Receptor Population Research Articles

A questionnaire based probabilistic risk assessment (PRA) of heavy metals in urban and suburban soils under different land uses and receptor populations

The choice of receptor populations is crucial for assessing the health risk posed by heavy metals in soil. A total of 457 questionnaires were conducted on 10 sensitive receptor populations, which were exposed to potentially contaminated soil under four land uses, to determine their exposure frequency (EF) and exposure time (ET) values. The exposure risk (ER: the probability of being exposed to target soil) varied between the receptor populations because of their different EF and ET values. Green maintenance workers in park soil, who had fixed outdoor working hours (6 or 7 days per week and 8 h per day), had the highest EF and ET values, while adults and children who visited parks only at weekends had the lowest EF values. Green maintenance workers, sanitation workers, and children faced the highest hazards (i.e., hazard index (HI) values) from heavy metals in soil in parks and residential areas, roadsides, and farmland, and were found to be the most sensitive receptor populations (MSRPs) for these land uses. The 90th percentile of the HI value for the four land uses based on MSRPs followed the order of roadsides (0.48) > parks (0.44) > residential areas (0.21) > suburban farmlands(0.18), and all were lower than 1, indicating a virtually safe level. Washing hands immediately and wearing shoes when working on farmland considerably reduced the HI values by 56.6%. The EF, ET, and body weight (BW) values were the most sensitive parameters in the health risk assessment. The results suggested that the type of receptor population will greatly affect the results of a health risk assessment for soil pollutants. It is necessary to select the risk receptors carefully based on the land use types.

Using Domain Based Latent Personal Analysis of B Cell Clone Diversity Patterns to Identify Novel Relationships Between the B Cell Clone Populations in Different Tissues.

The B cell population is highly diverse and very skewed. It is divided into clones (B cells with a common mother cell). It is thought that each clone represents an initial B cell receptor specificity. A few clones are very abundant, comprised of hundreds or thousands of B cells while the majority have only a few cells per clone. We suggest a novel method - domain-based latent personal analysis (LPA), a method for spectral exploration of entities in a domain, which can be used to find the spectral spread of sub repertoires within a person. LPA defines a domain-based spectral signature for each sub repertoire. LPA signatures consist of the elements, in our case - the clones, that most differentiate the sub repertoire from the person’s abundance of clones. They include both positive elements, which describe overabundant clones, and negative elements that describe missing clones. The signatures can also be used to compare the sub repertoires they represent to each other. Applying LPA to compare the repertoires found in different tissues, we reiterated previous findings that showed that gut and blood tissues have separate repertoires. We further identify a third branch of clonal patterns typical of the lymphatic organs (Spleen, MLN, and bone marrow) separated from the other two categories. We developed a python version of LPA analysis that can easily be applied to compare clonal distributions - https://github.com/ScanLab-ossi/LPA. It could also be easily adapted to study other skewed sequence populations used in the analysis of B cell receptor populations, for instance, k-mers and V gene usage. These analysis types should allow for inter and intra-repertoire comparisons of diversity, which could revolutionize the way we understand repertoire changes and diversity.

Metabolic Depletion of Sphingolipids Reduces Cell Surface Population of the Human Serotonin1A Receptor due to Impaired Trafficking.

Sphingolipids and their metabolites are increasingly implicated in the pathogenesis of many metabolic and neurological diseases. It has been postulated that sphingolipids coalesce with cholesterol to form laterally segregated lipid domains that are involved in protein sorting and trafficking. In this work, we have explored the effect of metabolic depletion of sphingolipids on cell surface expression of the human serotonin1A receptor, a neurotransmitter G protein-coupled receptor. We used fumonisin B1 (FB1), a fungal mycotoxin, to inhibit sphingolipid biosynthesis in HEK-293 cells stably expressing the human serotonin1A receptor. Our results obtained using flow cytometric analysis and confocal microscopic imaging show that the cell surface population of the serotonin1A receptor is reduced under sphingolipid-depleted condition. Western blot analysis confirmed that there was no significant difference in total cellular level of the serotonin1A receptor upon depletion of sphingolipids. Interestingly, the effect of FB1 on serotonin1A receptor population was reversed upon replenishment with sphingolipids. These results indicate that sphingolipid depletion does not alter total cellular receptor levels, but impairs serotonin1A receptor trafficking to the cellular plasma membrane. These results could provide mechanistic insights into the role of sphingolipids in modulation of neurotransmitter receptor signaling and trafficking in health and disease.

