours in a simple culture medium, where fibroin synthesis can be monitored through the incorporation of 14C alanine. The glands' response to a fibroin production stimulus does not reach the magnitude displayed by the large ampullates, but this is to be expected since their products supply different functions in this organism. This fibroin also seems to be elongated discontinuously. Translational pauses have been detected in the secretory epithelium of cylindrical and large ampullate glands of Nephila as well as in the silk glands of Bombyx mori. Since these glands produce the fibroin for the females egg case, they should prove to be an interesting model system.     

Ptr1 evolved convergently with RPS2 and Mr5 to mediate recognition of AvrRpt2 in diverse solanaceous species

The Ptr1 (Pseudomonas tomato race 1) locus in Solanum lycopersicoides confers resistance to strains of Pseudomonas syringae pv. tomato expressing AvrRpt2 and Ralstonia pseudosolanacearum expressing RipBN. Here we describe the identification and phylogenetic analysis of the Ptr1 gene. A single recombinant among 585 F2 plants segregating for the Ptr1 locus was discovered that narrowed the Ptr1 candidates to eight nucleotide‐binding leucine‐rich repeat protein (NLR)‐encoding genes. From analysis of the gene models in the S. lycopersicoides genome sequence and RNA‐Seq data, two of the eight genes emerged as the strongest candidates for Ptr1. One of these two candidates was found to encode Ptr1 based on its ability to mediate recognition of AvrRpt2 and RipBN when it was transiently expressed with these effectors in leaves of Nicotiana glutinosa. The ortholog of Ptr1 in tomato and in Solanum pennellii is a pseudogene. However, a functional Ptr1 ortholog exists in Nicotiana benthamiana and potato, and both mediate recognition of AvrRpt2 and RipBN. In apple and Arabidopsis, recognition of AvrRpt2 is mediated by the Mr5 and RPS2 proteins, respectively. Phylogenetic analysis places Ptr1 in a distinct clade compared with Mr5 and RPS2, and it therefore appears to have arisen by convergent evolution for recognition of AvrRpt2.