Study to Explore the Association of the Renin-Angiotensin System and Right Ventricular Function in Mechanically Ventilated Patients.

BACKGROUND: Right ventricular (RV) dysfunction is associated with pulmonary vasoconstriction in mechanically ventilated patients. Enhancing the activity of angiotensin-converting enzyme 2 (ACE2), a key enzyme of the renin-angiotensin system (RAS), using recombinant human ACE2 (rhACE2) could alleviate RAS-mediated vasoconstriction and vascular remodeling. METHODS: This prospective observational study investigated the association between concentrations of RAS peptides (Ang II or Ang(1-7)) and markers of RV function, as assessed by echocardiography (ratio of RV to left ventricular end-diastolic area, interventricular septal motion, and pulmonary arterial systolic pressure (PASP)). RESULTS: Fifty-seven mechanically ventilated patients were enrolled. Incidence rates of acute cor pulmonale (ACP) and pulmonary circulatory dysfunction (PCD) were consistent with previous studies. In the 45 evaluable participants, no notable or consistent changes in RAS peptides concentration were observed over the observation period, and there was no correlation between Ang II concentration and either PASP or RV size. The model of the predicted posterior distributions for the pre- and post-dose values of Ang II demonstrated no change in the likelihood of PCD after hypothetical dosing with rhACE2, thus meeting the futility criteria. Similar results were observed with the other RAS peptides evaluated. CONCLUSIONS: Pre-defined success criteria for an association between PCD and the plasma RAS peptides were not met in the mechanically ventilated unselected patients.

An open-label, dose-escalation study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of single doses of GSK2586881 in participants with pulmonary arterial hypertension.

Preclinical and early clinical studies suggest that angiotensin-converting enzyme type 2 activity may be impaired in patients with pulmonary arterial hypertension (PAH); therefore, administration of exogenous angiotensin-converting enzyme type 2 (ACE2) may be beneficial. This Phase IIa, multi-center, open-label, exploratory, single-dose, dose-escalation study (NCT03177603) assessed the potential vasodilatory effects of single doses of GSK2586881 (a recombinant human ACE2) on acute cardiopulmonary hemodynamics in hemodynamically stable adults with documented PAH who were receiving background PAH therapy. Successive cohorts of participants were administered a single intravenous dose of GSK2586881 of 0.1, 0.2, 0.4, or 0.8 mg/kg. Dose escalation occurred after four or more participants per cohort were dosed and a review of safety, tolerability, pharmacokinetics, and hemodynamic data up to 24 h postdose was undertaken. The primary endpoint was a change in cardiopulmonary hemodynamics (pulmonary vascular resistance, cardiac index, and mean pulmonary artery pressure) from baseline. Secondary/exploratory objectives included safety and tolerability, effect on renin-angiotensin system peptides, and pharmacokinetics. GSK2586881 demonstrated no consistent or sustained effect on acute cardiopulmonary hemodynamics in participants with PAH receiving background PAH therapy (N = 23). All doses of GSK2586881 were well tolerated. GSK2586881 was quantifiable in plasma for up to 4 h poststart of infusion in all participants and caused a consistent and sustained reduction in angiotensin II and a corresponding increase in angiotensin (1-7) and angiotensin (1-5). While there does not appear to be a consistent acute vasodilatory response to single doses of GSK2586881 in participants with PAH, the potential benefits in terms of chronic vascular remodeling remain to be determined.

Enhancement of Nonlinear Dielectric Properties in BiFeO3-BaTiO3 Ceramics by Nb-Doping.

