Utility of genetic screening in hypertrophic cardiomyopathy: prevalence and significance of novel and double (homozygous and heterozygous) beta-myosin mutations

Genetic screening of the beta-myosin heavy chain gene (MYH7) was evaluated in 100 consecutive unrelated patients with hypertrophic cardiomyopathy (HCM) and 200 normal unrelated subjects. Seventeen beta-myosin mutations were identified in 19 patients. Notably, 13, or 76%, were novel. Mutations were detected in both alleles in two patients: homozygous for Lys207Gln in one, and heterozygous for Pro211 Leu and Arg663His in another. No mutation was detected in the controls. MYH7-associated HCM was associated with more marked left atrial enlargement and syncope than non-MYH7-related HCM. Our findings indicate that: (1) screening methods should allow identification of novel mutations; and (2) more than one sarcomeric mutation may be present in a patient more commonly than is appreciated. Further studies are necessary to ascertain the clinical consequences of the novel and compound gene abnormalities, and to determine whether correlating functional domain to phenotype provides more useful information about the clinical significance of the molecular defects.     

Monitoring depth of anesthesia using combination of EEG measure and hemodynamic variables

Monitoring depth of anesthesia (DOA) via vital signs is a major ongoing challenge for anesthetists. A number of electroencephalogram (EEG)-based monitors such as the Bispectral (BIS) index have been proposed. However, anesthesia is related to central and autonomic nervous system functions whereas the EEG signal originates only from the central nervous system. This paper proposes an automated DOA detection system which consists of three steps. Initially, we introduce multiscale modified permutation entropy index which is robust in the characterization of the burst suppression pattern and combine multiscale information. This index quantifies the amount of complexity in EEG data and is computationally efficient, conceptually simple and artifact resistant. Then, autonomic nervous system activity is quantified with heart rate and mean arterial pressure which are easily acquired using routine monitoring machine. Finally, the extracted features are used as input to a linear discriminate analyzer (LDA). The method is validated with data obtained from 25 patients during the cardiac surgery requiring cardiopulmonary bypass. The experimental results indicate that an overall accuracy of 89.4 % can be obtained using combination of EEG measure and hemodynamic variables, together with LDA to classify the vital sign into awake, light, surgical and deep anesthetised states. The results demonstrate that the proposed method can estimate DOA more effectively than the commercial BIS index with a stronger artifact-resistance.     

Inhibition of cyclic AMP dependent protein kinase by vanadyl sulfate

Vanadium salts influence the activities of a number of mammalian enzymes in vitro but the mechanisms by which low concentrations of vanadium ameliorate the effects of diabetes in vivo remain poorly understood. The hypothesis that vanadium compounds act by inhibiting protein tyrosine phosphatases has attracted most support. The studies described here further evaluate the possibility that vanadyl sulfate trihydrate (VS) can also inhibit 3′,5′-cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA). Using conventional assay conditions, VS inhibited PKA only at high concentrations (IC₅₀>400 μM); however, PKA inhibition was seen at dramatically lower concentrations of VS (IC₅₀<10 μM) when sequestration of vanadyl ions was minimized. Vanadyl appears to be the effective PKA inhibitor because sodium orthovanadate did not inhibit PKA and inhibition by vanadyl was abolished by potential chelators such as ethylenediaminetetraacetic acid or glycyl peptides. PKA inhibition by vanadyl appears to be mixed rather than strictly competitive or uncompetitive and may replicate the inhibitory effects of high concentrations of Mg²⁺. The effect of vanadyl on PKA provides a possible explanation for the effects of vanadium salts on fat tissue lipolysis and perhaps on other aspects of energy metabolism that are controlled by cAMP-dependent mechanisms. Considering the high degree of conservation of the active sites of protein kinases, vanadyl may also influence other members of this large protein family. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Sequestration of dopamine D2 receptors depends on coexpression of G-protein-coupled receptor kinases 2 or 5.

We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [3H] sulpiride-binding activity from the cell surface and by transfer of [3H] spiperone-binding activity from the membrane fraction to the light vesicle fraction in sucrose-density gradients. In COS-7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway.     

Mutational analysis of third cytoplasmic loop domains in G-protein coupling of the HM1 muscarinic receptor.

We measured dose-response curves for carbachol stimulation of phosphatidyl inositol (PI) turnover with mutants of the Hm1 muscarinic cholinergic receptor having various deletions from amino acids 219 to 358 of the large third intracellular (i3) loop (208 to 366). These deletions had only small or no effects on the ability of Hm1 transfected into HEK 293 cells to stimulate PI turnover. This result indicates that only small regions of 9 to 11 amino acids adjacent to trans-membrane domains (TMDs) 5 and 6 can be directly involved in G protein coupling. Point mutations were constructed to test the role of charged amino acids in these junctions. A triple point mutation of Hm1 (E214 A/ E216K/ E221 K), which mimics the charge distribution in Hm2 (negatively coupled to cAMP) over the first 14 amino acids of i3, and a double point mutation in the N terminal junction, K359A/K361A, both failed to affect carbachol stimulated PI turnover. Therefore, charge distribution in the loop junctions appears to play a minor role in G protein coupling of Hm1 in HEK 293 cells.     

