Contribution of the cyclic nucleotide gated channel subunit, CNG-3, to olfactory plasticity in Caenorhabditis elegans.

In Caenorhabditis elegans, the AWC neurons are thought to deploy a cGMP signaling cascade in the detection of and response to AWC sensed odors. Prolonged exposure to an AWC sensed odor in the absence of food leads to reversible decreases in the animal's attraction to that odor. This adaptation exhibits two stages referred to as short-term and long-term adaptation. Previously, the protein kinase G (PKG), EGL-4/PKG-1, was shown necessary for both stages of adaptation and phosphorylation of its target, the beta-type cyclic nucleotide gated (CNG) channel subunit, TAX-2, was implicated in the short term stage. Here we uncover a novel role for the CNG channel subunit, CNG-3, in short term adaptation. We demonstrate that CNG-3 is required in the AWC for adaptation to short (thirty minute) exposures of odor, and contains a candidate PKG phosphorylation site required to tune odor sensitivity. We also provide in vivo data suggesting that CNG-3 forms a complex with both TAX-2 and TAX-4 CNG channel subunits in AWC. Finally, we examine the physiology of different CNG channel subunit combinations

Bcl-2 protein family: Implications in vascular apoptosis and atherosclerosis

Apoptosis has been recognized as a central component in the pathogenesis of atherosclerosis, in addition to the other human pathologies such as cancer and diabetes. The pathophysiology of atherosclerosis is complex, involving both apoptosis and proliferation at different phases of its progression. Oxidative modification of lipids and inflammation differentially regulate the apoptotic and proliferative responses of vascular cells during progression of the atherosclerotic lesion. Bcl-2 proteins act as the major regulators of extrinsic and intrinsic apoptosis signalling pathways and more recently it has become evident that they mediate the apoptotic response of vascular cells in response to oxidation and inflammation either in a provocative or an inhibitory mode of action. Here we address Bcl-2 proteins as major therapeutic targets for the treatment of atherosclerosis and underscore the need for the novel preventive and therapeutic interventions against atherosclerosis, which should be designed in the light of molecular mechanisms regulating apoptosis of vascular cells in atherosclerotic lesions

Lysophosphatidate Induces Chemo-Resistance by Releasing Breast Cancer Cells from Taxol-Induced Mitotic Arrest

Taxol is a microtubule stabilizing agent that arrests cells in mitosis leading to cell death. Taxol is widely used to treat breast cancer, but resistance occurs in 25-69% of patients and it is vital to understand how Taxol resistance develops to improve chemotherapy. The effects of chemotherapeutic agents are overcome by survival signals that cancer cells receive. We focused our studies on autotaxin, which is a secreted protein that increases tumor growth, aggressiveness, angiogenesis and metastasis. We discovered that autotaxin strongly antagonizes the Taxol-induced killing of breast cancer and melanoma cells by converting the abundant extra-cellular lipid, lysophosphatidylcholine, into lysophosphatidate. This lipid stimulates specific G-protein coupled receptors that activate survival signals.In this study we determined the basis of these antagonistic actions of lysophosphatidate towards Taxol-induced G2/M arrest and cell death using cultured breast cancer cells. Lysophosphatidate does not antagonize Taxol action in MCF-7 cells by increasing Taxol metabolism or its expulsion through multi-drug resistance transporters. Lysophosphatidate does not lower the percentage of cells accumulating in G2/M by decreasing exit from S-phase or selective stimulation of cell death in G2/M. Instead, LPA had an unexpected and remarkable action in enabling MCF-7 and MDA-MB-468 cells, which had been arrested in G2/M by Taxol, to normalize spindle structure and divide, thus avoiding cell death. This action involves displacement of Taxol from the tubulin polymer fraction, which based on inhibitor studies, depends on activation of LPA receptors and phosphatidylinositol 3-kinase.This work demonstrates a previously unknown consequence of lysophosphatidate action that explains why autotaxin and lysophosphatidate protect against Taxol-induced cell death and promote resistance to the action of this important therapeutic agent

Apoptosis of t(14;18)-positive lymphoma cells by a Bcl-2 interacting small molecule

Overexpression of Bcl-2 protein occurs via both t(14;18)-dependent and independent mechanisms and contributes to the survival and chemoresistance of non-Hodgkin lymphomas. HA14–1 is a nonpeptidic organic small molecule, which has been shown to inhibit the interaction of Bcl-2 with Bax, thereby interfering with the antiapoptotic function of Bcl-2. In this study, we sought to determine the in vitro efficacy of HA14–1 as a therapeutic agent for non-Hodgkin lymphomas expressing Bcl-2. Assessment of cell viability demonstrated that HA14–1 induced a dose- (IC50 = 10 μM) and time-dependent growth inhibition of a cell line (SudHL-4) derived from a t(14;18)-positive, Bcl-2-positive, non-Hodgkin lymphoma. HA14–1 effectively induced apoptosis via a caspase 3-mediated pathway but did not affect either the p38 MAPK or p44/42 MAPK pathways. Western blot analyses of Bcl-2 family proteins and other cell cycle-associated proteins were performed to determine the molecular sequelae of HA14–1-induced apoptosis. The results show down-regulation of Mcl-1 but up-regulation of p27kip1, Bad, Bcl-xL, and Bcl-2 proteins, without change in Bax levels during HA14–1-mediated apoptosis. Our findings further elucidate the cellular mechanisms accompanying Bcl-2 inhibition and demonstrate the potential of Bcl-2 inhibitors as therapeutic agents for the treatment of non-Hodgkin lymphomas

Fatigue, reduced sleep quality and restless legs syndrome in Charcot-Marie-Tooth disease: a web-based survey

To investigate the prevalence of fatigue, daytime sleepiness, reduced sleep quality, and restless legs syndrome (RLS) in a large cohort of patients with Charcot-Marie-Tooth disease (CMT) and their impact on health-related quality of life (HRQoL). Participants of a web-based survey answered the Epworth Sleepiness Scale, the Pittsburgh Sleep Quality Index, the Multidimensional Fatigue Inventory, and, if the diagnostic criteria of RLS were met, the International RLS Severity Scale. Diagnosis of RLS was affirmed in screen-positive patients by means of a standardized telephone interview. HRQoL was assessed by using the SF-36 questionnaire. Age- and sex-matched control subjects were recruited from waiting relatives of surgical outpatients. 227 adult self-reported CMT patients answered the above questionnaires, 42.9% were male, and 57.1% were female. Age ranged from 18 to 78 years. Compared to controls (n = 234), CMT patients reported significantly higher fatigue, a higher extent and prevalence of daytime sleepiness and worse sleep quality. Prevalence of RLS was 18.1% in CMT patients and 5.6% in controls (p = 0.001). RLS severity was correlated with worse sleep quality and reduced HRQoL. Women with CMT were affected more often and more severely by RLS than male patients. With regard to fatigue, sleep quality, daytime sleepiness, RLS prevalence, RLS severity, and HRQoL, we did not find significant differences between genetically distinct subtypes of CMT. HRQoL is reduced in CMT patients which may be due to fatigue, sleep-related symptoms, and RLS in particular. Since causative treatment for CMT is not available, sleep-related symptoms should be recognized and treated in order to improve quality of life

Interaction of SET domains with histones and nucleic acid structures in active chromatin

Changes in the normal program of gene expression are the basis for a number of human diseases. Epigenetic control of gene expression is programmed by chromatin modifications—the inheritable “histone code”—the major component of which is histone methylation. This chromatin methylation code of gene activity is created upon cell differentiation and is further controlled by the “SET” (methyltransferase) domain proteins which maintain this histone methylation pattern and preserve it through rounds of cell division. The molecular principles of epigenetic gene maintenance are essential for proper treatment and prevention of disorders and their complications. However, the principles of epigenetic gene programming are not resolved. Here we discuss some evidence of how the SET proteins determine the required states of target genes and maintain the required levels of their activity. We suggest that, along with other recognition pathways, SET domains can directly recognize the nucleosome and nucleic acids intermediates that are specific for active chromatin regions

The prognostic influence of bcl-2 in malignant glioma

The bcl-2 gene is one of a complex group of genes which control programmed cell death. Bcl-2 acts to extend cell survival by blocking apoptosis, and thereby may influence tumour prognosis. This study of 187 high grade gliomas reviews clinicopathological prognostic features and the relationship to bcl-2 expression. Bcl-2 immunostaining was assessed in 159 specimens from these patients, by scoring systems of 0 to 3 for intensity of scoring and proportion of cells staining. Age, histology, pre- and post-operative performance status were found to be strongly predictive of survival (log rank test P<0.0001). The type of surgery performed did not influence survival in this group of patients. The expression of bcl-2 had a significant relationship with survival (univariate Cox model P=0.0302, hazard ratio 0.8, 95% confidence interval 0.65–0.98), with increased staining associated with improved survival. Multivariate analysis showed performance status, histology and proportion of cells staining for bcl-2 to be independently predictive of survival. Bcl-2 staining was not related to histological grade of tumours

Engineering of Three-Finger Fold Toxins Creates Ligands with Original Pharmacological Profiles for Muscarinic and Adrenergic Receptors

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test “loop grafting,” a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α1A-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α1A-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles