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Drug-induced gastrointestinal toxicity (GIT) is a common treatment-emergent adverse event that can negatively impact dosing, thereby limiting efficacy and treatment options for patients. An in vitro assay of GIT is needed to address patient variability, mimic the microphysiology of the gut, and accurately predict drug-induced GIT. Primary human ileal organoids (termed 'enteroids') have proven useful for stimulating intestinal stem cell proliferation and differentiation to multiple cell types present in the gut epithelium. Enteroids have enabled characterization of gut biology and the signaling involved in the pathogenesis of disease. Here, enteroids were differentiated from four healthy human donors and assessed for culture duration-dependent differentiation status by immunostaining for gut epithelial markers lysozyme, chromogranin A, mucin, and sucrase isomaltase. Differentiated enteroids were evaluated with a reference set of 31 drugs exhibiting varying degrees of clinical incidence of diarrhea, a common manifestation of GIT that can be caused by drug-induced thinning of the gut epithelium. An assay examining enteroid viability in response to drug treatment demonstrated 90% accuracy for recapitulating the incidence of drug-induced diarrhea. The human enteroid viability assay developed here presents a promising in vitro model for evaluating drug-induced diarrhea., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Drug-induced gastrointestinal toxicities (DI-GITs) are among the most common adverse events in clinical trials. High prevalence of DI-GIT has persisted among new drugs due in part to the lack of robust experimental tools to allow early detection or to guide optimization of safer molecules. Developing in vitro assays for the leading GI toxicities (nausea, vomiting, diarrhoea, constipation, and abdominal pain) will likely involve recapitulating complex physiological properties that require contributions from diverse cell/tissue types including epithelial, immune, microbiome, nerve, and muscle. While this stipulation may be beyond traditional 2D monocultures of intestinal cell lines, emerging 3D GI microtissues capture interactions between diverse cell and tissue types. These interactions give rise to microphysiologies fundamental to gut biology. For GI microtissues, organoid technology was the breakthrough that introduced intestinal stem cells with the capability of differentiating into each of the epithelial cell types and that self-organize into a multi-cellular tissue proxy with villus- and crypt-like domains. Recently, GI microtissues generated using miniaturized devices with microfluidic flow and cyclic peristaltic strain were shown to induce Caco2 cells to spontaneously differentiate into each of the principle intestinal epithelial cell types. Second generation models comprised of epithelial organoids or microtissues co-cultured with non-epithelial cell types can successfully reproduce cross-'tissue' functional interactions broadening the potential of these models to accurately study drug-induced toxicities. A new paradigm in which in vitro assays become an early part of GI safety assessment could be realized if microphysiological systems (MPS) are developed in alignment with drug-discovery needs. Herein, approaches for assessing GI toxicity of pharmaceuticals are reviewed and gaps are compared with capabilities of emerging GI microtissues (e.g., organoids, organ-on-a-chip, transwell systems) in order to provide perspective on the assay features needed for MPS models to be adopted for DI-GIT assessment.

Kinase inhibitors have transformed the treatment of many cancers and are showing the same promise for other indications including inflammatory diseases. This class of drugs is one of the most predominant in the pharmaceutical industry, but development and clinical utility is often limited by a broad spectrum of cardiovascular (CV) toxicities including QT prolongation and arrhythmia, left ventricular dysfunction, congestive heart failure, ischemia and myocardial infarction, and hypertension. In this review article, we provide a broad overview of the spectrum of CV events detected in clinical trials of kinase inhibitors and the known and proposed on- and off-target links between kinase inhibitor targets and these specific cardiotoxicities. We also examine the unique features of kinase inhibitors that have impeded complete mechanistic understanding of kinase inhibitor-associated cardiotoxicities and contributed to the disconnect between preclinical predictions and clinical findings. We then discuss various in vitro models currently in use that are amenable for high-throughput screening as well as lower throughput models that are valuable for mechanistic insight. These physiologically relevant models, together with newer "omic"-wide approaches will help to increase our understanding of the mechanisms underlying kinase inhibitor-associated cardiotoxicity and enable rational design of kinase inhibitors in the future.

Drug-induced gastrointestinal toxicities (GITs) rank among the most common clinical side effects. Preclinical efforts to reduce incidence are limited by inadequate predictivity of in vitro assays. Recent breakthroughs in in vitro culture methods support intestinal stem cell maintenance and continual differentiation into the epithelial cell types resident in the intestine. These diverse cells self-assemble into microtissues with in vivo-like architecture. Here, we evaluate human GI microtissues grown in transwell plates that allow apical and/or basolateral drug treatment and 96-well throughput. Evaluation of assay utility focused on predictivity for diarrhea because this adverse effect correlates with intestinal barrier dysfunction which can be measured in GI microtissues using transepithelial electrical resistance (TEER). A validation set of widely prescribed drugs was assembled and tested for effects on TEER. When the resulting TEER inhibition potencies were adjusted for clinical exposure, a threshold was identified that distinguished drugs that induced clinical diarrhea from those that lack this liability. Microtissue TEER assay predictivity was further challenged with a smaller set of drugs whose clinical development was limited by diarrhea that was unexpected based on 1-month animal studies. Microtissue TEER accurately predicted diarrhea for each of these drugs. The label-free nature of TEER enabled repeated quantitation with sufficient precision to develop a mathematical model describing the temporal dynamics of barrier damage and recovery. This human 3D GI microtissue is the first in vitro assay with validated predictivity for diarrhea-inducing drugs. It should provide a platform for lead optimization and offers potential for dose schedule exploration., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology.)

Many drugs designed to inhibit kinases have their clinical utility limited by cardiotoxicity-related label warnings or prescribing restrictions. While this liability is widely recognized, designing safer kinase inhibitors (KI) requires knowledge of the causative kinase(s). Efforts to unravel the kinases have encountered pharmacology with nearly prohibitive complexity. At therapeutically relevant concentrations, KIs show promiscuity distributed across the kinome. Here, to overcome this complexity, 65 KIs with known kinome-scale polypharmacology profiles were assessed for effects on cardiomyocyte (CM) beating. Changes in human iPSC-CM beat rate and amplitude were measured using label-free cellular impedance. Correlations between beat effects and kinase inhibition profiles were mined by computation analysis (Matthews Correlation Coefficient) to identify associated kinases. Thirty kinases met criteria of having (1) pharmacological inhibition correlated with CM beat changes, (2) expression in both human-induced pluripotent stem cell-derived cardiomyocytes and adult heart tissue, and (3) effects on CM beating following single gene knockdown. A subset of these 30 kinases were selected for mechanistic follow up. Examples of kinases regulating processes spanning the excitation-contraction cascade were identified, including calcium flux (RPS6KA3, IKBKE) and action potential duration (MAP4K2). Finally, a simple model was created to predict functional cardiotoxicity whereby inactivity at three sentinel kinases (RPS6KB1, FAK, STK35) showed exceptional accuracy in vitro and translated to clinical KI safety data. For drug discovery, identifying causative kinases and introducing a predictive model should transform the ability to design safer KI medicines. For cardiovascular biology, discovering kinases previously unrecognized as influencing cardiovascular biology should stimulate investigation of underappreciated signaling pathways., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology.)

