Echocardiographic-determined septal morphology in Z-disc hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) can be classified into at least four major anatomic subsets based upon the septal contour, and the location and extent of hypertrophy: reverse curvature-, sigmoidal-, apical-, and neutral contour-HCM. Here, we sought to identify genetic determinants for sigmoidal-HCM and hypothesized that Z-disc-HCM may be associated preferentially with a sigmoidal phenotype. Utilizing PCR, DHPLC, and direct DNA sequencing, we performed mutational analysis of five genes encoding cardiomyopathy-associated Z-disc proteins. The study cohort consisted of 239 unrelated patients with HCM previously determined to be negative for mutations in the eight genes associated with myofilament-HCM. Blinded to the Z-disc genotype status, the septal contour was graded qualitatively using standard transthoracic echocardiography. Thirteen of the 239 patients (5.4%) had one of 13 distinct HCM-associated Z-disc mutations involving residues highly conserved across species and absent in 600 reference alleles: LDB3 (6), ACTN2 (3), TCAP (1), CSRP3 (1), and VCL (2). For this subset with Z-disc-associated HCM, the septal contour was sigmoidal in 11 (85%) and apical in 2 (15%). While Z-disc-HCM is uncommon, it is equal in prevalence to thin filament-HCM. In contrast to myofilament-HCM, Z-disc-HCM is associated preferentially with sigmoidal morphology.

HuR mRNA ligands expressed after seizure.

HuR is a ubiquitously expressed AU-rich element (ARE)-binding protein that interacts with and stabilizes selective early response gene (ERG) mRNAs after cell activation or stress. To date, approximately 20 mRNAs have been unambiguously defined as HuR ligands. Given the discordance between the large number of ERG mRNAs and those few defined as ligands, we applied in vitro selection to isolate a broad range of HuR mRNA ligands using postseizure mouse hippocampal tissue. Selected mRNAs were converted into cDNA libraries and sequenced. Using this approach, we have identified over 600 novel, putative HuR mRNA ligands. These genes code for a variety of proteins, including transcription factors, signaling molecules, and kinases, but many have unknown function. Consistent with the means of their selection, several of these HuR ligands are differentially expressed in hippocampus after seizure. These results demonstrate a biochemical approach to identify and characterize the diverse repertoire of ligands for HuR and other regulatory mRNA-binding proteins.

RhoB mRNA is stabilized by HuR after UV light.

RhoB is a small GTP-binding protein that is involved in apoptotic signal transduction. We have cloned the mouse RhoB mRNA including a 1377 nucleotide 3'-untranslated region (UTR) that contains six AU-rich elements (AREs) as well as several uridine-rich stretches. There is 94% homology overall between the mouse and rat RhoB genes and 92% homology between the mouse and a putative human clone. Ultraviolet light (UVL) induces RhoB production through regulated changes in gene transcription and mRNA stabilization although the latter mechanism is unknown. We observed that UVL increased the half-life of RhoB mRNA from 63 min to 3.3 h in NIH/3T3 cells and from 87 min to 2.7 h in normal human keratinocyte cells. In vitro mobility shift assays demonstrated that HuR bound the 3'-UTR of RhoB at three distinct locations (nucleotides 1342-1696, 1765-1920 and 1897-1977) suggesting a regulatory role for this RNA-binding protein. HuR immunoprecipitations were positive for RhoB mRNA indicating an in vivo association, and Western blot analysis and immunofluorescence demonstrated that HuR rapidly partitions from the nucleus to the cytoplasm after UVL. Therefore, we propose a model in which UVL induces stress-activated signal transduction leading to nuclear/cytoplasmic shuttling of HuR and subsequent stabilization of RhoB mRNA.

Automated quantitative analysis of the shift of frequency spectra generated by attenuated signals from contrast microbubbles.

The ultrasound-induced harmonic microbubble response spectrum is known to shift to lower frequencies with increasing tissue attenuation. We hypothesized that this shift could be reproducibly detected in received broadband radiofrequency spectra. We used an automatic Gaussian curve-fitting technique to measure the mean harmonic response generated by three different contrast agents at six incremental levels of attenuation. Analytical curve fitting identified a consistent, reproducible, and statistically significant shift in mean harmonic frequency with increasing attenuation. The presented method could be a step toward attenuation estimation by contrast harmonic imaging; optimization of harmonic signal reception by ultrasound systems; and, ultimately, automatic detection of contrast agents in tissue.