A hydrophobic area of the GABA ρ1 receptor containing phenylalanine 124 influences both receptor activation and deactivation.

Experimental evidence suggests that GABA ρ1 receptors are potential therapeutic targets for the treatment of a range of neurological conditions, including anxiety and sleep disorders. Homology modelling of the GABA ρ1 extracellular N-terminal domain has revealed a novel hydrophobic area that extends beyond, but not including the GABA-binding site. Phenylalanine 124 (F124) is predicted to be involved in maintaining the structural integrity of the orthosteric-binding site. We have assessed the activity of a series of GABA ρ1 receptors that incorporate a mutation at F124. Wild-type and mutant human GABA ρ1 subunits were expressed in Xenopus laevis oocytes and AD293 cells, and the pharmacology and kinetic properties of the receptors were measured using electrophysiological analysis. Mutation of F124 had minimal effect on receptor pharmacology. However, the rate of deactivation was significantly increased compared to wild type. This study provides further information about the role of residues within a novel hydrophobic area of the GABA ρ1 receptor. This knowledge can help future studies into the design of potent and subtype-selective ligands with therapeutic value.

Hypertrophic Cardiomyopathy: Prevalence, Hypertrophy Patterns, and Their Clinical and ECG Findings in a Hospital at Qatar.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a genetic disease associated with risk of morbidity and sudden cardiac death. The prevalence, hypertrophy patterns, mode of presentations, and different ECG findings vary in different regions of the world. To date, no data is present regarding these variables in Qatar. PATIENTS AND METHODS: A retrospective, cross sectional, descriptive analysis of all patients referred for echocardiography study at Hamad General Hospital, Qatar. The study period was from January 2008 till December 2010. AIMS: To study 1) the prevalence of HCM, 2) the different patterns of hypertrophy, and 3) the clinical and ECG presentations in this population. RESULTS: Out of the 29,286 cases evaluated, 38 patients were found to have HCM (0.13%). Their clinical, ECG, and echocardiography findings were analyzed. Mean age was 47 y, 35 males (92%) and 3 females (8%). Four patterns of hypertrophy were described; 17 (44.7%) had septal hypertrophy alone, 6 (15.8%) had septal and other segments hypertrophy but sparing the apex, 10 (26.3%) had apical segments along with any other segment hypertrophy, and 5 (13.2%) had apical hypertrophy alone. No obstruction was found in 19 (50%), left ventricular outflow (LVO) tract obstruction was found in 13 (34%), and mid cavity obstruction (MCO) in 6 (16%). Twenty one (55.3%) patients were referred because of chest pain, 15 (39.5%) with palpitations, 15 (39.5%) with shortness of breath, and 5 (13.2%) with syncope. Nine patients (23.7%) were asymptomatic and were referred because of cardiac murmur during routine examination. ECG evidence of LV hypertrophy was found in 29 (76.3%). CONCLUSION: The prevalence of HCM in our population group is 0.13% with a male predominance (12:1). There was a diversity of clinical presentation, ECG abnormalities and patterns of LV hypertrophy among HCM patients.

Pairing of heterochromatin in response to cellular stress.

We previously reported that exposure of human cells to DNA-damaging agents (X-rays and mitomycin C (MMC)) induces pairing of the homologous paracentromeric heterochromatin of chromosome 9 (9q12-13). Here, we show that UV irradiation and also heat shock treatment of human cells lead to similar effects. Since the various agents induce very different types and frequencies of damage to cellular constituents, the data suggest a general stress response as the underlying mechanism. Moreover, local UV irradiation experiments revealed that pairing of heterochromatin is an event that can be triggered without induction of DNA damage in the heterochromatic sequences. The repair deficient xeroderma pigmentosum cells (group F) previously shown to fail pairing after MMC displayed elevated pairing after heat shock treatment but not after UV exposure. Taken together, the present results indicate that pairing of heterochromatin following exposure to DNA-damaging agents is initiated by a general stress response and that the sensing of stress or the maintenance of the paired status of the heterochromatin might be dependent on DNA repair.

Mitomycin C-induced pairing of heterochromatin reflects initiation of DNA repair and chromatid exchange formation.

Chromatid interchanges induced by the DNA cross-linking agent mitomycin C (MMC) are over-represented in human chromosomes containing large heterochromatic regions. We found that nearly all exchange breakpoints of chromosome 9 are located within the paracentromeric heterochromatin and over 70% of exchanges involving chromosome 9 are between its homologues. We provide evidence that the required pairing of chromosome 9 heterochromatic regions occurs in G(0)/G(1) and S-phase cells as a result of an active cellular process initiated upon MMC treatment. By contrast, no pairing was observed for a euchromatic paracentromeric region of the equal-sized chromosome 8. The MMC-induced pairing of chromosome 9 heterochromatin is observed in a subset of cells; its percentage closely mimics the frequency of homologous interchanges found at metaphase. Moreover, the absence of pairing in cells derived from XPF patients correlates with an altered spectrum of MMC-induced exchanges. Together, the data suggest that the heterochromatin-specific pairing following MMC treatment reflects the initiation of DNA cross-link repair and the formation of exchanges.

Ionizing radiation-induced instant pairing of heterochromatin of homologous chromosomes in human cells.

Using fluorescence in situ hybridization with human band-specific DNA probes we examined the effect of ionizing radiation on the intra-nuclear localization of the heterochromatic region 9q12-->q13 and the euchromatic region 8p11.2 of similar sized chromosomes 9 and 8 respectively in confluent (G1) primary human fibroblasts. Microscopic analysis of the interphase nuclei revealed colocalization of the homologous heterochromatic regions from chromosome 9 in a proportion of cells directly after exposure to 4 Gy X-rays. The percentage of cells with paired chromosomes 9 gradually decreased to control levels during a period of one hour. No significant changes in localization were observed for chromosome 8. Using 2-D image analysis, radial and inter-homologue distances were measured for both chromosome bands. In unexposed cells, a random distribution of the chromosomes over the interphase nucleus was found. Directly after irradiation, the average inter-homologue distance decreased for chromosome 9 without alterations in radial distribution. The percentage of cells with inter-homologue distance <3 micro m increased from 11% in control cells to 25% in irradiated cells. In contrast, irradiation did not result in significant changes in the inter-homologue distance for chromosome 8. Colocalization of the heterochromatic regions of homologous chromosomes 9 was not observed in cells irradiated on ice. This observation, together with the time dependency of the colocalization, suggests an underlying active cellular process. The biological relevance of the observed homologous pairing remains unclear. It might be related to a homology dependent repair process of ionizing radiation induced DNA damage that is specific for heterochromatin. However, also other more general cellular responses to radiation-induced stress or change in chromatin organization might be responsible for the observed pairing of heterochromatic regions.