BiFeO3-BaTiO3 (BF-BT) ceramics exhibit great potential for diverse applications in high temperature piezoelectric transducers, temperature-stable dielectrics and pulsed-power capacitors. Further optimization of functional properties for different types of applications can be achieved by modification of processing parameters or chemical composition. In the present work, the influence of pentavalent niobium substitution for trivalent ferric ions on the structure, microstructure and dielectric properties of 0.7BF-0.3BT ceramics was investigated systematically. Doping with niobium led to incremental reductions in grain size (from 7.0 to 1.3 µm) and suppression of long-range ferroelectric ordering. It was found that core-shell type microstructural features became more prominent as the Nb concentration increased, which were correlated with the formation of distinct peaks in the dielectric permittivity-temperature relationship, at ~470 and 600 °C, which were attributed to the BT-rich shell and BF-rich core regions, respectively. Nb-doping of BF-BT ceramics yielded reduced electronic conductivity and dielectric loss, improved electrical breakdown strength and enhanced dielectric energy storage characteristics. These effects are attributed to the charge compensation of pentavalent Nb donor defects by bismuth vacancies, which suppresses the formation of oxygen vacancies and the associated electron hole conduction mechanism. The relatively high recoverable energy density (Wrec = 2.01 J cm-3) and energy storage efficiency (η = 68%) of the 0.7BiFeO3-0.3BaTiO3 binary system were achieved at 75 °C under an electric field of 15 kV mm-1. This material demonstrates the greatest potential for applications in energy storage capacitors and temperature-stable dielectrics.

Hereditary Mucin Deficiency Caused by Biallelic Loss of Function of MUC5B.

Rationale: Mucin homeostasis is fundamental to airway health. Upregulation of airway mucus glycoprotein MUC5B is observed in diverse common lung diseases and represents a potential therapeutic target. In mice, Muc5b is required for mucociliary clearance and for controlling inflammation after microbial exposure. The consequences of its loss in humans are unclear. Objectives: The goal of this study was to identify and characterize a family with congenital absence of MUC5B protein. Methods: We performed whole-genome sequencing in an adult proband with unexplained bronchiectasis, impaired pulmonary function, and repeated Staphylococcus aureus infection. Deep phenotyping over a 12-year period included assessments of pulmonary radioaerosol mucociliary clearance. Genotyping with reverse phenotyping was organized for eight family members. Extensive experiments, including immunofluorescence staining and mass spectrometry for mucins, were performed across accessible sample types. Measurements and Main Results: The proband, and her symptomatic sibling who also had extensive sinus disease with nasal polyps, were homozygous for a novel splicing variant in the MUC5B gene (NM_002458.2: c.1938 + 1G>A). MUC5B was absent from saliva, sputum, and nasal samples. Mucociliary clearance was impaired in the proband, and large numbers of apoptotic macrophages were present in sputum. Three siblings heterozygous for the familial MUC5B variant were asymptomatic but had a shared pattern of mild lung function impairments. Conclusions: Congenital absence of MUC5B defines a new category of genetic respiratory disease. The human phenotype is highly concordant with that of the Muc5b-/- murine model. Further study of individuals with decreased MUC5B production could provide unique mechanistic insights into airway mucus biology.

One-Year Readmission Following Undifferentiated Acute Hypercapnic Respiratory Failure.

Patients with acute hypercapnic respiratory failure (AHRF) often require hospitalization and respiratory support. Early identification of patients at risk of readmission would be helpful. We evaluated 1-y readmission and mortality rates of patients admitted for undifferentiated AHRF and identified the impact of initial severity on clinically important outcomes. We retrospectively analyzed patients who presented with AHRF to the emergency department of St Michael's Hospital in 2017. We collected data about patients' characteristics, hospital admission, readmission and mortality one year after the index admission. We analyzed predictors of readmission and mortality and conducted a survival analysis comparing patients who did and did not receive ventilatory support. A cohort of 212 patients with AHRF who survived their hospital admission were analyzed. At one year, 150 patients (70.8%) were readmitted and 19 (9%) had died. Main diagnoses included chronic obstructive pulmonary disease (60%), congestive heart failure (36%), asthma (22%) and obesity (19%), and these categories of patients had similar 1 y readmission rates. One third had more than one coexisting chronic illness. Although comorbidities were more frequent in readmitted patients, only a history of previous hospital admissions remained associated with 1 y readmission and mortality in multivariate analysis. Need for ventilatory support at admission was not associated with higher 1 y probability of readmission or death. Undifferentiated AHRF is the presentation of multiple chronic illnesses. Patients who survive one episode of AHRF and with previous history of admission have the highest risk of readmission and death regardless of whether they receive ventilatory support during index admission.