Efficient expression of concatenated α1β2δ and α1β3δ GABAA receptors, their pharmacology and stoichiometry.

GABAA receptors containing δ-subunits are notorious for being difficult to study in vitro due to heterogeneity of expressed receptor populations and low GABA-evoked current amplitudes. Thus, there are some published misconceptions and contradictory conclusions made regarding the pharmacology and stoichiometry of δ-containing receptors. The aim of this study was to obtain robust homogenous expression of α1βδ receptors for in-depth investigation. Novel δ-containing pentameric concatenated constructs were designed. The resulting α1β2δ and α1β3δ GABAA receptor concatemers were investigated by two-electrode voltage-clamp electrophysiology using Xenopus laevis oocytes. First, while homogenous α1βδ GABAA receptor pools could not be obtained by manipulating the ratio of injected cRNAs of free α1, β2/3, and δ subunits, concatenated pentameric α1β2δ and α1β3δ constructs resulted in robust expression levels of concatemers. Second, by using optimised constructs that give unidirectional assembly of concatemers, we found that the δ subunit cannot directly participate in GABA binding and receptor activation. Hence, functional δ-containing receptors are likely to all have a conventional 2α:2β:1δ stoichiometry arranged as βαβαδ when viewed counterclockwise from the extracellular side. Third, α1β2/3δ receptors were found to express efficiently in X. laevis oocytes but have a low estimated open probability of ~0.5% upon GABA activation. Because of this, these receptors are uniquely susceptible to positive allosteric modulation by, for example, neurosteroids. Our data answer important outstanding questions regarding the pharmacology and stoichiometry of α1δ-containing GABAA receptors and pave the way for future analysis and drug discovery efforts.

Development of Per and Polyfluoroalkyl Substances Ecological Risk-Based Screening Levels.

Evaluation of chemical risks to threatened and endangered species is a requirement for Superfund ecological risk assessments; however, screening levels to evaluate the potential for toxicity associated with ecological receptor exposure to per- and polyfluoroalkyl substances (PFAS) are lacking. Therefore, PFAS risk-based screening levels (RBSLs) were developed. Wildlife RBSLs were developed using surrogate receptors selected to be representative of threatened and endangered species with different habitat types, feeding guilds, and trophic levels. Published uptake and toxicity data were combined with receptor exposure factors to derive RBSLs for terrestrial and aquatic wildlife for several PFAS, including perfluorononanoic acid, perfluorooctanesulfonic acid, perfluorooctanoic acid, perfluorohexanoic acid, perfluorobutanesulfonic acid, and pentafluorobenzoic acid. Uptake information for surrogate PFAS were considered to calculate RBSLs for PFAS with toxicity data and insufficient bioaccumulation data to develop an RBSL. Both no-observed-adverse effect level (NOAEL)- and lowest-observed-adverse effect level-based wildlife RBSLs were calculated to allow for a range of risk estimates appropriate to individual threatened and endangered species and populations of nonlisted wildlife receptors, respectively. Recommended water quality RBSLs protective of aquatic life were developed for 23 PFAS based on published literature reviews and peer-reviewed aquatic toxicity studies and Great Lakes Initiative methodology. For wildlife receptors, NOAEL RBSLs ranged from to 0.013 to 340 mg/kg for soil, 0.0014 to 370 mg/kg for sediment, and 0.000075 to 1600 mg/L for surface water. For aquatic life, chronic RBSLs ranged from 0.00022 to 3.4 mg/L. For terrestrial plants and soil invertebrates, the no-observed-effect concentration screening levels range from 0.084 to 642 mg/kg and from 1 to 50 mg/kg, respectively. Environ Toxicol Chem 2021;40:921-936. © 2020 SETAC.