Antibody to Epitope Tag Induces Internalization of Human Muscarinic Subtype 1 Receptor

Abstract: We have studied the effect of an antibody against the epitope EYMPME on the internalization of the human muscarinic cholinergic receptor hm1 tagged with the epitope at the amino terminus. The antibody to the tag induces internalization of the hm1 receptor within minutes after exposure of human embryonic kidney 293 cells transfected with the tagged receptor. This antibody-induced internalization is reversible following removal of the antibody. In contrast to hm1 internalization induced by the agonist carbachol, internalization induced by antibody is not blocked by the muscarinic antagonist atropine. The mechanism of antibody-mediated internalization does not appear to involve receptor dimerization by the antibody, as Fab fragments derived from the antibody also induce internalization. The pathway of antibody-induced internalization, similar to the agonist-induced process, is mediated by clathrin-coated vesicles. Furthermore, antibody treatment does not result in any second messenger production, as measured by phosphoinositide accumulation. Our data show that internalization of a G protein-coupled receptor can be triggered by interaction of the amino terminus of the receptor with an exogenous ligand and can occur independently of second messenger production. This result suggests that the receptor can exist in multiple conformations, each mediating distinct downstream events.     

Characterization of the RDC1 gene which encodes the canine homolog of a proposed human VIP receptor. Expression does not correlate with an increase in VIP binding sites.

We have isolated a portion of the canine gene encoding the orphan receptor RDC1 [1]. The complete coding sequence is contained in a single exon, and an intron divides the 5' untranslated region of RDC1 mRNA. The RDC1 protein is 94% homologous to the gene product of GPRN1, which has been proposed to serve as a VIP receptor when expressed in CHO-K1 and COS-7 cells (Sreedharan, S.P. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 4986-4990). Northern analysis indicates that CHO-K1 cells endogenously express a 2.1 kb RDC1 mRNA. However, while CHO-K1 cells possess detectable low affinity [125I]VIP binding sites, VIP binding is not altered in membranes of CHO-K1 cells expressing varying amounts of the RDC1 gene construct. Further, endogenous VIP binding is not increased by transient expression of RDC1 in COS-7 cells. Taken together, the data suggest that RDC1 is not a canine homolog of the proposed VIP receptor.     

Rapid agonist-induced loss of 125I-beta-endorphin opioid receptor sites in NG108-15, but not SK-N-SH neuroblastoma cells.

We have measured mu and delta opioid receptor sites on intact SK-N-SH and NG108-15 neuroblastoma cells, respectively, in culture. Use of 125I-beta-endorphin (beta E) as a tracer, together with beta E(6-31) to block high-affinity non-opioid binding in both cell lines, permitted the measurement of cell surface mu and delta opioid receptor sites. Labeling was at delta sites in NG108-15 cells and predominantly at mu sites in SK-N-SH cells. Pretreatment with the mu and delta agonist, DADLE, caused a rapid loss of cell surface delta receptor sites in NG108-15 cells, but failed to reduce significantly mu receptor density in SK-N-SH cells.     

Three species of Ulva (Ulvophyceae) from the Persian Gulf as potential sources of protein,essential amino acids and fatty acids

Three species of the cosmopolitan genus Ulva (U. paschima , U. chaugulii and U. ohnoi ) from the Persian Gulf were identified using morphological and molecular markers, and were analyzed for total lipids, fatty acids, proteins and amino acid profiles. Our results show that the studied Ulva species have high protein content (9?25% dry weight), contain essential fatty acids and nearly all essential amino acids. This suggests Ulva could have potential as a form of human nutrition and animal feed with a low carbon footprint.     

Internalization of the Hm1 muscarinic cholinergic receptor involves the third cytoplasmic loop

The m1 muscarinic receptor was previously shown to stimulate phosphatidyl inositol (PI) turnover and to internalize rapidly upon agonist activation. Three receptor mutants with large deletions of the third cytoplasmic loop (i3) of human Hm1, leaving only 11 and 8 amino acids at the amino and carboxy terminal junctions of i3, respectively, retained full ability to stimulate PI turnover, when expressed in U293 cells, but receptor internalization was greatly reduced in two mutants with deletions reaching close to the NH2 terminal of i3. We propose that a receptor domain located toward the amino terminal junction of i3 plays a role in Hm1 internalization.