The expression levels from each respective initiation codon within the mouse MOR uORF region

<p><b>Copyright information:</b></p><p>Taken from "Translational repression of mouse mu opioid receptor expression via leaky scanning"</p><p></p><p>Nucleic Acids Research 2007;35(5):1501-1513.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865057.</p><p>© 2007 The Author(s).</p> () A schematic summary of various in-frame constructs. Mouse MOR uORF regions were fused in-frame to the luciferase gene. Initiation codons with vertical dotted lines indicate point mutations of ATG to ACG. () Autoradiogram of the proteins translated by a coupled transcription/translation system in the presence of [S]-methionine from representative translations. Proteins were separated by 10% SDS-PAGE (LUC, Fused Protein). Quantification of mRNA levels from the cell-free transcription/translation system was performed by RT-PCR (+/− RT). Expression levels from each initiation codon were normalized against those of SP6-LUC. Normalized mRNA levels (lower panel). M: 100 bp molecular weight markers. () Relative strength of each initiation context within the mouse MOR UTR as determined by translation. The LUC ATG context is designated 1.00

A novel multifunctional peptide oligomer of bacitracin with possible bioindustrial and therapeutic applications from a Korean food-source <i>Bacillus</i> strain - Fig 1

<p>(A) A neighbor-joining tree based on nearly complete 16S rRNA gene sequences showing relations between strain CS32 and related members of the genus <i>Bacillus</i>. The percentages at the nodes are the levels of bootstrap support based on neighbor-joining analyses of 1000 resampled datasets. The sequence of <i>Bacillus licheniformis</i> ATCC 14580(T) served as an outgroup. The scale bar: 0.01 nucleotide substitutions per position. (B) Production of CSP32 and the growth-inhibitory activity against <i>Mycobacterium smegmatis</i> ATCC 9341. The antibacterial activity is maximal at 60 h. The zone of inhibition against <i>Mycobacterium smegmatis</i> ATCC 9341 at every 12 h is shown. Cultivation was carried out in 250-mL flasks with 50 mL of a medium, at pH 7 and 37°C, with shaking at 180 rpm.</p

Effects of CSP32 on NO production and expression of inflammation-associated proteins in LPS-stimulated RAW 264.7 macrophage cells.

<p>The cells were treated with LPS (1 μg/mL) in the presence of various concentrations of CSP32 (10, 50, or 100 μg/mL) at 37°C for 24 h. (A) The amounts of NO were determined using the Griess reagent in the culture medium (Top panel). Equal amounts of cell lysates were resolved on SDS polyacrylamide gels, transferred to PVDF membranes, and probed with antibodies against iNOS and COX-2. β-Actin served as an internal control for western blot analysis (bottom panel). Effects of CSP32 on LPS-induced mRNA expression of iNOS, COX-2, TNF-α, IL-1β, and IL-6. (B) After LPS treatment for 2–12 h, the levels of iNOS, COX-2, TNF-α, IL-1β, and IL-6 mRNA were determined by RT-PCR. <i>GAPDH</i> served as an internal control for RT-PCR assays. Suppressive effects of CSP32 on TNF-α, IL-1β, and IL-6 production in LPS-stimulated RAW 264.7 macrophage cells. (C) The cells were incubated with the indicated concentrations of CSP32 for 30 min before treatment with LPS (1 μg/mL) for 24 h. After incubation for 24 h, the supernatant was collected, and the amounts of these proinflammatory cytokines were measured by ELISAs. Results represent the mean±S.D of three independent experiments performed in triplicate. * p<0.01; compared to LPS alone.</p

Inhibition of translation by the uORF is independent of its peptide sequence and )

<p><b>Copyright information:</b></p><p>Taken from "Translational repression of mouse mu opioid receptor expression via leaky scanning"</p><p></p><p>Nucleic Acids Research 2007;35(5):1501-1513.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865057.</p><p>© 2007 The Author(s).</p> Schematic representations of the DNA constructs used in this study. Shaded triangles indicate point mutations to the Stop (S) codon; vertical dotted lines indicate point mutations of ATG to ACG (M) ( and ). Forty-eight hours after transfection, cells were trypsinized and examined for luciferase and ß-galactosidase activities, as well as by RNA extraction and transcript quantification. Relative LUC activity and mRNA levels were expressed as the ratio LUC/ß-gal and LUC/LacZ, as described in Materials and Methods

Comparison of MS data with predicted molecular weight of each subunit.

<p>Comparison of MS data with predicted molecular weight of each subunit.</p

Toeprinting analyses of mouse MOR containing uORFs that regulate ribosome leaky scanning

<p><b>Copyright information:</b></p><p>Taken from "Translational repression of mouse mu opioid receptor expression via leaky scanning"</p><p></p><p>Nucleic Acids Research 2007;35(5):1501-1513.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865057.</p><p>© 2007 The Author(s).</p> Synthetic RNA (100 ng) transcripts were used to program translation mixtures derived from RRL. Mouse MOR-LUC RNA containing the wild-type uORFs [uAUG(+)] or deleted of all three uORFs [uAUG(−)] were incubated at 30°C for 15 min in reaction mixtures without cycloheximide (None) or supplemented with cycloheximide (Cyh) either prior to incubation (T0) or after 5 min of incubation at 30°C (T5). Control samples of RNA in reaction mixtures without extract (-EXT) or reaction mixtures containing extract without mRNA (-RNA) are indicated. The positions of the cDNA extension products corresponding to the mRNA 5′ end, uORF initiation (open triangles, asterisks) and LUC initiation codon (filled triangle) by radiolabeled mToe2 primer are indicated. During leaky scanning, ribosomes scan and load at either the upstream or downstream start codons. Adding cycloheximide at T0 traps ribosomes at the positions where they first load on the mRNA. Adding it during steady-state translation (T5) traps ribosomes where they are loaded, following the primary initiation event and subsequent reinitiation events

Comparative analysis of N-terminal amino acid sequences.

<p>Comparative analysis of N-terminal amino acid sequences.</p

Primers for RT-PCR.

<p>Primers for RT-PCR.</p