Background: We sought to determine if bilateral neck exploration (BNE) for hyperparathyroidism could be performed safely in an ambulatory setting (same-day discharge) when compared with focused parathyroidectomy., Methods: A retrospective review of 503 patients who underwent parathyroidectomy from 2010 to 2015 was performed. Focused parathyroidectomy was compared with BNE. Only patients with positive localization and no prior operations were included., Results: Forty-nine percent of patients underwent focused parathyroidectomy and 51% had BNE. BNE patients were more likely to have 1 or more glands removed (35% vs 14%, P < .01) and longer operative times (median 50 vs 41 minutes, P < .01). There were no differences in the rate of same-day discharge, transient hypocalcemia, emergency department visits, and readmissions., Conclusions: In this study, BNE for hyperparathyroidism was associated with excision of more parathyroid glands and slightly longer operative times. However, BNE had equal rates of same-day discharges and safety profile., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Concomitant thyroid pathology in patients with primary hyperparathyroidism is common. This study compares complications of patients who underwent parathyroidectomy to those who underwent parathyroidectomy with a concomitant thyroidectomy., Methods: A retrospective review of prospectively collected data on 709 patients who underwent parathyroidectomy was performed. Patients who had prior thyroid or parathyroid procedures were excluded. Chi-square, Fisher's exact, Student's t-test, and Wilcoxon rank-sum tests were used to compare cohorts., Results: Of the 641 patients included, 90% underwent parathyroidectomy alone and 10% underwent parathyroidectomy with a concomitant thyroidectomy. Overall, 49% had preoperative thyroid disease and 22% of patients with thyroid disease had a thyroid procedure. When compared with parathyroidectomy alone, parathyroidectomy with a concomitant thyroidectomy was associated with longer operative times (91 min versus 57 min, P < 0.001), increased rate of overnight stay (69% versus 17%, P < 0.001), and increased rate of transient hypocalcemia (15% versus 3%, P < 0.001)., Conclusions: Parathyroidectomy with a concomitant thyroidectomy is associated with longer operative times, increased rate of overnight stay, and increased transient hypocalcemia., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Knowledge of the intrinsic material properties of healthy and diseased arterial tissue components is of great importance in diagnostics. This study describes an in vitro comparison of 13 porcine carotid arteries using inflation testing combined with functional ultrasound and bi-axial tensile testing. The measured tissue behavior was described using both a linear, but geometrically non-linear, one-parameter (neo-Hookean) model and a two-parameter non-linear (Demiray) model. The shear modulus estimated using the linear model resulted in good agreement between the ultrasound and tensile testing methods, GUS = 25 ± 5.7 kPa and GTT = 23 ± 5.4 kPa. No significant correspondence was observed for the non-linear model aUS = 1.0 ± 2.7 kPa vs. aTT = 17 ± 8.8 kPa, p ∼ 0); however, the exponential parameters were in correspondence (bUS = 12 ± 4.2 vs. bTT = 10 ± 1.7, p > 0.05). Estimation of more complex models in vivo is cumbersome considering the sensitivity of the model parameters to small changes in measurement data and the absence of intraluminal pressure data, endorsing the use of a simple, linear model in vivo., (Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Cardiovascular (CV) toxicity is a leading cause of drug attrition and withdrawal. Introducing in vitro assays with higher throughput should permit earlier CV hazard identification and enable medicinal chemists to design-out liabilities. Heretofore, development of in vitro CV assays has been limited by the challenge of replicating integrated cardiovascular physiology while achieving the throughput and consistency required for screening. These challenges appear to be met with a combination of human stem cell-derived cardiomyocytes (CM) which beat spontaneously and monitoring the response with technology that can assess drug-induced changes in voltage dependent contraction such as cellular impedance which has been validated with excellent predictivity for drug-induced arrhythmia and contractility. Here, we review advances in cardiomyocyte impedance with emphasis on stem cell-derived cardiomyocyte models for toxicity screening. Key perspectives include: the electrical principles of impedance technology, impedance detection of cardiomyocyte beating, beat parameter selection/analysis, validation in toxicity and drug discovery, and future directions. As a conclusion, an in vitro screening cascade is proffered using the downstream, inclusive detection of CM impedance assays as a primary screen followed by complementary CM assays chosen to enable mechanism-appropriate follow-up. The combined approach will enhance testing for CV liabilities prior to traditional in vivo models.

Cardiovascular toxicity, a prominent reason for late-stage failures in drug development, has resulted in a demand for in vitro assays that can predict this liability in early drug discovery. Current in vitro cardiovascular safety testing primarily focuses on ion channel modulation and low throughput cardiomyocyte (CM) contractility measurements. We evaluated both human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and rat neonatal CMs (rat CMs) on the xCELLigence Cardio system which uses impedance technology to quantify CM beating properties in a 96-well format. Forty-nine compounds were tested in concentration-response mode to determine potency for modulation of CM beating, a surrogate biomarker for contractility. These compounds had previously been tested in vivo and in a low throughput in vitro optical-based contractility assay that measures sarcomere shortening in electrically paced dog CMs. In comparison with in vivo contractility effects, hiPSC-CM impedance had assay sensitivity, specificity, and accuracy values of 90%, 74%, and 82%, respectively. These values compared favorably to values reported for the dog CM optical assay (83%, 84%, and 82%) and were slightly better than impedance using rat CMs (77%, 74%, and 74%). The potency values from the hiPSC-CM and rat CM assays spanned four orders of magnitude and correlated with values from the dog CM optical assay (r(2 )= 0.76 and 0.70, respectively). The Cardio system assay has >5× higher throughput than the optical assay. Thus, hiPSC-CM impedance testing can help detect the human cardiotoxic potential of novel therapeutics early in drug discovery, and if a hazard is identified, has sufficient throughput to support the design-make-test-analyze cycle to mitigate this liability., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Resection of pheochromocytoma is often associated with hemodynamic instability (HDI). We examined patient and tumor factors that may influence HDI. The effect of pretreatment with nonselective α blockade phenoxybenzamine (PXB) versus selective α blockade on HDI and outcomes was also evaluated., Methods: The records of 91 patients who underwent adrenalectomy between 2002 and 2013 were retrospectively reviewed. HDI was determined by number of intraoperative episodes of systolic blood pressure (SBP) > 200 mmHg, those greater than or less than 30 % of baseline, heart rate > 110 bpm, and the need for postoperative vasopressors. Fishers exact, t test and regressions were performed., Results: Among 91 patients, 78 % received PXB, 18 % selective α blockade and 4 % no adrenergic blockade. Patient demographics, tumor factors and surgical approach were similar among the blockade groups. On multivariate analysis, increasing tumor size was associated with a significant rise in the number of episodes of SBP > 30 % [rate ratio (RR) 1.40] and an increased postoperative vasopressor requirement [odds ratio (OR) 1.23]. Open adrenalectomy and use of selective blockade were associated with an increased number of episodes of SBP > 200 mmHg (RR 27.8 and RR 20.9, respectively). Open adrenalectomy was also associated with increased readmissions (OR 12.3), complications (OR 5.6), use of postoperative vasopressors (OR 4.4) and hospital stay (4.6 days longer). There were no differences in other HDI measurements or postoperative outcomes among the blockade groups., Conclusions: Tumor size, open adrenalectomy, and type of α blockade were associated with intraoperative HDI during pheochromocytoma resection. Selective blockade was associated with significantly more episodes of intraoperative hypertension but no perioperative adverse outcomes.