Reciprocal stabilization of glycine receptors and gephyrin scaffold proteins at inhibitory synapses

Postsynaptic scaffold proteins immobilize neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptors (GlyRs). Gephyrin and GlyRs can also interact outside of synapses, where they form receptor-scaffold complexes. Although several models for the formation of postsynaptic scaffold domains in the presence of receptor-scaffold interactions have been advanced, a clear picture of the coupled dynamics of receptors and scaffold proteins at synapses is lacking. To characterize the GlyR and gephyrin dynamics at inhibitory synapses, we performed fluorescence time-lapse imaging after photoconversion to directly visualize the exchange kinetics of recombinant Dendra2-gephyrin in cultured spinal cord neurons. Immuno-immobilization of endogenous GlyRs with specific antibodies abolished their lateral diffusion in the plasma membrane, as judged by the lack of fluorescence recovery after photobleaching. Moreover, the cross-linking of GlyRs significantly reduced the exchange of Dendra2-gephyrin compared with control conditions, suggesting that the kinetics of the synaptic gephyrin pool is strongly dependent on GlyR-gephyrin interactions. We did not observe any change in the total synaptic gephyrin levels after GlyR cross-linking, however, indicating that the number of gephyrin molecules at synapses is not primarily dependent on the exchange of GlyR-gephyrin complexes. We further show that our experimental data can be quantitatively accounted for by a model of receptor-scaffold dynamics that includes a tightly interacting receptor-scaffold domain, as well as more loosely bound receptor and scaffold populations that exchange with extrasynaptic pools. The model can make predictions for single-molecule data such as typical dwell times of synaptic proteins. Taken together, our data demonstrate the reciprocal stabilization of GlyRs and gephyrin at inhibitory synapses and provide a quantitative understanding of their dynamic organization.

Cell type-specific genetic reconstitution of CB1 receptor subsets to assess their role in exploratory behaviour, sociability, and memory.

Several studies support the notion that exploratory behaviour depends on the functionality of the cannabinoid type 1 (CB1) receptor in a cell type-specific manner. Mice lacking the CB1 receptor in forebrain GABAergic or dorsal telencephalic glutamatergic neurons have served as essential tools revealing the necessary CB1 receptor functions in these two neuronal populations. However, whether these specific CB1 receptor populations are also sufficient within the endocannabinoid system for wild-type-like exploratory behaviour has remained unknown. To evaluate cell-type-specific sufficiency of CB1 receptor signalling exclusively in dorsal telencephalic glutamatergic neurons (Glu-CB1-RS) or in forebrain GABAergic neurons (GABA-CB1-RS), we utilised a mouse model in which CB1 receptor expression can be reactivated conditionally at endogenous levels from a complete CB1-KO background. The two types of conditional CB1-rescue mice were compared with CB1 receptor-deficient [no reactivation (Stop-CB1)] and wild-type [ubiquitous reactivation of endogenous CB1 receptor (CB1-RS)] controls to investigate the behavioural consequences. We evaluated social and object exploratory behaviour in four different paradigms. Remarkably, the reduced exploration observed in Stop-CB1 animals was rescued in Glu-CB1-RS mice and sometimes even surpassed CB1-RS (wild-type) exploration. In contrast, GABA-CB1-RS animals showed the lowest exploratory drive in all paradigms, with an even stronger phenotype than Stop-CB1 mice. Interestingly, these effects weakened with increasing familiarity with the environment, suggesting a causal role for altered neophobia in the observed phenotypes. Taken together, using our genetic approach, we were able to substantiate the opposing role of the CB1 receptor in dorsal telencephalic glutamatergic versus forebrain GABAergic neurons regarding exploratory behaviour.

