Effect of cell density on in vitro mouse immunoglobulin E production

In vitro immunoglobulin E (IgE) production was found to be sensitive to increasing cell concentration in culture wells. While class switching to IgE is intact as suggested by surface IgE staining, ELISPOT analysis provided evidence that the differentiation of IgE committed B cells to the plasma cell stage was arrested at high cell doses. In fact, splitting the cells at higher concentrations after culture initiation increased IgE production. Cells plated at higher doses were found to be more prone to apoptosis as assessed by Annexin staining. Interestingly, inhibiting apoptosis by the use of the caspase inhibitor DEVD significantly increased IgE levels implicating apoptosis in the preferential deletion of IgE expressing cells. These data not only highlight the caveat against using a single B-cell dose for IgE production in vitro but also suggest for the first time a possible IgE regulatory mechanism mediated by cell density

A Promoter in the Coding Region of the Calcium Channel Gene <i>CACNA1C</i> Generates the Transcription Factor CCAT

<div><p>The C-terminus of the voltage-gated calcium channel Ca_v1.2 encodes a transcription factor, the calcium channel associated transcriptional regulator (CCAT), that regulates neurite extension and inhibits Ca_v1.2 expression. The mechanisms by which CCAT is generated in neurons and myocytes are poorly understood. Here we show that CCAT is produced by activation of a cryptic promoter in exon 46 of <i>CACNA1C,</i> the gene that encodes Ca_V1.2. Expression of CCAT is independent of Ca_v1.2 expression in neuroblastoma cells, in mice, and in human neurons derived from induced pluripotent stem cells (iPSCs), providing strong evidence that CCAT is not generated by cleavage of Ca_V1.2. Analysis of the transcriptional start sites in <i>CACNA1C</i> and immune-blotting for channel proteins indicate that multiple proteins are generated from the 3′ end of the <i>CACNA1C</i> gene. This study provides new insights into the regulation of <i>CACNA1C,</i> and provides an example of how exonic promoters contribute to the complexity of mammalian genomes.</p></div

RNA tape sampling in cutaneous lupus erythematosus discriminates affected from unaffected and healthy volunteer skin

Objective Punch biopsy, a standard diagnostic procedure for patients with cutaneous lupus erythematosus (CLE) carries an infection risk, is invasive, uncomfortable and potentially scarring, and impedes patient recruitment in clinical trials. Non-invasive tape sampling is an alternative that could enable serial evaluation of specific lesions. This cross-sectional pilot research study evaluated the use of a non-invasive adhesive tape device to collect messenger RNA (mRNA) from the skin surface of participants with CLE and healthy volunteers (HVs) and investigated its feasibility to detect biologically meaningful differences between samples collected from participants with CLE and samples from HVs.Methods Affected and unaffected skin tape samples and simultaneous punch biopsies were collected from 10 participants with CLE. Unaffected skin tape and punch biopsies were collected from 10 HVs. Paired samples were tested using quantitative PCR for a candidate immune gene panel and semi-quantitative immunohistochemistry for hallmark CLE proteins.Results mRNA collected using the tape device was of sufficient quality for amplification of 94 candidate immune genes. Among these, we found an interferon (IFN)-dominant gene cluster that differentiated CLE-affected from HV (23-fold change; p&lt;0.001) and CLE-unaffected skin (sevenfold change; p=0.002), respectively. We found a CLE-associated gene cluster that differentiated CLE-affected from HV (fourfold change; p=0.005) and CLE-unaffected skin (fourfold change; p=0.012), respectively. Spearman’s correlation between per cent area myxovirus 1 protein immunoreactivity and IFN-dominant mRNA gene cluster expression was highly significant (dermis, rho=0.86, p&lt;0.001). In total, skin tape-derived RNA expression comprising both IFN-dominant and CLE-associated gene clusters correlated with per cent area immunoreactivity of some hallmark CLE-associated proteins in punch biopsies from the same lesions.Conclusions A non-invasive tape RNA collection technique is a potential tool for repeated skin biomarker measures throughout a clinical trial

CCAT Expression is Regulated During Brain Development.

<p>(A) Immunohistochemistry of E18, P1, and 3-week-old rat cortex, cerebellum, and thalamus showing developmental variation in the amount and distribution of CCAT nuclear staining. Cortex shows mostly cell body and dendritic staining of CCAT in the three developmental stages. Anti-CCAT is shown in red and nuclei in blue. (B) Immunohistochemistry of E18 mouse sagittal sections through the cortex, ventricle, and thalamus stained with anti-CCAT antibody. (C) Immunocytochemistry of cortical and thalamic neurons grown 5 days <i>in vitro</i> stained with anti-CCAT. Transcriptional assays of cortical (top) and thalamic (bottom) neuronal cultures transfected with the UAS-luciferase reporter along with the Gal4-tagged channels described in Fig. 1. Bars represent normalized transcription to Gal4 alone. (Means ± SD; * <0.005 and ** <0.0001 vs. M2011I-Gal4).</p

CCAT is Generated From an Independent Transcript <i>in vivo</i>.