Background: Endoscopic or open adrenalectomies are performed for variable pathologies. We investigated if adrenal pathology affects perioperative outcomes independent of operative approach., Methods: A multi-institutional retrospective review of 345 adrenalectomies was performed. A multivariate analysis was utilized., Results: Pathology groups included benign non-pheochromocytoma tumors (50.4%), pheochromocytomas (41%), adrenocortical carcinomas (5.2%), and metastatic tumors (3.4%). Controlling for age, body mass index, tumor size, procedure type, and pathology, pheochromocytomas exhibited greater blood loss (92 mL more, P = .007) and operative times (33 min more, P < .001) than benign non-pheochromocytoma tumors. Metastatic tumors demonstrated longer operative times (53 min more, P = .013). Open adrenalectomy was associated with greater blood loss (396 mL more, P = .001), transfusion requirement (P = .021), operative times (79 min more, P < .001), hospital stay (6.6 days more, P < .001) and complications (P < .001) when compared with endoscopic adrenalectomy., Conclusions: The type of adrenal pathology appears to influence blood loss and operative time but not complications in patients undergoing adrenalectomy. Open adrenalectomy remains a major driver of adverse perioperative outcomes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Vascular elastography techniques are promising tools for mechanical characterization of diseased arteries. These techniques are usually validated with simulations or phantoms or by comparing results with histology or other imaging modalities. In the study described here, vascular elastography was applied to porcine aortas in vitro during inflation testing (n = 10) and results were compared with those of standard bi-axial tensile testing, a technique that directly measures the force applied to the tissue. A neo-Hookean model was fit to the stress-strain data, valid for large deformations. Results indicated good correspondence between the two techniques, with GUS = 110 ± 11 kPa and GTT = 108 ± 10 kPa for ultrasound and tensile testing, respectively. Bland-Altman analysis revealed little bias (GUS-GTT = 2 ± 20 kPa). The next step will be the application of a non-linear material model that is also adaptable for in vivo measurements., (Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Cardiovascular (CV) toxicity is a leading contributor to drug attrition. Implementing earlier testing has successfully reduced human Ether-à-go-go-Related Gene-related arrhythmias. How- ever, analogous assays targeting functional CV effects remain elusive. Demand to address this gap is particularly acute for kinase inhibitors (KIs) that suffer frequent CV toxicity. The drug class also presents some particularly challenging requirements for assessing functional CV toxicity. Specifically, an assay must sense a downstream response that integrates diverse kinase signaling pathways. In addition, sufficient throughput is essential for handling inherent KI nonselectivity. A new opportunity has emerged with cellular impedance technology, which detects spontaneous beating cardiomyocytes. Impedance assays sense morphology changes downstream of cardiomyocyte contraction. To evaluate cardiomyocyte impedance assays for KI screening, we investigated two distinct KI classes where CV toxicity was discovered late and target risks remain unresolved. Microtubule-associated protein/microtubule affinity regulating kinase (MARK) inhibitors decrease blood pressure in dogs, whereas checkpoint kinase (Chk) inhibitors (AZD7762, SCH900776) exhibit dose-limiting CV toxicities in clinical trials. These in vivo effects manifested in vitro as cardiomyocyte beat cessation. MARK effects were deemed mechanism associated because beat inhibition potencies correlated with kinase inhibition, and gene knockdown and microtubule-targeting agents suppressed beating. MARK inhibitor impedance and kinase potencies aligned with rat blood pressure effects. Chk inhibitor effects were judged off-target because Chk and beat inhibition potencies did not correlate and knockdowns did not alter beating. Taken together, the data demonstrate that cardiomyocyte impedance assays can address three unmet needs-detecting KI functional cardiotoxicity in vitro, determining mechanism of action, and supporting safety structure-activity relationships.

Predicting human safety risks of novel xenobiotics remains a major challenge, partly due to the limited availability of human cells to evaluate tissue-specific toxicity. Recent progress in the production of human induced pluripotent stem cells (hiPSCs) may fill this gap. hiPSCs can be continuously expanded in culture in an undifferentiated state and then differentiated to form most cell types. Thus, it is becoming technically feasible to generate large quantities of human cell types and, in combination with relatively new detection methods, to develop higher-throughput in vitro assays that quantify tissue-specific biological properties. Indeed, the first wave of large scale hiSC-differentiated cell types including patient-derived hiPSCS are now commercially available. However, significant improvements in hiPSC production and differentiation processes are required before cell-based toxicity assays that accurately reflect mature tissue phenotypes can be delivered and implemented in a cost-effective manner. In this review, we discuss the promising alignment of hiPSCs and recently emerging technologies to quantify tissue-specific functions. We emphasize liver, cardiovascular, and CNS safety risks and highlight limitations that must be overcome before routine screening for toxicity pathways in hiSC-derived cells can be established., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Cardiovascular toxicity is a leading contributor to drug withdrawal and late-stage attrition. Earlier and broader screening is a validated approach to build-in cardiovascular safety as demonstrated with human Ether-à-go-go-related gene (hERG) screening to reduce drug-induced arrhythmia. There is an urgent need for novel in vitro assays to address other mechanistic aspects of cardiovascular function, including contractility, heart rate, toxicity, hypertrophy, and non-hERG arrhythmia. Recent advances in label-free cellular impedance technology now enable tracking of spontaneous, synchronized beating of cultured cardiomyocytes. Analysis of beating allows integrated detection that is downstream of electrical and mechanical aspects of contraction. Here, we evaluate impedance-based cardiomyocyte responses against criteria required for drug screening. The throughput and sensitivity allowed for rapid assay development. Critical variables for rat neonatal cardiomyocyte assays included cell density and serum levels. Once optimized, consistent, stable beating for at least 3 days was straight-forward to achieve. In tests of compounds spanning a breadth of target classes, the potency values showed excellent precision, wide dynamic range, and consistency across multiple experiments. Cardiomyocyte impedance assays can extract multiple beat-related parameters. In these experiments, rate, amplitude, and rise slope were examined and each yielded acceptable precision. Potency values calculated by beat rate and amplitude were highly correlated for most compounds although selected compounds displayed unique profiles indicative of different mechanisms. Tests with known cardiovascular active drugs revealed concordance with clinical findings. Thus, impedance assays combine novel features including sensitivity to contractile activity, versatile data analysis, and robust/translatable data in a format with sufficient throughput to become a valuable addition to the cardiovascular in vitro screening arsenal.

