Residues within the transmembrane domain of the glucagon-like peptide-1 receptor involved in ligand binding and receptor activation: modelling the ligand-bound receptor.

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9-39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9-39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

A naturally shaped silicone ventricle evaluated in a mock circulation loop: a preliminary study.

Mock circulation loops are used to evaluate the performance of cardiac assist devices prior to animal and clinical testing. A compressible, translucent silicone ventricle chamber that mimics the exact size, shape and motion of a failing heart is desired to assist in flow visualization studies around inflow cannulae during VAD support. The aim of this study was therefore to design and construct a naturally shaped flexible left ventricle and evaluate its performance in a mock circulation loop. The ventricle shape was constructed by the use of CT images taken from a patient experiencing cardiomyopathic heart failure and used to create a 3D image and subsequent mould to produce a silicone ventricle. Different cardiac conditions were successfully simulated to validate the ventricle performance, including rest, left heart failure and VAD support.

Magnetic Compton scattering study of the magnetocaloric material Gd(7)Pd(3).

The spin-dependent momentum density of Gd(7)Pd(3) was probed by the magnetic Compton scattering technique with elliptically polarized synchrotron radiation. A contribution to the spin moment from Pd 4d electrons was observed, at 2 and 280 K, alongside a large Gd 4f moment and a smaller Gd 5d moment. The total spin moment, at 2 K, was determined as 50.8 ± 0.7 µ(B) (f.u.)(-1). The Gd 4f contribution to the spin moment was determined as 43.4 ± 1.8 µ(B) (f.u.)(-1), the Gd 5d moment as 4.4 ± 0.7 µ(B) (f.u.)(-1) and the Pd 4d spin moment contribution as 2.9 ± 1.1 µ(B) (f.u.)(-1), where f.u. represents a formula unit. At 280 K the total spin moment was 27.3 ± 0.9 µ(B) (f.u.)(-1) with individual contributions determined as a Gd 4f spin moment of 23.8 ± 1.1 µ(B) (f.u.)(-1), a Gd 5d contribution of 2.2 ± 0.5 µ(B) (f.u.)(-1) and a Pd 5d spin moment of 1.2 ± 0.6 µ(B) (f.u.)(-1).

The orphan G-protein coupled receptor RDC1: evidence for a role in chondrocyte hypertrophy and articular cartilage matrix turnover.

OBJECTIVE: RDC1 is a class A orphan G-protein coupled receptor of unknown function. The purpose of this study was to identify compound RDC1 agonists and use these as tools to determine the effect of RDC1 activation in human chondrocytes and cartilage explant tissue. METHODS: Computational chemistry was employed to build a homology model of the RDC1 receptor. A virtual screen of in-house compounds was then performed and positive hits screened for their ability to invoke a Ca2+ response in a recombinant RDC1 HEK293 cell line, as measured by FLIPR. The effect of RDC1 activation on human chondrocytes and cartilage explant gene expression was determined by quantitative real-time polymerase chain reaction (PCR), and these effects validated as being mediated by RDC1 using siRNA antisense. RESULTS: Tissue expression profiling demonstrated that RDC1 expression was predominant in cartilage tissue. Treatment of human primary chondrocytes with RDC1 agonist induced a Ca2+ response, suggesting the receptor is active in this tissue type. Treatment for 24h with RDC1 agonist led to altered expression of a number of genes associated with chondrocyte hypertrophy and increased matrix degradation in human primary chondrocytes, and elevated total matrix metalloproteinase (MMP) activity in cartilage explant. Transfection with RDC1 siRNA caused a >90% reduction in human primary chondrocyte RDC1 expression and significantly reduced the impact of RDC1 agonist on the previously identified RDC1-regulated genes. CONCLUSIONS: RDC1 activation in human chondrocytes and cartilage explant leads to changes in gene expression and activity associated with chondrocyte hypertrophy, angiogenesis and increased matrix degradation, suggesting signalling via the RDC1 receptor may play an important role in the early development of osteoarthritis (OA).

In situ measurements of the sub-surface gamma dose from Chernobyl fallout.