Looking for Novelty in an "Old" Receptor: Recent Advances Toward Our Understanding of GABAARs and Their Implications in Receptor Pharmacology.

Diverse populations of GABAA receptors (GABAARs) throughout the brain mediate fast inhibitory transmission and are modulated by various endogenous ligands and therapeutic drugs. Deficits in GABAAR signaling underlie the pathophysiology behind neurological and neuropsychiatric disorders such as epilepsy, anxiety, and depression. Pharmacological intervention for these disorders relies on several drug classes that target GABAARs, such as benzodiazepines and more recently neurosteroids. It has been widely demonstrated that subunit composition and receptor stoichiometry impact the biophysical and pharmacological properties of GABAARs. However, current GABAAR-targeting drugs have limited subunit selectivity and produce their therapeutic effects concomitantly with undesired side effects. Therefore, there is still a need to develop more selective GABAAR pharmaceuticals, as well as evaluate the potential for developing next-generation drugs that can target accessory proteins associated with native GABAARs. In this review, we briefly discuss the effects of benzodiazepines and neurosteroids on GABAARs, their use as therapeutics, and some of the pitfalls associated with their adverse side effects. We also discuss recent advances toward understanding the structure, function, and pharmacology of GABAARs with a focus on benzodiazepines and neurosteroids, as well as newly identified transmembrane proteins that modulate GABAARs.

Establishment versus population growth in spatio-temporally varying environments.

We consider situations where repeated invasion attempts occur from a source population into a receptor population over extended periods of time. The receptor population contains two locations that provide different expected offspring numbers to invaders. There is demographic stochasticity in offspring numbers. In addition, temporal variation causes local invader fitnesses to vary. We show that effects of environmental autocorrelation on establishment success depend on spatial covariance of the receptor subpopulations. In situations with a low spatial covariance this effect is positive, whereas high spatial covariance and/or high migration probabilities between the subpopulations causes the effect to be negative. This result reconciles seemingly contradictory results from the literature concerning effects of temporal variation on population dynamics with demographic stochasticity. We study an example in the context of genetic introgression, where invasions of cultivar plant genes occur through pollen flow from a source population into wild-type receptor populations, but our results have implications in a wider range of contexts, such as the spread of exotic species, metapopulation dynamics and epidemics.

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics.

SummarySingle-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors in vivo in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.

Increased Adhesiveness of Red Blood Cells from Sickle Cell Trait Carriers after Exposure to Daunirubicin Breast Cancer Chemotherapeutic Agent