The κ-opioid receptor plays a central role in mediating the response to stressful life events. Inhibiting κ-opioid receptor signaling is proposed as a mechanism for treating stress-related conditions such as depression and anxiety. Preclinical testing consistently confirms that disruption of κ-opioid signaling is efficacious in animal models of mood disorders. However, concerns about the feasibility of developing antagonists into drugs stem from an unusual pharmacodynamic property of prototypic κ-opioid receptor-selective antagonists; they inhibit receptor signaling for weeks to months after a single dose. Several fundamental questions include - is it possible to identify short-acting antagonists; is long-lasting inhibition necessary for efficacy; and is it safe to develop long-acting antagonists in the clinic. Here, we test representative compounds (AZ-ECPC, AZ-MTAB, and LY-DMPF) from three new chemical series of κ-opioid receptor ligands for long-lasting inhibition. Each compound dose-dependently reversed κ-opioid agonist-induced diuresis. However, unlike the prototypic antagonist, nBNI, which fully inhibited evoked diuresis for at least four weeks, the new compounds showed no inhibition after one week. The two compounds with greater potency and selectivity were tested in prenatally-stressed rats on the elevated plus maze, an exploration-based model of anxiety. Spontaneous exploration of open arms in the elevated plus maze was suppressed by prenatal stress and restored with both compounds. These findings indicate that persistent inhibition is not an inherent property of κ-opioid-selective antagonists and that post-stress dosing with transient inhibitors can be effective in a mood disorder model. This further supports κ-opioid receptor as a promising target for developing novel psychiatric medications., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We investigate coherent Rayleigh-Brillouin spectroscopy as an efficient process to measure the bulk viscosity of gases at gigahertz frequencies. Scattered spectral distributions are measured using a Fizeau spectrometer. We discuss the statistical error due to the fluctuating mode structure of the used pump laser. Experiments were done for both polar and nonpolar gases and the bulk viscosity was obtained from the spectra using the Tenti S6 model. Results are compared to simple classical kinetic models of molecules with internal degrees of freedom. At the extremely high (gigahertz) frequencies of our experiment, most internal vibrational modes remain frozen and the bulk viscosity is dominated by the rotational degrees of freedom. Our measurements show that the molecular dipole moments have unexpectedly little influence on the bulk viscosity at room temperature and moderate pressure.

Initial high throughput screening efforts identified highly potent and selective kappa opioid receptor antagonist 3 (κ IC(50)=77 nM; μ:κ and δ:κ IC(50) ratios>400) which lacked CNS exposure in vivo. Modification of this scaffold resulted in development of a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides showing potent and selectivity κ antagonism as well as good brain exposure. Analog 6c (κ IC(50)=20 nM; μ:κ=36, δ:κ=415) was also shown to reverse κ-agonist induced rat diuresis in vivo., (Copyright © 2010. Published by Elsevier Ltd.)

Further structure activity relationship studies on a previously reported 8-azabicyclo[3.2.1]octan-3-yloxy-benzamide series of potent and selective kappa opioid receptor antagonists is discussed. Modification of the pendant N-substitution to include a cyclohexylurea moiety produced analogs with greater in vitro opioid and hERG selectivity such as 12 (kappa IC50=172 nM, mu:kappa ratio=93, delta:kappa ratio=>174, hERG IC50=>33 microM). Changes to the linker conformation and identity as well as to the benzamide ring moiety were also investigated., (Copyright (c) 2010. Published by Elsevier Ltd.)

A new class of instruments offers an unprecedented combination of label-free detection with exquisite sensitivity to live-cell responses. These instruments can quantify G-protein-coupled receptor (GPCR) signaling through G(s), G(i) and G(q) pathways and in some cases distinguish G-protein coupling, with sensitivity high enough to detect endogenous receptors. Here, we review emerging data evaluating impedance- and optical-based label-free instruments for GPCR drug discovery. In comparison with traditional GPCR assays, we highlight strengths, weaknesses and future opportunities for label-free biosensors. The ability to qualitatively distinguish G-protein coupling has groundbreaking potential for assessing functional selectivity, a concept that is changing the way GPCR pharmacology is defined and screening strategies are designed., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The diversity and impact of label-free technologies continues to expand in drug discovery. Two classes of label-free instruments, using either an electrical impedance-based or an optical-based biosensor, are now available for investigating the effects of ligands on cellular targets. Studies of GPCR function have been especially prominent with these instruments due to the importance of this target class in drug discovery. Although both classes of biosensors share similar high sensitivity to changes in cell shape and structure, it is unknown whether these biosensors yield similar results when comparing the same GPCR response. Furthermore, since cell morphology changes induced by GPCRs differ depending on which G-protein is activated, there is potential for these instruments to have differential sensitivities to G-protein signaling. Here 1 impedance (CellKey)- and 2 optical-based instruments (BIND and Epic) are compared using Gi-coupled (ACh M2), Gq-coupled (ACh M1), and Gs-coupled (CRF1) receptors. All 3 instruments were robust in agonist and antagonist modes yielding comparable potencies and assay variance. Both the impedance and optical biosensors showed similar high sensitivity for detecting an endogenous D1/D5 receptor response and a melanocortin-4 receptor inverse agonist (agouti-related protein). The impedance-based biosensor was uniquely able to qualitatively distinguish G-protein coupling and reveal dual signaling by CRF1. Finally, responses with a ligand-gated ion channel, TRPV1, were similarly detectable in each instrument. Thus, despite some differences, both impedance- and optical-based platforms offer robust live-cell, label-free assays well suited to drug discovery and typically yield similar pharmacological profiles for GPCR ligands.