Effects of Recombinant Human Angiotensin-Converting Enzyme 2 on Response to Acute Hypoxia and Exercise: A Randomised, Placebo-Controlled Study.

INTRODUCTION: Angiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin-angiotensin system (RAS) that has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS). Enhancing ACE2 activity using GSK2586881, a recombinant form of human ACE2, could be beneficial in diseases such as ARDS but may blunt the hypoxic pulmonary vasoconstriction (HPV) response and potentially impact systemic and tissue oxygenation. This study aimed to evaluate the effect of GSK2586881 0.8 mg/kg on HPV response in healthy adult volunteers during exercise under hypoxic conditions. METHODS: In this phase I, randomised, double-blind (sponsor open) study, GSK2586881 or placebo was administered as a single intravenous (IV) dose in a two-period crossover design. Treatment periods were separated by a washout period of 3-14 days. The primary endpoint was change from baseline in pulmonary artery systolic pressure (PASP) measured by echocardiography. Secondary endpoints included RAS peptides and oxygen saturation. RESULTS: Seventeen adults aged 18-40 years were randomised to treatment. There were no clinically relevant differences (defined as a reduction of ≥ 5 mmHg) in change from baseline in PASP between GSK2586881 and placebo. GSK2586881 was well tolerated, with no serious adverse events, no worsening of hypoxaemia and no evidence of immunogenicity. The study was terminated early after review of the data, which showed that the predefined success criteria had not been met. Following GSK2586881 administration, levels of the RAS peptide angiotensin II decreased while angiotensin (1-7) increased, as expected, indicating that GSK2586881 was pharmacologically active. CONCLUSIONS: A single IV dose of GSK2586881 0.8 mg/kg was well tolerated but did not impact the acute HPV response in healthy volunteers.

Can Adding Constitutive Receptor Activity Redefine Biased Signaling Quantification?

Biased signaling, the differential activation of distinct signaling pathways, is currently at the center of pharmacology. Reliable characterization of biased ligands requires robust scales applicable to all ligand classes: agonists, neutral antagonists, and inverse agonists. To this end, constitutive receptor activity should be included in the models.

A method for the quantification of biased signalling at constitutively active receptors.

BACKGROUND AND PURPOSE: Biased agonism, the ability of an agonist to differentially activate one of several signal transduction pathways when acting at a given receptor, is an increasingly recognized phenomenon at many receptors. The Black and Leff operational model lacks a way to describe constitutive receptor activity and hence inverse agonism. Thus, it is impossible to analyse the biased signalling of inverse agonists using this model. In this theoretical work, we develop and illustrate methods for the analysis of biased inverse agonism. EXPERIMENTAL APPROACH: Methods were derived for quantifying biased signalling in systems that demonstrate constitutive activity using the modified operational model proposed by Slack and Hall. The methods were illustrated using Monte Carlo simulations. KEY RESULTS: The Monte Carlo simulations demonstrated that, with an appropriate experimental design, the model parameters are 'identifiable'. The method is consistent with methods based on the measurement of intrinsic relative activity (RAi ) (ΔΔlogR or ΔΔlog(τ/Ka )) proposed by Ehlert and Kenakin and their co-workers but has some advantages. In particular, it allows the quantification of ligand bias independently of 'system bias' removing the requirement to normalize to a standard ligand. CONCLUSIONS AND IMPLICATIONS: In systems with constitutive activity, the Slack and Hall model provides methods for quantifying the absolute bias of agonists and inverse agonists. This provides an alternative to methods based on RAi and is complementary to the ΔΔlog(τ/Ka ) method of Kenakin et al. in systems where use of that method is inappropriate due to the presence of constitutive activity.

Accuracy and safety of pin placement during lateral versus dorsal stabilization of lumbar spinal fracture-luxation in dogs.