Methods of estimating external radiation exposure of soil-dwelling organisms are currently of much research and regulatory interest. In this paper, we report the first in situ measurements of the sub-surface gamma dose rate for 137Cs contaminated land that quantify variation in dose rate with depth. Two contrasting sites have been investigated. The first site comprised a mineral type soil with a low percentage of organic matter and the second site chosen was in a peat-bog. The different soil compositions afford different 137Cs mobility and this results in variations in the measured gamma dose-rate with soil depth. For each site the paper reports the measured dose rates, the 137Cs activity depth profile, the 137Cs inventory and a description of the soil-characteristics. It is suggested that these data can be used to produce estimates of the sub-surface gamma dose rate in other sites of 137Cs contamination.

A new method to account for the depth distribution of 137Cs in soils in the calculation of external radiation dose-rate.

This work reports a new method for calculating the external dose-rate as a function of height above land that has been contaminated with a surface deposition of (137)Cs. Unlike previous work this method accounts for vertical migration of (137)Cs using the Advection Dispersion Equation (ADE) with appropriate parameters. The results have been successfully verified with field measurements from the (137)Cs contaminated regions within the Republic of Belarus. The method also correctly predicts the observed variation of dose-rate with elevation above the soil surface and it is shown how this method can be used to predict the reduction in surface dose-rate after remediation measures such as deep ploughing have taken place.

An analysis of the environmental mobility of radiostrontium from weapons testing and Chernobyl in Finnish river catchments.

The mobility of radiostrontium within the Arctic environment and surrounding area has been studied by analysing the mobility of 90Sr in river catchments that are within Finland. The environmental mobility of 90Sr deposited by both nuclear weapons testing and the Chernobyl accident has been investigated in five Finnish river catchments. Different models assessing the time-dependent mobility of 90Sr have been evaluated. No significant differences were found between the mobility of 90Sr from nuclear weapons tests and from the Chernobyl accident. Model parameters obtained by fitting to the measurements of the deposition and runoff rates of the nuclear weapons test fallout gave predictions which were consistent with the mid- and long-term contamination by the Chernobyl fallout. A comparison of 90Sr with 137Cs showed that they had similar mobility on deposition but, as time passed, the relative mobility of 90Sr increased with respect to 137Cs over a period of 5-8 years. Once the relative migration of 90Sr with respect to 137Cs reached equilibrium, its runoff rate was, on average, approximately an order of magnitude greater than 137Cs.

The ligand-receptor-G-protein ternary complex as a GTP-synthase. steady-state proton pumping and dose-response relationships for beta -adrenoceptors.

Steady-state solutions are developed for the rate of G alpha.GTP production in a synthase model of the ligand-receptor-G-protein ternary complex activated by a ligand-receptor proton pumping mechanism. The effective rate, k(31), defining the proton transfer, phosphorylation and G alpha.GTP release is a controlling rate of the synthase in the presence of a ligand with an efficient mode of signal activation, the ligand-receptor interaction taking place under effectively equilibrium conditions. The composite rate, however, becomes an amplifying factor in any dose-response relationship. The amplification is a triple product of the rate, k(31), the equilibrium constant associated with the activation of the proton signal, K(act)and the fraction of agonist conformer transmitting the signal, f(*). Where the rate of activation of the proton signal becomes critically inefficient, the rate of activation, k(act 1)replaces k(31)K(act). A correlation between beta(1)-adrenergic receptor-stimulated GDP release and adenylate cyclase activation shows that this correlation is not unique to an exchange reaction. Within the initiating Tyr-Arg-Tyr receptor proton shuttle mechanism, the position of Arg(r156) paralleldictates the high-(R(p)) and low-(R(u)) ligand-binding affinities. These states are close to R(*)and R(0)of the equilibrium model (De Lean et al., 1980, J. Biol. Chem.255, 7108-7117). An increased rate of hydrogen ion diffusion into a receptor mutant can give rise to constitutive activity while increased rates of G-protein release and changes in receptor state balance can contribute to the resultant level of action. Constitutive action will arise from a faster rate of G-protein release alone if proton diffusion in the wild-type receptor contributes to a basal level of G-protein activation. Competitive ligand-receptor occupancy for constitutive mutants shows that, where the rate of G-protein activation from the proportion of ligand-occupied receptors is less than the equivalent rate that would be generated from this fraction by proton diffusion, inverse agonism will occur. Rate-dependent dose-responses developed for the proposed synthase mechanism give explicit definition to the operational model for partial agonism (Black & Leff, 1983, Proc. Roy. Soc. Lond. B220, 141-162). When comparable ligands have effectively identical conformational states at the transition state for signal activation, the antagonist component of the binding "in vitro" can be derived by multiplying the apparent binding constant by (1-e) where e is the maximum stimulatory response. This component should be consistent throughout the tissues.