G-protein-coupled receptors can couple to different signal transduction pathways in different cell types (termed cell-specific signaling) and can activate different signaling pathways depending on the receptor conformation(s) stabilized by the activating ligand (functional selectivity). These concepts offer potential for developing pathway-specific drugs that increase efficacy and reduce side effects. Despite significant interest, functional selectivity has been difficult to exploit in drug discovery, in part due to the burden of multiple assays. Cellular impedance assays use an emerging technology that can qualitatively distinguish Gs, Gi/o, and Gq signaling in a single assay and is thereby suited for studying these pharmacological concepts. Cellular impedance confirmed cell-specific Gs and Gq coupling for the melanocortin-4 receptor and dual Gi and Gs signaling with the cannabinoid-1 (CB1) receptor. The balance of Gi versus Gs signaling depended on the cell line. In CB1-HEKs, Giand Gs-like responses combined to yield a novel impedance profile demonstrating the dynamic nature of these traces. Cellspecific signaling was observed with endogenous D1 receptor in U-2 cells and SK-N-MC cells, yet the pharmacological profile of partial and full agonists was similar in both cell lines. We conclude that the dynamic impedance profile encodes valuable relative signaling information and is sufficiently robust to help evaluate cell-specific signaling and functional selectivity.

Cellular dielectric spectroscopy (CDS) is an emerging technology capable of detecting a range of whole-cell responses in a label-free manner. A new CDS-based instrument, CellKey, has been developed that is optimized for G-protein coupled receptor (GPCR) detection and has automated liquid handling in microplate format, thereby making CDS accessible to lead generation/optimization drug discovery. In addition to having sufficient throughput, new assay technologies must pass rigorous standards for assay development, signal window, dynamic range, and reproducibility to effectively support drug discovery SAR studies. Here, the authors evaluated CellKey with 3 different G(i)-coupled GPCRs for suitability in supporting SAR studies. Optimized assay conditions compatible with the precision, reproducibility, and throughput required for routine screening were quickly achieved for each target. Across a 1000-fold range in compound potencies, CellKey results correlated with agonist and antagonist data obtained using classical methods ([(35)S]GTPgammaS binding and cAMP production). For partial agonists, relative efficacy measurements also correlated with GTPgammaS data. CellKey detection of positive allosteric modulators appeared superior to GTPgammaS methodology. Agonist and antagonist activity could be accurately quantified under conditions of low receptor expression. CellKey is a new technology platform that uses label-free detection in a homogeneous assay that is unaffected by color quenching and is easily integrated into existing microtiter-based compound testing and data analysis procedures for drug discovery.

Aquaporin-4 water channels and the inwardly rectifying potassium channels Kir4.1 are coexpressed in a highly polarized manner at the perivascular and subvitreal endfeet of retinal Müller cells and astrocytes. The present study was aimed at resolving the anchoring mechanisms responsible for the coexpression of these molecules. Both aquaporin-4 and Kir4.1 contain PDZ-domain binding motifs at their C-termini and it was recently shown that mice with targeted disruption of the dystrophin gene display altered distribution of aquaporin-4 and Kir4.1 in the retina. To test our hypothesis that alpha-syntrophin (a PDZ-domain containing protein of the dystrophin associated protein complex) is involved in aquaporin-4 and Kir4.1 anchoring in retinal cells, we studied the expression pattern of these molecules in alpha-syntrophin null mice. Judged by quantitative immunogold cytochemistry, deletion of the alpha-syntrophin gene causes a partial loss (by 70%) of aquaporin-4 labeling at astrocyte and Müller cell endfeet but no decrease in Kir4.1 labeling at these sites. These findings suggest that alpha-syntrophin is not involved in the anchoring of Kir4.1 and only partly responsible for the anchoring of aquaporin-4 in retinal endfeet membranes. Furthermore we show that wild type and alpha-syntrophin null mice exhibit strong beta1 syntrophin labeling at perivascular and subvitreal Müller cell endfeet, raising the possibility that beta1 syntrophin might be involved in the anchoring of Kir4.1 and the alpha-syntrophin independent pool of aquaporin-4.

Genetic studies implicating the region of human chromosome 18p11.2 in susceptibility to bipolar disorder and schizophrenia have observed parent-of-origin effects that may be explained by genomic imprinting. We have identified a transcriptional variant of the GNAL gene in this region, employing an alternative first exon that is 5' to the originally identified start site. This alternative GNAL transcript encodes a longer functional variant of the stimulatory G-protein alpha subunit, Golf. The isoforms of Golf display different expression patterns in the CNS and functionally couple to the dopamine D1 receptor when heterologously expressed in Sf9 cells. In addition, there are CpG islands in the vicinity of both first exons that are differentially methylated, a hallmark of genomic imprinting. These results suggest that GNAL, and possibly other genes in the region, is subject to epigenetic regulation and strengthen the case for a susceptibility gene in this region.

Background: Acute respiratory tract infections (ARTIs) are responsible for considerable morbidity in the community, but little is known about the presence of respiratory pathogens in asymptomatic individuals. We hypothesized that asymptomatic persons could have a subclinical infection and thus act as a source of transmission., Methods: During the period of 2000-2003, all patients with ARTI who visited their sentinel general practitioner had their data reported to estimate the incidence of ARTI in Dutch general practices. A random selection of these patients (case patients) and an equal number of asymptomatic persons visiting for other complaints (control subjects) were included in a case-control study. Nose and throat swabs of participants were tested for a broad range of pathogens., Results: The overall incidence of ARTI was 545 cases per 10,000 person-years, suggesting that, in the Dutch population, an estimated 900,000 persons annually consult their general practitioner for respiratory complaints. Rhinovirus was most common in case patients (24%), followed by influenza virus type A (11%) and coronavirus (7%). Viruses were detected in 58% of the case patients, beta -hemolytic streptococci group A were detected in 11%, and mixed infections were detected in 3%. Pathogens were detected in approximately 30% of control subjects, particularly in the youngest age groups., Conclusion: This study confirms that most ARTIs are viral and supports the reserved policy of prescribing antibiotics. In both case and control subjects, rhinovirus was the most common pathogen. Of bacterial infections, only group A beta-hemolytic streptococci were more common in case patients than in control subjects. Furthermore, we demonstrated that asymptomatic persons might be a neglected source of transmission.