OBJECTIVE: To determine the accuracy and safety of pin placement for lateral vertebral stabilization to the reference dorsal stabilization. STUDY DESIGN: A randomized noninferiority trial. SAMPLE POPULATION: Twenty Greyhound cadaveric lumbar spines (L1-L6). METHODS: One hundred and fifty-nine lumbar vertebral pins placed in 80 vertebrae were assessed; these pins were distributed approximately equally between the dorsal and lateral approaches, and between 2 surgeons. Pin angle accuracy, bone purchase distance, and distances from pin to the spinal canal and the aorta were measured for each pin. RESULTS: The lateral approach was superior for pin angle accuracy and bone purchase. The mean angle of deviation was 15.3° with the dorsal approach and 7.0° with the lateral approach. The mean bone purchase was 16.7 mm with the dorsal approach and 22.2 mm with the lateral approach. Pins were placed at a mean of 2.3 mm from the spinal canal with the dorsal approach and 1.7 mm with the lateral approach. Pins were placed at a mean of 3.8 mm from the aorta with the dorsal approach and 8.0 mm with the lateral approach. The percentage of pins breaching the spinal canal was 14% with the dorsal approach and 19% with the lateral approach. Fourteen percent of pins placed via the dorsal approach breached the aorta, whereas no pins placed via the lateral approach breached the aorta. CONCLUSION: Relative to the dorsal approach, the lateral approach improves angle accuracy, bone purchase, and distance between pins, and the aorta and is noninferior with regards to the distance between pins and the spinal canal.

Intracellular interactions of umeclidinium and vilanterol in human airway smooth muscle.

BACKGROUND: Intracellular mechanisms of action of umeclidinium (UMEC), a long-acting muscarinic receptor antagonist, and vilanterol (VI), a long-acting ß2-adrenoceptor (ß2R) agonist, were investigated in target cells: human airway smooth-muscle cells (ASMCs). MATERIALS AND METHODS: ASMCs from tracheas of healthy lung-transplant donors were treated with VI, UMEC, UMEC and VI combined, or control compounds (salmeterol, propranolol, ICI 118.551, or methacholine [MCh]). Cyclic adenosine monophosphate (cAMP) was measured using an enzyme-linked immunosorbent assay, intracellular free calcium ([Ca2+]i) using a fluorescence assay, and regulator of G-protein signaling 2 (RGS2) messenger RNA using real-time quantitative polymerase chain reaction. RESULTS: VI and salmeterol (10-12-10-6 M) induced cAMP production from ASMCs in a concentration-dependent manner, which was greater for VI at all concentrations. ß2R antagonism by propranolol or ICI 118.551 (10-12-10-4 M) resulted in concentration-dependent inhibition of VI-induced cAMP production, and ICI 118.551 was more potent. MCh (5×10-6 M, 30 minutes) attenuated VI-induced cAMP production (P<0.05), whereas pretreatment with UMEC (10-8 M, 1 hour) restored the magnitude of VI-induced cAMP production. ASMC stimulation with MCh (10-11-5×10-6 M) resulted in a concentration-dependent increase in [Ca2+]i, which was attenuated with UMEC pretreatment. Reduction of MCh-induced [Ca2+]i release was greater with UMEC + VI versus UMEC. UMEC enhanced VI-induced RGS2 messenger RNA expression. CONCLUSION: These data indicate that UMEC reverses cholinergic inhibition of VI-induced cAMP production, and is a more potent muscarinic receptor antagonist when in combination with VI versus either alone.

Unravelling intrinsic efficacy and ligand bias at G protein coupled receptors: A practical guide to assessing functional data.

Stephenson's empirical definition of an agonist, as a ligand with binding affinity and intrinsic efficacy (the ability to activate the receptor once bound), underpins classical receptor pharmacology. Quantifying intrinsic efficacy using functional concentration response relationships has always presented an experimental challenge. The requirement for realistic determination of efficacy is emphasised by recent developments in our understanding of G protein coupled receptor (GPCR) agonists, with recognition that some ligands stabilise different active conformations of the receptor, leading to pathway-selective, or biased agonism. Biased ligands have potential as therapeutics with improved selectivity and clinical efficacy, but there are also pitfalls to the identification of pathway selective effects. Here we explore the basics of concentration response curve analysis, beginning with the need to distinguish ligand bias from other influences of the functional system under study. We consider the different approaches that have been used to quantify and compare biased ligands, many of which are based on the Black and Leff operational model of agonism. Some of the practical issues that accompany these analyses are highlighted, with opportunities to improve estimates in future, particularly in the separation of true agonist intrinsic efficacy from the contributions of system dependent coupling efficiency. Such methods are by their nature practical approaches, and all rely on Stephenson's separation of affinity and efficacy parameters, which are interdependent at the mechanistic level. Nevertheless, operational analysis methods can be justified by mechanistic models of GPCR activation, and if used wisely are key elements to biased ligand identification.