Introduction: African-Americans (AA) with breast cancer continue to experience worse morbidity and mortality than white patients. In addition to barriers to care, an active area of investigation has been the etiology of the increased risk adverse events (AEs) related to chemotherapy. An emerging question is if sickle cell trait (i.e., heterozygotes for hemoglobin beta gene), present in an estimated 1 in 12 of the AA population, confers increased susceptibility to chemotherapy-related complications. We posit that conditions of high physiological stress might be manifest during systemic treatment with toxic agents resulting in alterations of red blood cells (RBCs). Using an experimental biomechanical model system, we hypothesized that RBCs from SCT carriers would be prone to increased adhesiveness after exposure to a common systemic anti-cancer agent used in breast cancer. Increased adhesiveness of RBCs can be a precipitating factor of blocked vasculature and subsequent sickling crises, potentially leading to AEs during treatment. Methods: Our study testedex vivoRBCs from two groups of healthy female participants: 20 African-American sickle cell trait carriers (AA-SCT); and 15 white subjects with wild-type hemoglobin (W-WT). Isolated RBCs were treated with scaled Daunorubicin (DNR) doses. The unbinding forces between αvβ3 ligands and intercellular adhesion molecule-4 (ICAM-4) receptors are reported using the frequency distribution, which states the percentage of events whose unbinding forces are within each width of the bin. We then compared the median values of the forces measured in experiments without and with treatment for each drug. The collective frequency (CF%) is related to the population of active ICAM-4 receptors and is defined as the percentage of all unbinding events divided by the total number of measurements, which is 32 × 32 =1024 for each cell multiplied by the number of tested cells for each blood sample. Results: For AA-SCT RBCs, pre-treatment baseline CF of active ICAM-4 receptors was 4.88 ± 0.87%, which was significantly increased after administration of DNR (13.13 ± 2.30%, p<0.0001). In contrast, treatment of W-WT RBCs held the CF of active ICAM-4 receptors at a comparable level as untreated RBCs (9.54 ± 1.48%; 7.47 ± 1.07%, p=0.50, respectively). Conclusion: Our findings could support hypotheses for adhesion-related RBC clumping among AAs with SCT during systemic treatment with anti-neoplastic agents, and, putatively, resultant AEs. Given past studies showing African-American women with breast cancer have higher rates of self-withdrawal from treatment, further exploration with additional systemic agents are warranted. Our novel study is limited by a small sample size as well lack of potential confounding factors in analyses. Future studies are planned with additional agent(s) and AAs without SCT. Should it be confirmed that SCT carrier status predicts RBC alterations during systemic treatment for breast cancer, and are linked to subsequent adverse events, it could lead to precision treatment planning. Disclosures Andemariam: Novartis:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding;Hemanext:Membership on an entity's Board of Directors or advisory committees;Global Blood Therapeutics:Consultancy, Membership on an entity's Board of Directors or advisory committees;Vertex:Honoraria;Imara:Research Funding;Emmaus:Membership on an entity's Board of Directors or advisory committees;bluebird bio:Consultancy, Membership on an entity's Board of Directors or advisory committees;NovoNordisk:Consultancy, Membership on an entity's Board of Directors or advisory committees;CRISPR/Vertex:Consultancy, Membership on an entity's Board of Directors or advisory committees;CHNCT:Consultancy;Accordant:Membership on an entity's Board of Directors or advisory committees;Guidepoint:Honoraria;Sanofi Genzyme:Consultancy, Membership on an entity's Board of Directors or advisory committees;Terumo BCT:Consultancy, Membership on an entity's Board of Directors or advisory committees;Roche:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;Cyclerion:Consultancy, Membership on an entity's Board of Directors or advisory committees;Pfizer:Consultancy, Membership on an entity's Board of Directors or advisory committees.

Two Distinct Populations of α1α6-Containing GABAA-Receptors in Rat Cerebellum.

GABAA receptors are pentameric GABA-gated chloride channels. The existence of 19 different subunits (six α, three β, three γ, δ, ε, θ, π, and three ρ) in mammalian systems gives rise to an enormous theoretical diversity of GABAA receptor subtypes with distinct subunit composition and unique pharmacological properties. These receptors are already now the drug targets of several clinically used compounds, such as benzodiazepines, anesthetics, and many more. There is a constant quest to identify novel molecules and possible future drug targets: Currently, α6-containing GABAA receptors are being discussed in the context of treating sensorimotor gating deficits in neuropsychiatric disorders, such as tic disorders and schizophrenia. Therefore, we aim to learn more about α6-containing GABAA receptors. They are mostly expressed in the cerebellar granule cell layer, where they form the following subtypes: α6βxγ2, α1α6βxγ2, α6βxδ, and α1α6βxδ. In former studies, α1α6βxγ2-containing GABAA receptors were considered a single receptor population. In the current study, we investigate the possibility, that this population can consist of two subgroups with alternative arrangements depending if α1 neighbors γ2 (forming a “diazepam-sensitive” receptor), or if α6 neighbors γ2 (forming a “diazepam-insensitive” receptor) and aimed to prove the existence of both subtypes in native tissue. We performed immunoprecipitation experiments on rat cerebellar lysates using α1- or α6 subunit-specific antibodies followed by radioligand binding assays with either 3H-flunitrazepam or 3H-Ro 15-4513. Indeed, we were able to prove the existence of two distinct populations of α1α6-containing GABAA-receptors and could quantify the different receptor populations: α1βxγ2 receptors constitute approximately 60% of all γ2-containing receptors in the rat cerebellum, α6βxγ2 about 20%, and both isoforms of α1α6βxγ2 9–15% each. The simple classification of GABAA-receptors into αx-containing subtypes seems not to reflect the complexity of nature; those receptors are more diverse than previously thought.