The syntrophins and dystrobrevins are members of the dystrophin-associated protein complex, and are thought to function as modular adaptors for signalling proteins recruited to the sarcolemmal membrane. We have characterised the expression of the syntrophins (alpha-, beta1-, and beta2-) and alpha-dystrobrevin by immunohistochemistry in normal human muscle and in biopsies from 162 patients with myopathies of unknown aetiology (with normal staining for dystrophin and other dystrophin-associated proteins). Unlike mice, beta2-syntrophin is expressed at the sarcolemma in post-natal human skeletal muscle. Deficiency of alpha-dystrobrevin +/- beta2-syntrophin was present in 16/162 (10%) patients, compared to age-matched controls. All patients presented with congenital-onset hypotonia and weakness, although there was variability in clinical severity. Two major clinical patterns emerged: patients with deficiency of beta2-syntrophin and alpha-dystrobrevin presented with severe congenital weakness and died in the first year of life, and two patients with deficiency of alpha-dystrobrevin had congenital muscular dystrophy with complete external ophthalmoplegia. We have sequenced the coding regions of alpha-dystrobrevin and beta2-syntrophin in these patients, and identified a new isoform of dystrobrevin, but have not identified any mutations. This suggests that disease causing mutations occur outside the coding region of these genes, in gene(s) encoding other components of the syntrophin-dystrobrevin subcomplex, or in gene(s) responsible for their post-translational modification and normal localisation.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the abnormal expansion of a polyglutamine tract in the huntingtin protein. We have developed PC12 cell lines in which the expression of an N-terminal truncation of huntingtin (N63) with either wild type (23Q) or expanded polyglutamine (148Q) can be induced by the removal of doxycycline. Differentiated PC12 cells induced to express N63-148Q showed cellular toxicity reaching up to 50% at 6 days post-induction. Histone acetyltransferase (HAT) activity and global histone acetylation was significantly decreased in cells expressing truncated huntingtin with mutant but not normal huntingtin. These data suggest that altered chromatin modification via reduction in coactivator activity may cause neuronal transcriptional dysregulation and contribute to cellular toxicity.

Expanded polyglutamine repeats have been proposed to cause neuronal degeneration in Huntington's disease (HD) and related disorders, through abnormal interactions with other proteins containing short polyglutamine tracts such as the transcriptional coactivator CREB binding protein, CBP. We found that CBP was depleted from its normal nuclear location and was present in polyglutamine aggregates in HD cell culture models, HD transgenic mice, and human HD postmortem brain. Expanded polyglutamine repeats specifically interfere with CBP-activated gene transcription, and overexpression of CBP rescued polyglutamine-induced neuronal toxicity. Thus, polyglutamine-mediated interference with CBP-regulated gene transcription may constitute a genetic gain of function, underlying the pathogenesis of polyglutamine disorders.

Huntington's disease is caused by an expanded CAG trinucleotide repeat coding for a polyglutamine stretch within the huntingtin protein. Currently, the function of normal huntingtin and the mechanism by which expanded huntingtin causes selective neurotoxicity remain unknown. Clues may come from the identification of huntingtin-associated proteins (HAPs). Here, we show that huntingtin copurifies with a single novel 40-kDa protein termed HAP40. HAP40 is encoded by the open reading frame factor VIII-associated gene A (F8A) located within intron 22 of the factor VIII gene. In transfected cell extracts, HAP40 coimmunoprecipitates with full-length huntingtin but not with an N-terminal huntingtin fragment. Recombinant HAP40 is cytoplasmic in the presence of huntingtin but is actively targeted to the nucleus in the absence of huntingtin. These data indicate that HAP40 is likely to contribute to the function of normal huntingtin and is a candidate for involvement in the aberrant nuclear localization of mutant huntingtin found in degenerating neurons in Huntington's disease.

The autosomal dominant hereditary spastic paraplegias (AD-HSP) are a heterogeneous group of degenerative disorders of the central motor system, characterized by progressive spasticity of the lower limbs. Five loci for pure AD-HSP have been identified to date: SPG3 at 14q, SPG4 at 2p, SPG6 at 15q, SPG8 at 8q, and more recently SPG10 at 12q. We have analyzed a Brazilian family with 16 affected individuals by pure AD-HSP who developed progressive gait disturbance with onset at age 18-26 years. Linkage analysis performed with 13 relatives (6 affected and 7 normal) excluded SPG3, SPG4, and SPG6 as candidate regions. However, positive LOD scores were obtained with markers flanking the candidate region for the SPG8 locus [maximum two point Lod score (Zmax) = 3.3 at theta = 0 for D8S1804]. In this region lies the syntrophin beta 1 gene (SNT2B1), a widely expressed dystrophin-associated protein and therefore a good positional and functional candidate for this disease. Immunohistochemical and Western Blot (WB) studies showed that the distribution, expression, and apparent molecular weight of the beta 1 syntrophin protein were comparable to those of normal control individuals. Therefore, it is unlikely that defects in this protein are related to SPG8, at least in the present family., (Copyright 2000 Wiley-Liss, Inc.)

At least eight neurodegenerative diseases result from expansions of polyglutamine tracts encoded by CAG trinucleotide repeats. Although polyglutamine diseases typically have onset after age 50 in humans, these diseases can be modeled in animals and in cell culture by using highly expanded repeats to accelerate the pathogenesis. Unfortunately, current methods for preparing recombinant constructs with large glutamine tracts either alter the coding region adjacent to the repeat or yield highly unstable pure CAG repeats. We have developed a technique for expanding repeats that results in a more stable mix of CAG and CAA glutamine codons. We expect this technique to allow rapid preparation of highly expand repeats suitable for stable animal and cell culture models for any of the polyglutamine repeat diseases.

Huntington's disease is a neurodegenerative disorder resulting from expansion of the polyglutamine region in huntingtin. Although huntingtin is normally cytoplasmic, in affected brain regions proteolytic fragments of mutant huntingtin containing the polyglutamine repeat form intranuclear inclusions. Here, we examine the contribution of nuclear localization to toxicity by transiently transfecting neuro-2a cells with an N-terminal huntingtin fragment similar in size to that believed to be present in patients. The huntingtin fragment, HD-N63, was targeted either to the cytoplasm with a nuclear export signal (NES) or to the nucleus with a nuclear localization signal (NLS). The NES decreased the number of cells with aggregates in the nucleus while an NLS had the opposite effect. By cotransfecting HD-N63 with GFP as a marker, we observed direct cell loss with constructs containing expanded polyglutamine repeats. Compared to unmodified HD-N63-75Q, adding an NES reduced cell loss by 57% while an NLS increased cell loss by 111%. These results indicate that nuclear localization of mutant huntingtin fragments plays an important role in cell toxicity., (Copyright 1999 Academic Press.)

