Developing and mature human granulocytes express ELP 6 in the cytoplasm.

BACKGROUND: c3orf75 is a conserved open reading frame within the human genome and has recently been identified as the Elongator subunit, ELP6 [1]. The Elongator enzyme complex has diverse roles, including translational control, neuronal development, cell migration and tumorigenicity [2]. OBJECTIVE: To identify genes expressed early in human eosinophil development. METHODS: Eosinophilopoiesis was investigated by gene profiling of IL-5 stimulated CD34+ cells; ELP6 mRNA is upregulated. A monoclonal antibody was raised to the recombinant protein predicted by the open reading frame. RESULTS: ELP6 transcripts are upregulated in a human tissue culture model of eosinophil development during gene profiling experiments. Transcripts are expressed in most tissue types, as shown by reverse-transcriptase PCR. Western blot experiments show that human ELP6 is a 30 kDa protein expressed in the bone marrow, as well as in many other tissues. Flow cytometry experiments of human bone marrow mononuclear cells show that ELP6 is expressed intracellularly, in developing and mature human neutrophils, eosinophils and monocytes. CONCLUSIONS: ELP6 is expressed intracellularly in developing and mature granulocytes and monocytes but not in lymphocytes and erythrocytes.

RAZOR EX anthrax air detection system.

The RAZOR EX Anthrax Air Detection System, developed by Idaho Technology, Inc. (ITI), is a qualitative method for the detection of Bacillus anthracis spores collected by air collection devices. This system comprises a DNA extraction kit, a freeze-dried PCR reagent pouch, and the RAZOR EX real-time PCR instrument. Each pouch contains three assays, which distinguish potentially virulent B. anthracis from avirulent B. anthracis and other Bacillus species. These assays target the pXO1 and pXO2 plasmids and chromosomal DNA. When all targets are detected, the instrument makes an "anthrax detected" call, meaning that virulence genes of the anthrax bacillus are present. This report describes results from AOAC Method Developer (MD) and Independent Laboratory Validation (ILV) studies, which include matrix, inclusivity/exclusivity, environmental interference, upper and lower LOD of DNA, robustness, product consistency and stability, and instrument variation testing. In the MD studies, the system met the acceptance criteria for sensitivity and specificity, and the performance was consistent, stable, and robust for all components of the system. For the matrix study, the acceptance criteria of 95/96 expected calls was met for three of four matrixes, clean dry filters being the exception. Ninety-four of the 96 clean dry filter samples tested gave the expected calls. The nucleic acid limit of detection was 5-fold lower than AOAC's acceptable minimum detection limit. The system demonstrated no tendency for false positives when tested with Bacillus cereus. Environmental substances did not inhibit accurate detection of B. anthracis. The ILV studies yielded similar results for the matrix and inclusivity/exclusivity studies. The ILV environmental interference study included environmental substances and environmental organisms. Subsoil at a high concentration was found to negatively interfere with the pXO1 reaction. No interference was observed from the environmental organisms. The nucleic acid LOD, however, was 10 times higher (1 pg/reaction, equivalent to about 200 spores) than that found in the MD study. These results indicate that the RAZOR System is a sensitive and specific system that accurately identifies B. anthracis in aerosol matrixes and in the presence of interfering substances, and that the method can be performed by an independent laboratory and achieve similar results.

EGO, a novel, noncoding RNA gene, regulates eosinophil granule protein transcript expression.

Gene expression profiling of early eosinophil development shows increased transcript levels of proinflammatory cytokines, chemokines, transcription factors, and a novel gene, EGO (eosinophil granule ontogeny). EGO is nested within an intron of the inositol triphosphate receptor type 1 (ITPR1) gene and is conserved at the nucleotide level; however, the largest open reading frame (ORF) is 86 amino acids. Sucrose density gradients show that EGO is not associated with ribosomes and therefore is a noncoding RNA (ncRNA). EGO transcript levels rapidly increase following interleukin-5 (IL-5) stimulation of CD34(+) hematopoietic progenitors. EGO RNA also is highly expressed in human bone marrow and in mature eosinophils. RNA silencing of EGO results in decreased major basic protein (MBP) and eosinophil derived neurotoxin (EDN) mRNA expression in developing CD34(+) hematopoietic progenitors in vitro and in a CD34(+) cell line model. Therefore, EGO is a novel ncRNA gene expressed during eosinophil development and is necessary for normal MBP and EDN transcript expression.