Nitric oxide pathway-related gene alterations in inflammatory bowel disease.

Objective: To reveal specific gene activation in nitric oxide (NO)-related inflammation we studied differential gene expression in inflammatory bowel disease (IBD).

Methods: Total RNA was isolated from 20 biopsies of inflamed mucosa from Crohn's disease (CD) and ulcerative colitis (UC) patients each as well as from six controls, labeled with (32)P-dCTP and hybridized to a human NO gene array. Significant genes were analyzed for functional gene interactions and heatmaps generated by hierarchical clustering.

Phosphorylation of skeletal muscle calsequestrin enhances its Ca2+ binding capacity and promotes its association with junctin.

Calcium signaling, intrinsic to skeletal and cardiac muscle function, is critically dependent on the amount of calcium stored within the sarcoplasmic reticulum. Calsequestrin, the main calcium buffer in the sarcoplasmic reticulum, provides a pool of calcium for release through the ryanodine receptor and acts as a luminal calcium sensor for the channel via its interactions with triadin and junctin. We examined the influence of phosphorylation of calsequestrin on its ability to store calcium, to polymerise and to regulate ryanodine receptors by binding to triadin and junctin.

ARVC-related mutations in divergent region 3 alter functional properties of the cardiac ryanodine receptor.

Two single-nucleotide polymorphisms in the type 2 ryanodine receptor (RyR2) leading to the nonsynonymous amino acid replacements G1885E and G1886S are associated with arrhythmogenic right ventricular cardiomyopathy in patients who are carrying both of the corresponding RyR2 alleles. The functional properties of HEK293 cell lines isogenically expressing RyR2 mutants associated with arrhythmogenic right ventricular cardiomyopathy, RyR2-G1885E, RyR2-G1886S, RyR2-G1886D (mimicking a constitutively phosphorylated Ser(1886)), and the double mutant RyR2-G1885E/G1886S were investigated by analyzing the intracellular Ca(2+) release activity resulting from store-overload-induced calcium release. The substitution of serine for Gly(1886) caused a significant increase in the cellular Ca(2+) oscillation activity compared with RyR2 wild-type-expressing HEK293 cells.

Characterization of human cardiac calsequestrin and its deleterious mutants.

Mutations of conserved residues of human cardiac calsequestrin (hCSQ2), a high-capacity, low-affinity Ca2+-binding protein in the sarcoplasmic reticulum, have been associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). In order to understand the molecular mechanism and pathophysiological link between these CPVT-related missense mutations of hCSQ2 and the resulting arrhythmias, we generated three CPVT-causing mutants of hCSQ2 (R33Q, L167H, and D307H) and two non-pathological mutants (T66A and V76M) and investigated the effect of these mutations. In addition, we determined the crystal structure of the corresponding wild-type hCSQ2 to gain insight into the structural effects of those mutations.

Composite polymorphisms in the ryanodine receptor 2 gene associated with arrhythmogenic right ventricular cardiomyopathy.

Objective: Mutations in the cardiac ryanodine receptor (RYR2) gene have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC). The molecular mechanisms by which genetic modifications lead to ARVC are still not well understood.

Methods: ARVC patients were screened for mutations in the RYR2 gene by denaturing HPLC and DNA sequencing.