An increasing number of neurodegenerative disorders have been found to be caused by expanding CAG triplet repeats that code for polyglutamine. Huntington's disease (HD) is the most common of these disorders and dentatorubral-pallidoluysian atrophy (DRPLA) is very similar to HD, but is caused by mutation in a different gene, making them good models to study. In this review, we will concentrate on the roles of protein aggregation, nuclear localization and proteolytic processing in disease pathogenesis. In cell model studies of HD, we have found that truncated N-terminal portions of huntingtin (the HD gene product) with expanded repeats form more aggregates than longer or full length huntingtin polypeptides. These shorter fragments are also more prone to aggregate in the nucleus and cause more cell toxicity. Further experiments with huntingtin constructs harbouring exogenous nuclear import and nuclear export signals have implicated the nucleus in direct cell toxicity. We have made mouse models of HD and DRPLA using an N-terminal truncation of huntingtin (N171) and full-length atrophin-1 (the DRPLA gene product), respectively. In both models, diffuse neuronal nuclear staining and nuclear inclusion bodies are observed in animals expressing the expanded glutamine repeat protein, further implicating the nucleus as a primary site of neuronal dysfunction. Neuritic pathology is also observed in the HD mice. In the DRPLA mouse model, we have found that truncated fragments of atrophin-1 containing the glutamine repeat accumulate in the nucleus, suggesting that proteolysis may be critical for disease progression. Taken together, these data lead towards a model whereby proteolytic processing, nuclear localization and protein aggregation all contribute to pathogenesis.

alpha-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex. Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length alpha-dystrobrevin-1 (84 kD), and COOH-terminal truncated alpha-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that alpha-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds. alpha-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts. In contrast, alpha-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin. In yeast two-hybrid experiments and coimmunoprecipitation of in vitro-translated proteins, alpha-dystrobrevin-2 binds dystrophin, whereas alpha-dystrobrevin-1 binds both dystrophin and utrophin. alpha-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin. In contrast, alpha-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants. Thus, the distinct distributions of alpha-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved. These results show that alternative splicing confers distinct properties of association on the alpha-dystrobrevins.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expanding CAG repeat coding for polyglutamine in the huntingtin protein. Recent data have suggested the possibility that an N-terminal fragment of huntingtin may aggregate in neurons of patients with HD, both in the cytoplasm, forming dystrophic neurites, and in the nucleus, forming intranuclear neuronal inclusion bodies. An animal model of HD using the short N-terminal fragment of huntingtin has also been found to have intranuclear inclusions and this same fragment can aggregate in vitro . We have now developed a cell culture model demonstrating that N-terminal fragments of huntingtin with expanded glutamine repeats aggregate both in the cytoplasm and in the nucleus. Neuroblastoma cells transiently transfected with full-length huntingtin constructs with either a normal or expanded repeat had diffuse cytoplasmic localization of the protein. In contrast, cells transfected with truncated N-terminal fragments showed aggregation only if the glutamine repeat was expanded. The aggregates were often ubiquitinated. The shorter truncated product appeared to form more aggregates in the nucleus. Cells transfected with the expanded repeat construct but not the normal repeat construct showed enhanced toxicity to the apoptosis-inducing agent staurosporine. These data indicate that N-terminal truncated fragments of huntingtin with expanded glutamine repeats can aggregate in cells in culture and that this aggregation can be toxic to cells. This model will be useful for future experiments to test mechanisms of aggregation and toxicity and potentially for testing experimental therapeutic interventions.

Dystrophin, the protein disrupted in Duchenne muscular dystrophy, is one of several related proteins that are key components of the submembrane cytoskeleton. Three dystrophin-related proteins (utrophin, dystrophin-related protein-2 (DRP2), and dystrobrevin) have been described. Here, we identify a human gene on chromosome 2p22-23 that encodes a novel protein, beta-dystrobrevin, with significant homology to the other known dystrobrevin (now termed alpha-dystrobrevin). Sequence alignments including this second dystrobrevin strongly support the concept that two distinct subfamilies exist within the dystrophin family, one composed of dystrophin, utrophin, and DRP2 and the other composed of alpha- and beta-dystrobrevin. The possibility that members of each subfamily form distinct protein complexes was examined by immunopurifying dystrobrevins and dystrophin. A beta-dystrobrevin antibody recognized a protein of the predicted size (71 kDa) that copurified with the dystrophin short form, Dp71. Thus, like alpha-dystrobrevin, beta-dystrobrevin is likely to associate directly with dystrophin. alpha- and beta-dystrobrevins failed to copurify with each other, however. These results suggest that members of the dystrobrevin subfamily form heterotypic associations with dystrophin and raise the possibility that pairing of a particular dystrobrevin with dystrophin may be regulated, thereby providing a mechanism for assembly of distinct submembrane protein complexes.

The syntrophins are a multigene family of intracellular dystrophin-associated proteins comprising three isoforms, alpha1, beta1, and beta2. Based on their domain organization and association with neuronal nitric oxide synthase, syntrophins are thought to function as modular adapters that recruit signaling proteins to the membrane via association with the dystrophin complex. Using sequences derived from a new mouse beta1-syntrophin cDNA, and previously isolated cDNAs for alpha1- and beta2-syntrophins, we prepared isoform-specific antibodies to study the expression, skeletal muscle localization, and dystrophin family association of all three syntrophins. Most tissues express multiple syntrophin isoforms. In mouse gastrocnemius skeletal muscle, alpha1- and beta1-syntrophin are concentrated at the neuromuscular junction but are also present on the extrasynaptic sarcolemma. beta1-syntrophin is restricted to fast-twitch muscle fibers, the first fibers to degenerate in Duchenne muscular dystrophy. beta2-syntrophin is largely restricted to the neuromuscular junction. The sarcolemmal distribution of alpha1- and beta1-syntrophins suggests association with dystrophin and dystrobrevin, whereas all three syntrophins could potentially associate with utrophin at the neuromuscular junction. Utrophin complexes immunoisolated from skeletal muscle are highly enriched in beta1- and beta2-syntrophins, while dystrophin complexes contain mostly alpha1- and beta1-syntrophins. Dystrobrevin complexes contain dystrophin and alpha1- and beta1-syntrophins. From these results, we propose a model in which a dystrophin-dystrobrevin complex is associated with two syntrophins. Since individual syntrophins do not have intrinsic binding specificity for dystrophin, dystrobrevin, or utrophin, the observed preferential pairing of syntrophins must depend on extrinsic regulatory mechanisms.