Whole-Exome Sequencing and Targeted Copy Number Analysis in Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is an autosomal-recessive disorder resulting from loss of normal ciliary function. Symptoms include neonatal respiratory distress, chronic sinusitis, bronchiectasis, situs inversus, and infertility. Clinical features may be subtle and highly variable, making the diagnosis of PCD challenging. The diagnosis can be confirmed with ciliary ultrastructure analysis and/or molecular genetic testing of 32 PCD-associated genes. However, because of this genetic heterogeneity, comprehensive molecular genetic testing is not considered the standard of care, and the most efficient molecular approach has yet to be elucidated. Here, we propose a cost-effective and time-efficient molecular genetic algorithm to solve cases of PCD. We conducted targeted copy number variation (CNV) analysis and/or whole-exome sequencing on 20 families (22 patients) from a subset of 45 families (52 patients) with a clinical diagnosis of PCD who did not have a molecular genetic diagnosis after Sanger sequencing of 12 PCD-associated genes. This combined molecular genetic approach led to the identification of 4 of 20 (20%) families with clinically significant CNVs and 7 of 20 (35%) families with biallelic pathogenic mutations in recently identified PCD genes, resulting in an increased molecular genetic diagnostic rate of 55% (11/20). In patients with a clinical diagnosis of PCD, whole-exome sequencing followed by targeted CNV analysis results in an overall molecular genetic yield of 76% (34/45).

Antagonism of angiotensin 1-7 prevents the therapeutic effects of recombinant human ACE2.

UNLABELLED: Activation of the angiotensin 1-7/Mas receptor (MasR) axis counteracts angiotensin II (Ang II)-mediated cardiovascular disease. Recombinant human angiotensin-converting enzyme 2 (rhACE2) generates Ang 1-7 from Ang II. We hypothesized that the therapeutic effects of rhACE2 are dependent on Ang 1-7 action. Wild type male C57BL/6 mice (10-12 weeks old) were infused with Ang II (1.5 mg/kg/d) and treated with rhACE2 (2 mg/kg/d). The Ang 1-7 antagonist, A779 (200 ng/kg/min), was administered to a parallel group of mice. rhACE2 prevented Ang II-induced hypertrophy and diastolic dysfunction while A779 prevented these beneficial effects and precipitated systolic dysfunction. rhACE2 effectively antagonized Ang II-mediated myocardial fibrosis which was dependent on the action of Ang 1-7. Myocardial oxidative stress and matrix metalloproteinase 2 activity was further increased by Ang 1-7 inhibition even in the presence of rhACE2. Activation of Akt and endothelial nitric oxide synthase (eNOS) by rhACE2 were suppressed by the antagonism of Ang 1-7 while the activation of pathological signaling pathways was maintained. Blocking Ang 1-7 action prevents the therapeutic effects of rhACE2 in the setting of elevated Ang II culminating in systolic dysfunction. These results highlight a key cardioprotective role of Ang 1-7, and increased Ang 1-7 action represents a potential therapeutic strategy for cardiovascular diseases. KEY MESSAGES: Activation of the renin-angiotensin system (RAS) plays a key pathogenic role in cardiovascular disease. ACE2, a monocarboxypeptidase, negatively regulates pathological effects of Ang II. Antagonizing Ang 1-7 prevents the therapeutic effects of recombinant human ACE2. Our results highlight a key protective role of Ang 1-7 in cardiovascular disease.