Relationship of the functional oropharynx to craniofacial morphology.

The association between the functional oropharyngeal airway (defined as the minimal sagittal dimension at right angles to the airstream) and craniofacial morphology was investigated using 16 craniofacial variables taken from lateral cephalometric radiographs. The sample consisted of 70 subjects (31 males and 39 females) 10 to 13 years of age. There was no difference in ages between males and females, and no correlation with age except upper face height. Oropharyngeal airway was positively correlated with length of the mandible (Gon-Men), the distance between the third cervical vertebra and the hyoid bone (C3-Hy), and cranial base angle (NSBa). Although short mandibular length is a characteristic finding in patients with obstructive sleep apnea, none of the subjects in this study had this diagnosis.

Kinetic and structural characteristics of the inhibition of enoyl (acyl carrier protein) reductase by triclosan.

Triclosan is used widely as an antibacterial agent in dermatological products, mouthwashes, and toothpastes. Recent studies imply that antibacterial activity results from binding to enoyl (acyl carrier protein) reductase (EACPR, EC 1.3.1.9). We first recognized the ability of triclosan to inhibit EACPR from Escherichia coli in a high throughput screen where the enzyme and test compound were preincubated with NAD(+), which is a product of the reaction. The concentration of triclosan required for 50% inhibition approximates to 50% of the enzyme concentration, indicating that the free compound is depleted by binding to EACPR. With no preincubation or added NAD(+), the degree of inhibition by 150 nM triclosan increases gradually over several minutes. The onset of inhibition is more rapid when NAD(+) is added. Gel filtration and mass spectrometry show that inhibition by triclosan is reversible. Steady-state assays were designed to avoid depletion of free inhibitor and changes in the degree of inhibition. The results suggest that triclosan binds to E-NAD(+) complex, with a dissociation constant around 20-40 pM. Triclosan follows competitive kinetics with respect to NADH, giving an inhibition constant of 38 pM at zero NADH and saturating NAD(+). Uncompetitive kinetics are observed when NAD(+) is varied, giving an inhibition constant of 22 pM at saturating NAD(+). By following regain of catalytic activity after dilution of EACPR that had been preincubated with triclosan and NAD(+), the rate constant for dissociation of the inhibitor (k(off)) is measured as 1.9 x 10(-4) s(-1). The association rate constant (k(on)) is estimated as 2.6 x 10(7) s(-1) M(-1) by monitoring the onset of inhibition during assays started by addition of EACPR. As expected, the ratio k(off)/k(on) = 7.1 pM is similar to the inhibition constants from the steady-state studies. The crystal structure of E. coli EACPR in a complex with coenzyme and triclosan has been determined at 1.9 A resolution, showing that this compound binds in a similar site to the diazaborine inhibitors. The high affinity of triclosan appears to be due to structural similarity to a tightly bound intermediate in catalysis.

The dawn of rapid maxillary expansion.

The first report of lateral maxillary expansion by separation of the maxilla, written by Angell and published in 1860, was discredited. Applying our present-day knowledge of the technique to the original documents indicates that the case history agrees in general with current observations. The arguments mounted against Angell, especially by McQuillen, may be dismissed as irrelevant and Angell's thesis is upheld. In addition, good reason exists to accept three further "firsts" in this unprecedented work: (1) The significance of the first permanent molars in occlusal development, (2) the use of a double-action jackscrew, and (3) the use of a retention plate.

Trimeric G alpha beta gamma protein coupling to a 3D model of G protein-coupled receptors confirming a new molecular mechanism for activation.

The present-day model for G protein activation and the associated theory on how a G protein-coupled receptor may activate the G protein are summarized. Experimental data are outlined which seem not to be in accordance with this present-day model. An alternative molecular mechanism for ternary complex activation is presented together with a three-dimensional model for a receptor coupled to the appropriate trimeric G protein. This 3D structure confirms our new molecular mechanism of activation.

Gender, social mobility and psychiatric diagnoses.