Identification of destabilizing SNPs in SARS-CoV2-ACE2 protein and spike glycoprotein: implications for virus entry mechanisms

COVID-19 an outbreak of a novel corona virus originating from Wuhan, China in December 2019 has now spread across the entire world and has been declared a pandemic by WHO. Angiotensin converting enzyme 2 (ACE2) is a receptor protein that interacts with the spike glycoprotein of the host to facilitate the entry of coronavirus (SARS-CoV-2) hence causing the disease (COVID-19). Our experimental design is based on bioinformatics approach that combines sequence, structure and consensus based tools to label a protein coding single nucleotide polymorphism (SNP) as damaging/deleterious or neutral. The interaction of wildtype ACE2-spike glycoprotein and their variants were analyzed using docking studies. The mutations W461R, G405E and F588S in ACE2 receptor protein and population specific mutations P391S, C12S and G1223A in the spike glycoprotein were predicted as highly destabilizing to the structure of the bound complex. So far, no extensive in silico study has been reported that identifies the effect of SNPs on Spike glycoprotein-ACE2 interaction exploring both sequence and structural features. To this end, this study conducted an in-depth analysis that facilitates in identifying the mutations that blocks the interaction of two proteins that can result in stopping the virus from entering the host cell. Communicated by Ramaswamy H. Sarma

Exploring Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Autoproteolysis Process by Molecular Simulations: Hints for Drug Design.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a notable target for the treatment of hypercholesterolemia because it regulates the population of the low-density lipoprotein receptor (LDLR) on liver cells. The PCSK9 zymogen is a serine protease that spontaneously undergoes a double self-cleavage step. Available X-ray structures depict the PCSK9 mature state, but the atomic details of the zymogen state of the enzyme are still unknown. Additionally, why the protease activity of PCSK9 is blocked after the second autoprocessing step remains unclear, as this deviates from other members of the PCSK family. By performing constant-pH molecular dynamics (MD) simulations, we investigated the protonation state of the catalytic triad of PCSK9 and found that it strongly influences the catalytic properties of the enzyme. Moreover, we determined the final step of the maturation process by classical and steered MD simulations. This study could facilitate the identification of ligands capable of interfering with the PCSK9 maturation process.

Position Review: Functional Selectivity in Mammalian Olfactory Receptors.

There is increasing appreciation that G-protein-coupled receptors (GPCRs) can initiate diverse cellular responses by activating multiple G proteins, arrestins, and other biochemical effectors. Structurally different ligands targeting the same receptor are thought to stabilize the receptor in multiple distinct active conformations such that specific subsets of signaling effectors are engaged at the exclusion of others, creating a bias toward a particular outcome, which has been referred to as ligand-induced selective signaling, biased agonism, ligand-directed signaling, and functional selectivity, among others. The potential involvement of functional selectivity in mammalian olfactory signal transduction has received little attention, notwithstanding the fact that mammalian olfactory receptors comprise the largest family of mammalian GPCRs. This position review considers the possibility that, although such complexity in G-protein function may have been lost in the specialization of olfactory receptors to serve as sensory receptors, the ability of olfactory receptor neurons (ORNs) to function as signal integrators and growing appreciation that this functionality is widespread in the receptor population suggest otherwise. We pose that functional selectivity driving 2 opponent inputs have the potential to generate an output that reflects the balance of ligand-dependent signaling, the direction of which could be either suppressive or synergistic and, as such, needs to be considered as a mechanistic basis for signal integration in mammalian ORNs.

