A 43-Nucleotide RNACis-Acting Element Governs the Site-Specific Formation of the 3′ End of a Poxvirus Late mRNA

AbstractThe 3′ ends of late mRNAs of theatigene, encoding the major component of the A-type inclusions, are generated by endoribonucleolytic cleavage at a specific site in the primary transcript [Antczaket al.,(1992),Proc. Natl. Acad. Sci. USA89, 12033–12037]. In this study, sequence analysis of cDNAs of the 3′ ends ofatimRNAs showed these mRNAs are 3′ polyadenylated at the RNA cleavage site. This suggests thatatimRNA 3′ end formation involves cleavage of a late transcript, with subsequent 3′ polyadenylation of the 5′ cleavage product. The RNAcis-acting element, the AX element, directing orientation-dependent formation of these mRNA 3′ ends, was mapped to a 345-bpAluI–XbaI fragment. Deletion analyses of this fragment showed that the boundaries of the AX element are within −5 and +38 of the RNA cleavage site. Scanning mutagenesis showed that the AX element contains at least two subelements: subelement I, 5′-UUUAU↓CCGAUAAUUC-3′, containing the cleavage site (↓), separated from the downstream subelement II, 5′-AAUUUCGGAUUUGAAUGC-3′, by a 10-nucleotide region, whose composition may be altered without effect on RNA 3′ end formation. These features, which differ from those of other elements controlling RNA processing, suggest that the AX element is a component of a novel mechanism of RNA 3′ end formation

Expression of CD44 molecules and CD44 ligands during human thymic fetal development: expression of CD44 isoforms is developmentally regulated

It has recently been recognized that CD44 comprises a large family of alternatively spliced forms.In the thymus, CD44 has been postulated to play an important role in immature T cell migration and maturation. In this paper, we have studied the expression of CD44 molecules and two CD44 ligands, hyaluronan (HA) and fibronectin (FN), during human thymic fetal development. We found that mAbs against all CD44 isoforms (A3D8 or A1G3) reacted with both thymic epithelial (TE) cells and thymocytes beginning at the time of initial colonization of the human thymus by hematopoietic stem cells at 8.2 weeks of fetal gestation. However, mAbs specific for splice variants of CD44 containing membrane-proximal inserts (11.24, 11.10 and 11.9) reacted only with terminally differentiated TE cells in and around Hassall's bodies beginning at 16-19 weeks of fetal gestation. Studies of differentiated versus undifferentiated TE cells in vitro confirmed the selective expression of CD44 variant isoforms on terminally differentiated TE cells. Expression of HA and FN was determined by fluorescence microscopy using either biotlnylated-HA binding protein or an anti-FN mAb. We found that whereas FN was present throughout the human fetal thymus beginning at 8.2 weeks, HA was not present until 16 weeks of gestational age. These data demonstrate the differential expression of standard versus variant CD44 isoforms during thymic ontogeny and implicate CD44 interactions with ligands other than HA as important in the earlier stages of humanthymus developmen

Optimization of Rolling-Circle Amplified Protein Microarrays for Multiplexed Protein Profiling

Protein microarray-based approaches are increasingly being used in research and clinical applications to either profile the expression of proteins or screen molecular interactions. The development of high-throughput, sensitive, convenient, and cost-effective formats for detecting proteins is a necessity for the effective advancement of understanding disease processes. In this paper, we describe the generation of highly multiplexed, antibody-based, specific, and sensitive protein microarrays coupled with rolling-circle signal amplification (RCA) technology. A total of 150 cytokines were simultaneously detected in an RCA sandwich immunoassay format. Greater than half of these proteins have detection sensitivities in the pg/mL range. The validation of antibody microarray with human serum indicated that RCA-based protein microarrays are a powerful tool for high-throughput analysis of protein expression and molecular diagnostics

Dual targeting of CCR2 and CX3CR1 in an arterial injury model of vascular inflammation

<p>Abstract</p> <p>Objectives</p> <p>The chemokine receptors CCR2 and CX3CR1 are important in the development of coronary artery disease. The purpose of this study is to analyze the effect of a novel CCR2 inhibitor in conjunction with CX3CR1 deletion on vascular inflammation.</p> <p>Methods</p> <p>The novel CCR2 antagonist MRL-677 was characterized using an in vivo model of monocyte migration. To determine the relative roles of CCR2 and CX3CR1 in vascular remodeling, normal or CX3CR1 deficient mice were treated with MRL-677. After 14 days, the level of intimal hyperplasia in the artery was visualized by paraffin sectioning and histology of the hind limbs.</p> <p>Results</p> <p>MRL-677 is a CCR2 antagonist that is effective in blocking macrophage trafficking in a peritoneal thioglycollate model. Intimal hyperplasia resulting from vascular injury was also assessed in mice. Based on the whole-blood potency of MRL-677, sufficient drug levels were maintained for the entire 14 day experimental period to afford good coverage of mCCR2 with MRL-677. Blocking CCR2 with MRL-677 resulted in a 56% decrease in the vascular injury response (n = 9, p < 0.05) in normal animals. Mice in which both CCR2 and CX3CR1 pathways were targeted (CX3CR1 KO mice given MRL-677) had an 88% decrease in the injury response (n = 6, p = 0.009).</p> <p>Conclusion</p> <p>In this study we have shown that blocking CCR2 with a low molecular weight antagonist ameliorates the inflammatory response to vascular injury. The protective effect of CCR2 blockade is increased in the presence of CX3CR1 deficiency suggesting that CX3CR1 and CCR2 have non-redundant functions in the progression of vascular inflammation.</p

