A run of 11 adenine or thymine residues at the 5' end of an out-of-frame lacZ gene causes a high level of beta-galactosidase expression in E. coli. This effect was not observed for a run of guanine residues. Reverse transcription of mRNA isolated from E. coli containing the run of 11 A's reveals heterogeneity of transcript length while reverse transcription of mRNA isolated from S. cerevisiae containing the same gene shows no heterogeneity. Protein sequencing of the beta-galactosidase molecules derived from the out-of-frame construct containing a run of adenines reveals the addition of a lysine at the run. A new method was developed where messages small enough to allow resolution of single nucleotide differences on an acrylamide gel are electrophoresed, electroblotted onto nylon and probed. This confirmed the reverse transcription results and showed that additional residues can be added to transcripts derived from DNA containing 10 or 11 thymine residues. A mechanism for slippage is discussed where the A-U rich RNA-DNA hybrid can denature during elongation and rehybridize in an offset position, causing the addition of extra residues to the transcript.

Encapsulation with various dextrose equivalent (DE) hydrolyzed starches affected stability of a-and P-carotene in spray-dried carrot powders. Degradation of Q-and p-carotene during storage of the powders at temperatures ranging from 37 to 65'C followed first-order kinetics and both degraded at the same rate. Hydrolyzed starch of 36.5DE was superior to 4, 15, and 25DE in improving retention of the carotenes. Carotenes encapsulated with 36.5DE hydrolyzed starch had a predicted half-life of 450 days at 21°C compared to 2 days for carrot juice spray dried alone. Increasing the proportion of carrier decreased the carotene degradation rate and similarly decreased surface carotene. Air was critical in carotene stability, but exposure of encapsulated carrot powders to light did not accelerate degradation.

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 IntroductionEosinophils are tissue-dwelling hematopoietic cells that likely play a role in parasitic immunity and allergic disease, such as asthma. 1 Activated eosinophils secrete toxic basic proteins such as major basic protein (MBP) and are postulated to cause bronchial hyperreactivity, damage of the bronchial mucosa, and remodeling of the airways. 1 Mice lacking eosinophils fail to show hallmarks of asthma such as airway hyperresponsiveness, tissue remodeling, and mucous metaplasia. 2,3 A more complete understanding of eosinophil development could lead to drugs targeting eosinophil progenitors prior to migration to the asthmatic lung.Eosinophils develop in the bone marrow from hematopoietic stem cells and migrate mainly to the gut or to sites of inflammation. Eosinophils, neutrophils, and monocytes have a common progenitor in the myeloid pathway of development. The combinatorial interactions of several transcription factors, including GATA-1, PU.1, and the CCAAT enhancer binding proteins, c/EBP␣ and ⑀, are important to eosinophil development. [4][5][6][7] High levels of PU.1 specify myeloid differentiation by antagonizing GATA-1 in the earliest stages of stem cell commitment. 8 In particular, a highaffinity GATA-1 binding site within the GATA-1 promoter appears to be critical for eosinophil development; deletion of this binding site in mice specifically abolishes the entire eosinophil lineage. 9 During later stages of eosinophil development, an intermediate level of GATA-1 in synergy with PU.1 directs the formation of the eosinophil lineage by activating dual binding sites in the MBP promoter. [10][11][12] GATA-1 also activates the eotaxin receptor CC chemokine receptor-3 (CCR3) promoter and the interleukin-5 (IL-5) receptor ␣ (IL-5R␣) gene. 13 The CCAAT enhancer binding protein, c/EBP␣, is important in early myeloid development, whereas c/EBP⑀ plays a later role. 14 Mouse knockouts of c/EBP⑀ affect both neutrophil and eosinophil development at the myelocyte to metamyelocyte stage. 14 Other genes involved in eosinophil development include the helix-loop-helix transcription factors, Id 1 and 2, and FOG (friend of GATA). FOG inhibits eosinophil development by intera...

Background-Mepolizumab, a monoclonal anti-interleukin-5 (IL-5) antibody, is an effective corticosteroid-sparing agent for patients with F/P-negative hypereosinophilic syndrome. Lymphocytic variant hypereosinophilic syndrome (L-HES) is characterized by marked overproduction of IL-5 by dysregulated T-cells.

Eosinophil cationic proteins influence several biological functions of the respiratory epithelium, yet their direct contribution to airway remodeling has not been established. We show that incubation of the human bronchial epithelial cell line, BEAS-2B, or primary cultured human bronchial epithelial cells, normal human bronchial epithelial cells, with subcytotoxic concentrations (0.1, 0.3, and 1 μM) of major basic protein (MBP), or eosinophil peroxidase (EPO), augmented the transcripts of endothelin-1, TGF-α, TGF-β1, platelet-derived growth factor (PDGF)-β, epidermal growth factor receptor, metalloproteinase (MMP)-9, fibronectin, and tenascin. A down-regulation of MMP-1 gene expression was observed exclusively in BEAS-2B cells. Cationic protein-induced transcriptional effects were followed by the release of endothelin-1, PDGF-AB in the supernatants by ELISA, and by a down- and up-regulation, respectively, in the levels of MMP-1 and MMP-9 in cell lysates, by Western blot. Cell stimulation with the synthetic polycation, poly-l-arginine, reproduced some but not all effects of MBP and EPO. Finally, simultaneous cell incubation with the polyanion molecules, poly-l-glutamic acid or heparin, restored MMP-1 gene expression but incompletely inhibited MBP- and EPO-induced transcriptional effects as well as endothelin-1 and PDGF-AB release, suggesting that cationic proteins act partially through their cationic charge. We conclude that eosinophil-derived cationic proteins are able to stimulate bronchial epithelium to synthesize factors that influence the number and behavior of structural cells and modify extracellular matrix composition and turnover.