Guanylin, a peptide purified from rat jejunum, is thought to regulate water and electrolyte balance in the intestine. We show here, using a combination of Northern blots, Western blots, and functional assays, that guanylin and its receptor (GCC) are not distributed in parallel within the rat intestine. To investigate the possibility that there might be a second intestinal peptide that serves as a ligand for GCC, we assayed tissue extracts for the ability to stimulate cyclic GMP synthesis in a GCC-expression cell line. Duodenal extracts display a peak of biological activity that is not present in colon and that does not comigrate with guanylin or proguanylin. The activity co-purifies with a novel peptide (TIATDECELCINVACTGC) that has high homology with uroguanylin, a peptide initially purified from human and opossum urine. A rat uroguanylin cDNA clone was found to encode a propeptide whose C-terminus corresponds to our purified peptide. Northern blots with probes generated from this clone reveal that prouroguanylin mRNA is strongly expressed in proximal small intestine, but virtually absent from colon, corroborating our biochemical measurements. Taken together, these studies demonstrate an intestinal origin for uroguanylin, and show that within the intestine its distribution is complementary to that of guanylin.

Becker muscular dystrophy is an X-linked disease due to mutations of the dystrophin gene. We now show that neuronal-type nitric oxide synthase (nNOS), an identified enzyme in the dystrophin complex, is uniquely absent from skeletal muscle plasma membrane in many human Becker patients and in mouse models of dystrophinopathy. An NH2-terminal domain of nNOS directly interacts with alpha 1-syntrophin but not with other proteins in the dystrophin complex analyzed. However, nNOS does not associate with alpha 1-syntrophin on the sarcolemma in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a ternary interaction of nNOS, alpha 1-syntrophin, and the central domain of dystrophin in vivo, a conclusion supported by developmental studies in muscle. These data indicate that proper assembly of the dystrophin complex is dependent upon the structure of the central rodlike domain and have implications for the design of dystrophin-containing vectors for gene therapy.

Dystrophin-related and -associated proteins are important in the formation and maintenance of the mammalian neuromuscular junction. We have characterized mouse cDNA clones encoding isoforms of the dystrophin-homologous 87-kDa postsynaptic protein, dystrobrevin. In Torpedo, the 87-kDa protein is multiply phosphorylated and closely associated with proteins in the postsynaptic cytoskeleton, including the acetylcholine receptor. In contrast to Torpedo, where only a single transcript is seen, the mouse expresses several mRNAs encoding different isoforms. A 6.0-kilobase transcript in brain encodes a 78-kDa protein (dystrobrevin-2) that has a different C terminus, lacking the putative tyrosine kinase substrate domain. In skeletal and cardiac muscle, transcripts of 1.7 and 3.3/3.5 kilobases predominate and encode additional isoforms. Alternative splicing within the coding region and differential usage of untranslated regions produce additional variation. Multiple dystrobrevin-immunoreactive proteins copurify with syntrophin from mouse tissues. In skeletal muscle, dystrobrevin immunoreactivity is restricted to the neuromuscular junction and sarcolemma. The occurrence of many dystrobrevin isoforms is significant because alternative splicing and phosphorylation often have profound effects upon the biological activity of synaptic proteins.

Neuronal nitric oxide synthase (nNOS) is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. Here, we show that the N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in postsynaptic density-95 protein (PSD-95) and a related novel protein, PSD-93.nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interactions also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin-associated protein. nNOS isoforms lacking a PDZ domain, identified in nNOSdelta/delta mutant mice, do not associate with PSD-95 in brain or with skeletal muscle sarcolemma. Interaction of PDZ-containing domains therefore mediates synaptic association of nNOS and may play a more general role in formation of macromolecular signaling complexes.

The syntrophins are a multigene family of proteins which bind C-terminal domains of dystrophin, utrophin and homologs thereof. We report here that antibodies specific for one isoform, beta 2-syntrophin, labeled only the neuromuscular junction (NMJ) in rat skeletal muscle. Anti-alpha 1-syntrophin antibodies gave strong labeling of the sarcolemma and NMJ in normal rat and mouse muscle, and similar but much weaker labeling in dystrophin-minus mdx muscle. beta 2-Syntrophin therefore appears to be specific to the NMJ in normal muscle, as is utrophin, and may be involved in acetylcholine receptor clustering. alpha 1-Syntrophin appears to be associated mainly with dystrophin, as expected, but a small portion must be associated with another protein, possibly homologs of the electric tissue 87K protein.

Syntrophin, a 58 kd extrinsic membrane protein, is concentrated at postsynaptic sites at the neuromuscular junction and may be involved in clustering acetylcholine receptors. In muscle and nonmuscle tissues, syntrophin is associated with dystrophin, utrophin, and two homologs of the dystrophin carboxy-terminal region. We have isolated three cDNAs encoding Torpedo and mouse syntrophins. The Torpedo cDNA encodes a full-length protein, and on Northern blots recognizes a 3.5 kb mRNA. The two mouse syntrophin cDNAs are products of separate genes but encode proteins that share 50% identity. Syntrophin-1 mRNA (2.2 kb) is expressed at highest levels in skeletal muscle. Syntrophin-2 mRNAs (2.2, 5.0, and 10 kb) are expressed in all mouse tissues examined. These patterns of expression suggest that syntrophin-1 and syntrophin-2 may associate with different members of the dystrophin family.

Bone marrow contains a small population of primitive erythroid progenitor cells which can be detected by their capacity to form large numbers of erythroid progeny in viscous cultures containing erythropoietin (EP). These cells have been termed erythroid 'burst-forming units' (BFUe). The present study demonstrates that expression of the erythroid differentiation potential of BFUe requires the presence of an activity additional to EP. This activity has been designated as BFA (burst feeder activity). It is shown that the number of BFUe detected and their apparent sensitivity to EP are directly related to the BFA concentration of the cultures. BFA was found to be associated with a population of bone marrow cells of high buoyant density and small volume, which are sensitive to irradiation. The radiation dose-effect curve provided strong evidence that bone marrow BFA is independent of cell proliferation; this was supported by showing that BFA is unaffected by in vivo treatment with hydroxyurea. The findings are compatible with a two-step regulation model for erythroid differentiation in which BFA-induced progeny of BFUe acquire sensitivity to EP.

Medium conditioned by human peripheral blood leukocytes (HLCM) was studied for its in vitro effects on haemopoietic progenitor cells (CFU-s and CFU-c) present in mouse bone marrow. HLCM has poor colony stimulating activity in semi-solid cultures of mouse bone marrow cells, but invariably increases the number of colonies obtained in the presence of plateau levels of semi-purified colony stimulating factor (CSF). In liquid cultures, HLCM appears to contain a potent initiator of DNA synthesis in CFU-s, an activity which coincides with an increased CFU-s maintenance and causes a three- to four-fold increase in CFU-c number. It is apparent from this study that HLCM, in addition to stimulating colony formation in cultures of human bone marrow cells, has a profound in vitro effect on primitive haemopoietic progenitor cells of the mouse, which cannot be attributed to CSF.