Data from a Swedish cohort born in 1953 and studied through to 1983 is used to examine the relationship between the incidence of psychiatric disorder, parental socio-economic status and intergenerational social mobility. No difference is found in the over-all incidence of in-patient treatment between men and women, but there are considerable differences in the incidence of individual diagnoses. As found in other studies, rates of schizophrenia and substance abuse are greater among men than women, while rates of neurosis are greater among women. The data generally support the drift explanation of inequalities in health rather than the social causation hypothesis, but there is some variation by both gender and diagnosis. Little association is found between parental status, measured when cohort members were aged 10, and the incidence of disorder, except in the case of substance abuse, but there is a strong association between disorder and own status, measured at age 27 yr. By far the highest rates of disorder are found among those members of the cohort who are not in the workforce. Both schizophrenia and neurosis exhibit strong drift effects; there is some evidence that the children of higher status parents have a heightened risk of being diagnosed as schizophrenic; in the case of substance abuse both downwards social mobility and low class origins appear to be implicated in the cumulative incidence of in-patient treatment.

The entropic penalty of ordered water accounts for weaker binding of the antibiotic novobiocin to a resistant mutant of DNA gyrase: a thermodynamic and crystallographic study.

Novobiocin is an antibiotic which binds to a 24 kDa fragment from the B subunit of DNA gyrase. Naturally occurring resistance arises from mutation of Arg-136 which hydrogen bonds to the coumarin ring of novobiocin. We have applied calorimetry to characterize the binding of novobiocin to wild-type and R136H mutant 24 kDa fragments. Upon mutation, the Kd increases from 32 to 1200 nM at 300 K. The enthalpy of binding is more favorable for the mutant (DeltaH degrees shifts from -12.1 to -17.5 kcal/mol), and the entropy of binding is much less favorable (TDeltaS degrees changes from -1.8 to -9.4 kcal/mol). Both of these changes are in the direction opposite to that expected if the loss of the Arg residue reduces hydrogen bonding. The change in heat capacity at constant pressure upon binding (DeltaCp) shifts from -295 to -454 cal mol-1 K-1. We also report the crystal structure, at 2.3 A resolution, of a complex between the R136H 24 kDa fragment and novobiocin. Although the change in DeltaCp often would be interpreted as reflecting increased burial of hydrophobic surface on binding, this structure reveals a small decrease. Furthermore, an ordered water molecule is sequestered into the volume vacated by removal of the guanidinium group. There are large discrepancies when the measured thermodynamic parameters are compared to those estimated from the structural data using empirical relationships. These differences seem to arise from the effects of sequestering ordered water molecules upon complexation. The water-mediated hydrogen bonds linking novobiocin to the mutant protein make a favorable enthalpic contribution, whereas the immobilization of the water leads to an entropic cost and a reduction in the heat capacity of the system. Such a negative contribution to DeltaCp, DeltaH degrees , and TDeltaS degrees appears to be a general property of water molecules that are sequestered when ligands bind to proteins.

High-energy magnetic compton scattering from iron.

The magnetic Compton profile of Fe [111] was measured using circularly polarized synchrotron radiation at incident energies of 84.4, 167.2 and 256.0 keV on the high-energy beamline at the European Synchrotron Radiation Facility. It was found that the momentum resolution of these experiments, which use semiconductor detectors, improves by almost a factor of two over what was previously possible by this technique at photon energies of approximately (1/10)mc(2). It was also observed that all three spectra reduced to the magnetic Compton profile, describing the spin-dependent ground-state momentum density, and that within the experimental error the integrated intensity of the magnetic effect scaled as predicted by the cross section derived in the limit of energies much less than the rest energy of the electron. The magnetic Compton profile of Fe [111], measured using 167.2 keV incident energy and with momentum resolution of 0.42 a.u., was compared with the prediction from a full-potential linearized augmented-plane-wave model profile. The fine structure predicted by theory was confirmed by the experimental profile at this improved resolution.

Teenage mothers and the adult mental health of their sons: evidence from a Stockholm cohort

Data from a Swedish male cohort born in 1953 is used to investigate the effects of teenage motherhood on later mental health and illness. Measures of coping ability and psychiatric impairment at the age of 19 years and of hospitalization with a psychiatric diagnosis at ages 20-30 show that the sons of teenage mothers have a poorer health record than those born to older women. The differences disappear, however, when controls are introduced for the marital and socio-economic status of the boys' mothers, and the data suggest that teenage motherhood is not associated with poor outcomes in the absence of other predisposing factors.