Bending strength and stiffness of canine cadaver spines after fixation of a lumbar spinal fracture-luxation using a novel unilateral stabilization technique compared to traditional dorsal stabilization.

OBJECTIVE: To 1) assess the bending strength and stiffness of canine cadaver spines after fixation of a lumbar spinal fracture-luxation using a novel unilateral stabilization technique with pins and polymethyl methacrylate (PMMA) and 2) compare the results to a reference standard dorsal pin and PMMA technique. STUDY DESIGN: A randomized non-inferiority trial. SAMPLE POPULATION: Cadaveric lumbar spines (L1-L6) from 20 Greyhounds. METHODS: Specimens were paired to match bodyweight and vertebral size. A standardized fracture/luxation was performed between L3 and L4. One spine within each pair was randomly assigned the unilateral fixation technique and the other received the reference standard dorsal fixation technique. Four-point bending of each specimen in flexion was performed by applying load to pins placed transversely into vertebrae L1, L2, L5, and L6. During testing, angular bending strength and stiffness were measured as a function of flexion angle. Margins for non-inferiority were defined a priori. Strength and stiffness of the specimens for each technique were compared statistically. RESULTS: Lower limits of 95% confidence intervals were above the defined margins for non-inferiority. Thus, based on these margins, for strength and stiffness, unilateral fixation was not inferior to dorsal fixation. CONCLUSIONS: This novel unilateral approach to lumbar spinal fixation yielded comparable strength and stiffness when tested for bending in flexion to that of reference standard dorsal approach. This approach is therefore a suitable alternative to the dorsal approach in appropriate lumbar spinal fracture configurations.

Smoking enhances the proinflammatory effects of nucleotides on cytokine release from human lung.

Nucleotides have effects on immune cells which are complex but generally proinflammatory, and have been suggested to play a role in smoking-related lung diseases. However, there have been no studies directly measuring functional responses to nucleotides in human lungs taken from smokers. We used fragments of post mortem human lung from smokers and non-smokers, incubated them with a range of nucleotides (4-1000 µM) in the presence of lipopolysaccharide (LPS; 10 µg/ml) for 24 hours and measured cytokines (IL-1ß, IFNγ, IL-17, TNFα, IL-6, IL-8, IL-2 and IL-10) in the supernatants using multiplex immunoassays. Although the basal cytokine levels in the smokers were generally higher in the smokers than the non-smokers, there were no significant differences in either the basal release or the LPS-stimulated release of any of the cytokines when lungs from smokers and non-smokers were compared. There were no significant effects of ATP, ADP, AMP, UTP, α,ß-methylene-ATP, P1, P4-diATP, 2-methylthio-ATP or Bz-ATP on the release of cytokines from the lungs. However, the stable ATP analogue ATPγS increased the release of IL-1ß and IFNγ, and the effect was greatly increased in lungs from smokers. In non-smokers but not in smokers ATPγS increased the release of IL-17. Overall these results clearly demonstrate for the first time that in normal human lung a stable ATP analogue can enhance LPS-induced pro-inflammatory cytokine release, and that these effects are greatly altered by a prior history of smoking. This provides strong support for the suggestion that nucleotides are involved in the pathogenesis of smoking-related diseases.

Distinct conformations of the chemokine receptor CCR4 with implications for its targeting in allergy.

CC chemokine receptor 4 (CCR4) is expressed by Th2 and regulatory T cells and directs their migration along gradients of the chemokines CCL17 and CCL22. Both chemokines and receptor are upregulated in allergic disease, making CCR4 a therapeutic target for the treatment of allergy. We set out to assess the mechanisms underlying a previous report that CCL22 is a dominant ligand of CCR4, which may have implications for its therapeutic targeting. Human T cells expressing endogenous CCR4 and transfectants engineered to express CCR4 were assessed for receptor function, using assays of calcium release, chemotaxis, receptor endocytosis, and ligand binding. Despite the two ligands having equal potency in calcium flux and chemotaxis assays, CCL22 showed dominance in both receptor endocytosis assays and heterologous competitive binding assays. Using two different CCR4-specific Abs, we showed that CCR4 exists in at least two distinct conformations, which are differentially activated by ligand. A major population is activated by both CCL17 and CCL22, whereas a minor population is activated only by CCL22. Mutation of a single C-terminal residue K310 within a putative CCR4 antagonist binding site ablated activation of CCR4 by CCL17, but not by CCL22, despite having no effect on the binding of either ligand. We conclude that CCL17 and CCL22 are conformationally selective ligands of CCR4 and interact with the receptor by substantially different mechanisms. This finding suggests that the selective blockade of CCR4 in allergy may be feasible when one CCR4 ligand dominates, allowing the inhibition of Th2 signaling via one ligand while sparing regulatory T cell recruitment via another.

Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

The Chloroplast 2010 Project (http://www.plastid.msu.edu/) identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles were identified.

Application of receptor theory to allosteric modulation of receptors.

In this chapter, three topics are considered. The allosteric two-state model (ATSM) is compared with explicit multiconformational models. This demonstrates that the ATSM encapsulates the common behaviors of any model with at least two active and two inactive conformations: the "states" of the model represent ensembles of active and inactive conformations. A matrix representation of multiconformational models is introduced to provide a compact notation for models with arbitrarily large numbers of conformations. Allosteric modulation is further explored in the context of an operational model of receptor activity which includes constitutive receptor activity. Fitting this model allows the apparent affinity, intrinsic efficacy, and cooperativity constants of a pair of allosteric ligands to be determined. It is also demonstrated that, within certain limits, it is possible to estimate the parameters of the ATSM. Finally, a novel operational model is developed that may allow the analysis of protean ligands. This model requires a nonlinear stimulus function and two parameters to define the efficacy of a ligand. Expressions describing competitive and allosteric interactions under this model are developed and the results of applying null analyses to the data are determined.

Synthesis and structure-activity relationships of indazole arylsulfonamides as allosteric CC-chemokine receptor 4 (CCR4) antagonists.

A series of indazole arylsulfonamides were synthesized and examined as human CCR4 antagonists. Methoxy- or hydroxyl-containing groups were the more potent indazole C4 substituents. Only small groups were tolerated at C5, C6, or C7, with the C6 analogues being preferred. The most potent N3-substituent was 5-chlorothiophene-2-sulfonamide. N1 meta-substituted benzyl groups possessing an α-amino-3-[(methylamino)acyl]-group were the most potent N1-substituents. Strongly basic amino groups had low oral absorption in vivo. Less basic analogues, such as morpholines, had good oral absorption; however, they also had high clearance. The most potent compound with high absorption in two species was analogue 6 (GSK2239633A), which was selected for further development. Aryl sulfonamide antagonists bind to CCR4 at an intracellular allosteric site denoted site II. X-ray diffraction studies on two indazole sulfonamide fragments suggested the presence of an important intramolecular interaction in the active conformation.

Antagonism of human CC-chemokine receptor 4 can be achieved through three distinct binding sites on the receptor.

Chemokine receptor antagonists appear to access two distinct binding sites on different members of this receptor family. One class of CCR4 antagonists has been suggested to bind to a site accessible from the cytoplasm while a second class did not bind to this site. In this report, we demonstrate that antagonists representing a variety of structural classes bind to two distinct allosteric sites on CCR4. The effects of pairs of low-molecular weight and/or chemokine CCR4 antagonists were evaluated on CCL17- and CCL22-induced responses of human CCR4(+) T cells. This provided an initial grouping of the antagonists into sets which appeared to bind to distinct binding sites. Binding studies were then performed with radioligands from each set to confirm these groupings. Some novel receptor theory was developed to allow the interpretation of the effects of the antagonist combinations. The theory indicates that, generally, the concentration-ratio of a pair of competing allosteric modulators is maximally the sum of their individual effects while that of two modulators acting at different sites is likely to be greater than their sum. The low-molecular weight antagonists could be grouped into two sets on the basis of the functional and binding experiments. The antagonistic chemokines formed a third set whose behaviour was consistent with that of simple competitive antagonists. These studies indicate that there are two allosteric regulatory sites on CCR4.