An integrative expression vector for <Emphasis Type="Italic">Actinosynnema pretiosum</Emphasis>

Background  

The Actinomycete Actinosynnema pretiosum ssp. auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent. One way to increase AP-3 production would be to constitutively express selected genes so as to relieve bottlenecks in the biosynthetic pathway; however, an integrative expression vector for A. pretiosum is lacking. The aim of this study was to construct a vector for heterologous gene expression in A. pretiosum.     

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.     

Identification of the 80-kDa LPS-binding protein (LMP80) as decay-accelerating factor (DAF, CD55)

The activation of immunocompetent cells by lipopolysaccharide (LPS) during severe Gram-negative infections is responsible for the pathophysiological reactions, possibly resulting in the clinical picture of sepsis. Monocytes recognize LPS mainly through the LPS receptor CD14, however, other cellular binding structures have been assumed to exist. In previous studies, we have described an 80-kDa LPS-binding membrane protein (LMP80), which is present on human monocytes as well as endothelial cells. Here we demonstrate that LMP80 is widely distributed and that it forms complexes together with LPS and sCD14. Furthermore, we report on the biochemical purification of LMP80 and its identification as decay-accelerating factor, CD55, by amino acid sequencing and cloning techniques. Our results imply a new feature of CD55 as a molecule which interacts with LPS/sCD14 complexes. However, the involvement of CD55 in LPS-induced signaling remains to be elucidated.     

The CD11a partner in Sus scrofa lymphocyte function-associated antigen-1 (LFA-1): mRNA cloning, structure analysis and comparison with mammalian homologues

Background  

Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2), the most abundant and widely expressed beta2-integrin, is required for many cellular adhesive interactions during the immune response. Many studies have shown that LFA-1 is centrally involved in the pathogenesis of several diseases caused by Repeats-in-toxin (RTX) -producing bacteria.