Recycling of the Membrane-anchored Chemokine, CX 3 CL1

CX(3)CL1 (fractalkine) plays an important role in inflammation by acting as both chemoattractant and as an adhesion molecule. As for other chemokines, expression of CX(3)CL1 is known to be regulated at the level of transcription and translation. The unique transmembrane structure of CX(3)CL1 raises the possibility of additional functional regulation by altering its abundance at the cell surface. This could be accomplished in principle by changes in traffic between subcellular compartments. To analyze this possibility we examined the subcellular distribution of CX(3)CL1 in human ECV-304 cells stably expressing untagged or green fluorescent protein-tagged forms of the chemokine. CX(3)CL1 was present in two distinct compartments, diffusely on the plasma membrane and in a punctate juxtanuclear compartment. The latter shared some features with, yet was distinct from the conventional endocytic pathway and may represent a specialized recycling subcompartment. Accordingly, surface CX(3)CL1 was found to be in dynamic equilibrium with the juxtanuclear vesicular compartment. Intracellular CX(3)CL1 co-localized with the SNARE (soluble N-ethylmaleimide factor attachment protein receptor) proteins syntaxin-13 and VAMP-3. Cleavage of VAMP-3 by tetanus toxin or impairment of syntaxin-13 function by expression of a dominant-negative allele inhibited the ability of internalized CX(3)CL1 to traffic back to the plasma membrane. These data demonstrate the existence of a dynamic, SNARE-mediated recycling of CX(3)CL1 from the cell surface to and from an endomembrane storage compartment. The intracellular storage depot may serve as a source of the chemokine that could be rapidly mobilized by stimuli

Ozone enhancement of lower airway allergic inflammation is prevented by γ-tocopherol

Ozone is a commonly encountered environmental oxidant which has been linked to asthma exacerbation in epidemiological studies. Ozone induces airway inflammation and enhances response to inhaled allergen. It has been suggested that antioxidant therapy may minimize the adverse effects of ozone in asthma. We have previously shown that the antioxidant gamma-tocopherol (γT), an isoform of vitamin E, also has anti-inflammatory effects. We employed a Brown Norway rat model of ozone-enhanced allergic responses to test the therapeutic effects of γT on O3-induced airway inflammation. Ovalbumin (OVA) -sensitized rats were intranasally challenged with 0 or 0.5% OVA on Days 1 and 2, and exposed to 0 or 1 ppm ozone (8h/day) on Days 4 and 5. Rats were also given 0 or 100 mg/kg γT on Days 2 through 5. Pulmonary tissue and bronchoalveolar lavage fluid (BALF) were collected on Day 6. OVA challenge caused increased total cells (267% increase) and eosinophils (4000%) in BALF that was unaffected by ozone exposure. Morphometric evaluation of lung tissue revealed increases in intraepithelial mucosubstances (IM) (300%) and subepithelial eosinophils (400%) in main axial airways. Ozone exposure of allergic rats enhanced IM increases in proximal axial airways (200%), induced cys-leukotrienes, MCP-1 and IL-6 production in BALF, and upregulated expression of IL-5 and IL-13 mRNA. γT treatment had no effect on IM increases by allergen, but blocked enhancement by ozone. γT attenuated both OVA- or ozone –stimulated eosinophilic infiltration, and increases of BALF cys-leukotrienes, MCP-1 and IL-6, as well as IL-5 and IL-13 mRNA. These data demonstrate broad anti-inflammatory effects of a γT and suggest it may be an effective therapy of allergic airway inflammation

Enhanced Th17-Cell Responses Render CCR2-Deficient Mice More Susceptible for Autoimmune Arthritis

CCR2 is considered a proinflammatory mediator in many inflammatory diseases such as rheumatoid arthritis. However, mice lacking CCR2 develop exacerbated collagen-induced arthritis. To explore the underlying mechanism, we investigated whether autoimmune-associated Th17 cells were involved in the pathogenesis of the severe phenotype of autoimmune arthritis. We found that Th17 cells were expanded approximately 3-fold in the draining lymph nodes of immunized CCR2−/− mice compared to WT controls (p = 0.017), whereas the number of Th1 cells and regulatory T cells are similar between these two groups of mice. Consistently, levels of the Th17 cell cytokine IL-17A and Th17 cell-associated cytokines, IL-6 and IL-1β were approximately 2–6-fold elevated in the serum and 22–28-fold increased in the arthritic joints in CCR2−/− mice compared to WT mice (p = 0.04, 0.0004, and 0.01 for IL-17, IL-6, and IL-1β, respectively, in the serum and p = 0.009, 0.02, and 0.02 in the joints). Furthermore, type II collagen-specific antibodies were significantly increased, which was accompanied by B cell and neutrophil expansion in CCR2−/− mice. Finally, treatment with an anti-IL-17A antibody modestly reduced the disease severity in CCR2−/− mice. Therefore, we conclude that while we detect markedly enhanced Th17-cell responses in collagen-induced arthritis in CCR2-deficient mice and IL-17A blockade does have an ameliorating effect, factors additional to Th17 cells and IL-17A also contribute to the severe autoimmune arthritis seen in CCR2 deficiency. CCR2 may have a protective role in the pathogenesis of autoimmune arthritis. Our data that monocytes were missing from the spleen while remained abundant in the bone marrow and joints of immunized CCR2−/− mice suggest that there is a potential link between CCR2-expressing monocytes and Th17 cells during autoimmunity

Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics

Background Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. Results Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. Conclusion The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings