Rescue of misrouted GnRHR mutants reveals its constitutive activity

G protein-coupled receptors (GPCR) play central roles in almost all physiological functions, and mutations in GPCR are responsible for over 30 hereditary diseases associated with loss or gain of receptor function. Gain of function mutants are frequently described as having constitutive activity (CA), that is, they activate effectors in the absence of agonist occupancy. Although many GPCR have mutants with CA, the GnRH receptor (GnRHR) was not, until 2010, associated with any CA mutants. The explanation for the failure to observe CA appears to be that the quality control system of the cell recognizes CA mutants of GnRHR as misfolded and retains them in the endoplasmic reticulum. In the present study, we identified several human (h)GnRHR mutants with substitutions in transmembrane helix 6 (F(272)K, F(272)Q, Y(284)F, C(279)A, and C(279)S) that demonstrate varying levels of CA after being rescued by pharmacoperones from different chemical classes and/or deletion of residue K(191), a modification that increases trafficking to the plasma membrane. The movement of the mutants from the endoplasmic reticulum (unrescued) to the plasma membrane (after rescue) is supported by confocal microscopy. Judging from the receptor-stimulated inositol phosphate production, mutants F(272)K and F(272)Q, after rescue, display the largest level of CA, an amount that is comparable with agonist-stimulated activation. Because mutations in other GPCR are, like the hGnRHR, scrutinized by the quality control system, this general approach may reveal CA in receptor mutants from other systems. A computer model of the hGnRHR and these mutants was used to evaluate the conformation associated with CA.     

Light Intensity Physical Activity and Sedentary Behavior in Relation to Body Mass Index and Grip Strength in Older Adults: Cross-Sectional Findings from the Lifestyle Interventions and Independence for Elders (LIFE) Study

BackgroundIdentifying modifiable determinants of fat mass and muscle strength in older adults is important given their impact on physical functioning and health. Light intensity physical activity and sedentary behavior are potential determinants, but their relations to these outcomes are poorly understood. We evaluated associations of light intensity physical activity and sedentary time—assessed both objectively and by self-report—with body mass index (BMI) and grip strength in a large sample of older adults.MethodsWe used cross-sectional baseline data from 1130 participants of the Lifestyle Interventions and Independence for Elders (LIFE) study, a community-dwelling sample of relatively sedentary older adults (70-89 years) at heightened risk of mobility disability. Time spent sedentary and in light intensity activity were assessed using an accelerometer worn for 3–7 days (Actigraph GT3X) and by self-report. Associations between these exposures and measured BMI and grip strength were evaluated using linear regression.ResultsGreater time spent in light intensity activity and lower sedentary times were both associated with lower BMI. This was evident using objective measures of lower-light intensity, and both objective and self-reported measures of higher-light intensity activity. Time spent watching television was positively associated with BMI, while reading and computer use were not. Greater time spent in higher but not lower intensities of light activity (assessed objectively) was associated with greater grip strength in men but not women, while neither objectively assessed nor self-reported sedentary time was associated with grip strength.ConclusionsIn this cross-sectional study, greater time spent in light intensity activity and lower sedentary times were associated with lower BMI. These results are consistent with the hypothesis that replacing sedentary activities with light intensity activities could lead to lower BMI levels and obesity prevalence among the population of older adults. However, longitudinal and experimental studies are needed to strengthen causal inferences.     

Molecularly imprinted composite cryogels for hemoglobin depletion from human blood

A molecularly imprinted composite cryogel (MICC) was prepared for depletion of hemoglobin from human blood prior to use in proteome applications. Poly(hydroxyethyl methacrylate) based MICC was prepared with high gel fraction yields up to 90%, and characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, swelling studies, flow dynamics and surface area measurements. MICC exhibited a high binding capacity and selectivity for hemoglobin in the presence of immunoglobulin G, albumin and myoglobin. MICC column was successfully applied in fast protein liquid chromatography system for selective depletion of hemoglobin for human blood. The depletion ratio was highly increased by embedding microspheres into the cryogel (93.2%). Finally, MICC can be reused many times with no apparent decrease in hemoglobin adsorption capacity. Copyright © 2014 John Wiley & Sons, Ltd.     

DNA damage causes TP53-dependent coupling of self-renewal and senescence pathways in embryonal carcinoma cells

Comment on: Wu R, et al. Clin Cancer Res 2011; 17:7359–72     

Immunity status against coxsackie viruses B in 25 patients with acute myocarditis

Viruses are an important cause of myocarditis, particularly the enterovirus group B coxsackievirus. Viral infection may be suspected on the basis of history and presentation and can be proved by direct or serological identification of virus. Twenty-five patients were diagnosed with acute myocarditis and were investigated with a serologic test battery covering Coxsackie viruses group B types 1 to 5 at the National Reference Center for Enteroviruses in Cantacuzino Institute Bucharest, Romania. A possible Coxsakie B virus etiology could be documented in 11 from 25 cases with acute myocarditis and high titers against Coxsackie virus B type 2 (1 patient), type 3 (5 patients) and type 5 (in 4 patients) were detected. In one HIV positive patient (17 years old), a concomitant infection with Coxsackie virus B types 2 and 4 was detected. The earlier detection of enterovirus myocarditis could be followed by antiviral therapies with a potential therapeutic role.     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

The role of estrogens in the regulation of lactate dehydrogenase activity and its submolecular organization in rat anterior pituitary

Estrogens increase LDH activity and decrease H/M subunit ratio in rat anterior pituitary in both the experimental circumstances and the physiological conditions. The cellular messengers mediating estrogenic effect are structure- and stereospecific. The activity increasing and subunit ratio decreasing potency of the three tested estrogens was of following decreasing order: 17 beta-estradiol, estrone, and estriol. 17 alpha-estradiol did not affect activity parameters and submolecular organisation of the enzyme. The estrogen induced activity increase is consequence of the enhanced de novo enzyme protein synthesis which could be inhibited by Actinomycin-D. The lack of adrenocorticoids did not involve the alteration of LDH activity and H/M ratio in female rat anterior pituitary. Accordingly, these steroids do not mediate estrogenic action. 17 beta-estradiol had a substantial increasing effect on LDH activity in the subrenal implanted pituitary homografts and decreased H/M subunit ratio. Pituitary LDH activity in androgenized female rats decreased only after the removal of the polycystic ovaries. The two latter observations suggest that hypothalamic hormones are not involved in the regulation of pituitary LDH activity and its submolecular organization. De novo synthesis of LDH enzyme protein and the regulation its submolecular organization is induced by the direct action on anterior pituitary cells of the estrogens.