Functions of interleukin-6 in ovulation of female climbing perch, Anabas testudineus

In mammals, interleukin 6 (IL-6) has an important function during ovulation, however, the functions of IL-6 in fish have not been elucidated. In the present study, there was quantification of de novo synthesis of ovarian IL-6 and tumor necrosis factor-alpha (TNFα) in control and hCG-treated fish and results were compared with those from an in vitro study where there was evaluation of the regulatory functions of gonadotropins and TNFα of IL-6 secretions. Relatively greater concentrations of ovarian IL-6 at the post-GVBD (post-germinal vesicle breakdown) stage indicates IL-6 modulates ovulatory processes. The hCG-induced increase in relative abundance of IL-6 (in vitro) mRNA transcript and secretion from the ovary were attenuated when there was administration of the inhibitor of TNFα secreting enzyme, TAPI-I, which indicates TNFα modulates IL-6 secretion. Treatments with IL-6 induced a marked increase in ovulation rate in vitro when there was induction of activating matrix metalloproteinase (MMP). Furthermore, treatment with IL-6 resulted in production of prostaglandin as indicated by the IL-6 induced increase in the abundance of ptgs2 mRNA transcript in the ovary of Anabas testudineus. Furthermore, results indicate the source of IL-6 in the ovary is the granulosa cells with secretion of IL-6 being induced by the additions of hCG and TNFα in the medium. There was also an IL-6-induced increase in abundance of receptors (IL-6 Rα and gp130) to which it binds indicating IL-6 autoregulates this population of receptors. Results from this study, for the first time, elucidate the reproductive functions of IL-6 in a teleost fish.

CD14 Gene (−159 C>T) Polymorphism and its Surface Expression on Monocytes in Pulmonary Tuberculosis Patients

BACKGROUND: Tuberculosis (TB) is an infectious disease that affects millions of people around the world. The innate immune response against TB starts by interaction of several receptors on monocytes with mycobacterium tuberculosis (MTB). CD14 is one of these receptors present on the monocytes which facilitate the entry of MTB into the cell. Certain polymorphisms in CD14 gene, for example, CD14 (−159 C>T) in the promotor region have suggested susceptibility of TB.
 AIM: This study was designed to determine and compare CD14 (−159 C>T) gene polymorphism and its surface expression in pulmonary TB patients (before and during anti-TB treatment) and healthy controls.
 MATERIALS AND METHODS: The study population comprised three groups (pulmonary TB patients before treatment, pulmonary TB patients during treatment, and healthy controls) whereas 53 blood samples were collected from each group. The percentage of monocytes and CD14 mean fluorescence intensity (MFI) was measured by flow cytometry whereas polymerase chain reaction-restriction fragment length polymorphism was used to determine gene polymorphism.
 RESULTS: CD14 MFI was significantly high in healthy controls than in TB patients (432 as compared to 193 and 365, p < 0.0001). There was no significant difference in CD14 single nucleotide polymorphism allele frequencies or genotypes between TB patients and healthy controls.
 CONCLUSION: CD14 gene (−159 C>T) polymorphism was not associated with pulmonary TB disease in a sample of Pakistani population and surface expression of CD14 receptor on peripheral blood monocytes decreases with active TB disease and during treatment.

Involvement of Histamine 2 Receptor in Alpha 1 Adrenoceptor Mediated Cardiac Hypertrophy and Oxidative Stress in H9c2 Cardio Myoblasts.

Despite the involvement of ɑ1adrenergic (ɑ1AR) and Histamine 2 receptors (H2R) in cardiac hypertrophy (CH), their relationship is yet to be studied. Our study investigated interrelationship between them using in vitro CH model. H9c2 cardiomyoblasts were exposed to phenylephrine (ɑ1AR agonist-50μM) in the presence, the absence of famotidine (H2R antagonist-10μM) and BAY 11-7082 (NF-kB inhibitor-10μM). The impact of ɑ1AR stimulation on H2R expression and oxidative stress was assessed. Hypertrophic indices were assessed from activities of enzymatic mediators of cardiac hypertrophy, total protein content, BNP levels and cell volume. Additionally, the inverse agonistic property of famotidine and NFkB activity was also studied. ɑ1AR-induced H2R expression, oxidative stress and hypertrophic indices were significantly abolished by famotidine and pharmacological inhibitor of NFkB. Increase in constitutive activity of H2R was noticed correlating with increased receptor population. These results suggest involvement of NFkB-mediated upregulation of H2R in ɑ1AR-mediated CH.

The distinctive assembly pattern of ε subunit in ternary α1β3ε and binary β3ε GABAA receptors

Among all GABAA receptor subunits, ε subunit is a more recent discovery. ε subunit-containing GABAA receptors exhibit spontaneous channel activity, rapid desensitisation, low sensitivity to Zn2+, smaller GABAmediated current amplitudes and an insensitivity to benzodiazepines, despite displaying an agonistic effect at higher benzodiazepine concentrations. The promiscuous role of the ε subunit, co-assembling with other subunits forming GABAARs, may add to the complexities of the pharmacological properties of GABAARs; however, these varying pharmacological responses can be used to distinguish varying subunit combinations of these receptors. Using two electrode voltage-clamped electrophysiology, we investigated the GABAA α1β3ε and β3ε receptors and explored the effects of different stoichiometries of these receptor subtypes by varying the relative ratios of α1/β3/ε (for α1β3ε receptors) and β3/ε (for β3ε receptors) subunit complementary RNA injections into Xenopus laevis oocytes. We discovered the existence of different populations of GABAA α1β3ε and β3ε receptors, due to subunit ratio variation, in which receptors formed at each injection ratio showed different level of GABA sensitivities, spontaneous current activities and Zn2+ mediated current inhibition. These unique pharmacological features are tightly associated with various subtypes of GABAA receptors contributed by the unique assembly pattern of ε subunit.

Pharmacology of Metabotropic Glutamate Receptor 2 Homo‐ and Heterodimers

Metabotropic Glutamate Receptors (mGluRs) are a family of class C g‐protein coupled receptors (GPCRs) that respond to glutamate as their native agonist. The eight members of the mGluR family are widely expressed across the nervous and are primarily charged with shaping and fine‐tuning neuronal excitability and synaptic efficacy. Like most class C GPCRs, mGluRs are obligate dimer proteins. All mGluRs maintain the capacity to homodimerize, but recent work has demonstrated that heterodimeric mGluRs can form and exist natively. Further, studies of the mGluR2‐4 heterodimer have demonstrated that this heterodimeric receptor exhibits a distinct and unpredicted pharmacological profile compared to either the mGluR2 homodimer or the mGluR4 homodimer. These findings underscore the importance of better understanding mGluR heterodimer pharmacology, particularly with preclinical studies showing promising results for leveraging mGluRs as therapeutic targets for a wide variety of conditions. To develop a better understanding of the behavior of mGluR2, we used a multifaceted approach to investigate mGluR2s dimerization preferences, whole cell signaling, and individual receptor signaling when expressed by itself, or with other mGluRs. To test dimerization preference, we utilize a split luciferase assay. To test whole cell signaling, we utilize a bioluminescent resonance energy transfer (BRET) based G‐protein Beta‐Gamma sensor. Finally, to detect and determine the pharmacological profile of specific mGluR2 heterodimers, we combine the split luciferase technique with BRET, allowing us to isolate signals from a specific dimer within a population of receptors. These studies will help define the pharmacological profile of various mGluR2 containing heterodimers, as well as develop new tools to help further interrogate the pharmacology of other dimer complexes. These new tools are also high‐throughput compatible, allowing for the development of more advanced drug screens to identify new molecules that specifically target mGluR heterodimers.Support or Funding InformationUniversity of Rochester Harold C Hodge Memorial Fund and the University of Rochester Drug Targets and Mechanism Pilot Award