<p>(A–B) Northern blot analysis of mRNA extracted from cortex, diencephalon, and cerebellum from E18, P1, and adult rats. The membranes were hybridized with radioactively labeled DNA probe to either exon 47 (A) or exons 21–24 (B) of the channel. Bottom panel shows the same membrane labeled with a probe to the 18S ribosomal RNA as a loading control. (C) Graphs showing the normalized expression of the full-length and 2.2 Kb band in the cortex, midbrain, and cerebellum at 3 developmental stages: E18, P1, and adult. Each line represents an independent experiment. The northern blot shown in A corresponds to the blue tracing. Signals were normalized to the 18S RNA signal. (D–E) Dot plots showing the normalized expression of exons 2, 8, 44, and 47 per cell in mouse neurons. For all comparisons of exon 47 to the other exons, p<0.001 (***) according to one-way ANOVA with post-hoc Bonferroni correction for multiple comparisons. Exons 2, 8, and 44 are not significantly different from each other. (F) Graphs showing fractions of human IPSC-derived neurons expressing exon 47 exclusively or both exons 47 and 8 (n = 269). (G) Fraction of exon 47-containing human IPSC-derived neurons expressing either exon 47 exclusively or co-expressing exon 8.</p

CCAT is Translated from an Independent Transcript Driven by an Exonic Promoter.

<p>(A) Northern blot analysis of mRNA extracted from Neuro2A cells expressing Ca_v1.2-Gal4 channel constructs with or without CMV promoter. The first lane contains mRNA extracted from untransfected cells. The membranes were hybridized with a radioactively labeled RNA probe to Exon 47 of the channel. (B) Western blot of Neuro2A cells expressing Ca_v1.2-Gal4 channel constructs with or without CMV promoter. Upper panel shows full-length channels. Bottom panels shows Gal4-tagged CCAT. Membranes were immunoblotted with a Gal4 antibody. (C) UAS reporter activity of Neuro2A cells expressing Ca_v1.2-Gal4 channel constructs with or without CMV promoter. (Means ± SD; *<0.0001 vs. Gal4). (D) Schematic representation of Firefly reporter constructs designed to map the region within the coding sequence of the channel responsible for the promoter activity. Full-length construct has the complete sequence of the channel upstream of the luciferase coding sequence. E1-45 construct includes all exons up to exon 45. E46-47 contains exon 46 and 47 upstream of luciferase. (E) Luciferase activity as a surrogate measure of luciferase expression from constructs depicted in D. Graph compares expression level in Neuro2A cells transfected with either full-length, E1-45, E46-47, E46, or E47 constructs. (Means ± SD). (F) Schematic representation of the minigenes. A 4 Kb genomic segment containing the last two exons and introns of <i>CACNA1C</i> was fused to the coding sequence of Gal4. WT is the wild-type minigene. FS is a negative control where a G has been inserted between exon 47 and Gal4. Δ238bp lacks the 238bp region identified in E. Met 2011 has been mutated to isoleucine in M2011. (G) Mean luciferase activity (± SD) in Neuro2A cells expressing WT, FS, Δ238bp, and M2011I Gal4 minigenes along with a UAS-luciferase reporter. (* <0.0001 vs. WT).</p

Transcriptional Start Sites in the 3′ End of <i>CACNA1C</i> Produce Multiple Proteins.

<p>(A) Schematic representation of <i>CACNA1C</i> showing the location of TSS's found and depictions of the proteins predicted to be expressed from these transcripts. (B) Table showing a summary of transcriptional start sites and nearby CAGE tags. Chromosomal addresses for experimental TSS are given as the corresponding location in the Mouse July 2007 genome assembly. NT (Not tested), NC (non-coding). (C) HEK cells expressing Mem-CCAT GFP and CCAT GFP constructs. (D-E) Western blot analysis of membrane fractions (D) and nuclear fractions (E) obtained from 11.5 dpc heterozygous (N/+) or homozygous (N/N) Ca_v1.2 knockout embryos probed with the anti-CCAT antibody. Bottom panels show loading controls. (F) Immunohistochemistry of 11.5 dpc Ca_v1.2 null embryos reveals strong nuclear staining with the anti-CCAT antibody (red) in the developing somites. Nuclei are shown in blue.</p