Replacement of lysine-181 by aspartic acid in the third transmembrane region of endothelin type B receptor reduces its affinity to endothelin peptides and sarafotoxin 6c without affecting G protein coupling.

A conserved aspartic acid residue in the third transmembrane region of many of the G protein-coupled receptors has been shown to play a role in ligand binding. In the case of endothelin receptors, however, a lysine residue replaces this conserved aspartic acid residue. To access the importance of this residue in ligand binding, we have replaced it with an aspartic acid in the rat endothelin type B (ETb) receptor by PCR mediated mutagenesis. The binding characteristics and functional properties of both the wild type and mutant receptors were determined in COS-7 cells transiently expressing the cloned receptor cDNAs. Using 125I-ET-1 as the radioactive peptide ligand in displacement binding studies, the wild type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three endothelin peptides (ET-1, ET-2, and ET-3) and sarafotoxin 6c (SRTX 6c). Interestingly, the mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM) but displayed a much larger increase in IC50 value for SRTX 6c (> 10 uM). The lysine mutant receptor still elicited full inositol phosphate (IP) turnover responses in the presence of saturating concentrations of endothelins (10 nM of ET-1, 100 nM of ET-2, or 1 uM of ET-3), indicating that the mutation (K181D) did not affect the coupling of mutant receptor to the appropriate G protein. These results demonstrate that lysine-181 on the receptor is important for binding ET peptides; however, it is required for binding the ETb selective agonist-SRTX 6c.     

Mutations of aspartate 103 in the Hm2 receptor and alterations in receptor binding properties of muscarinic agonists

Aspartate 103 (D103) in the third transmembrane domain of the Hm2 receptor was mutated to glutamate (D103E), asparagine (D103N), or alanine (D103A). As measured by [3H]-NMS, no significant binding was observed in D103A, while a 2-fold decrease in ligand affinity was seen in D103E and a 32-fold decrease in affinity was found in the D103N mutant. Examination of reference agonists showed greater loss of affinity in D103N than in D103E with the rank order of change being: L-607,207>carbachol>arecoline>pilocarpine>oxotremorine>McN-A-343. Of the novel 1-azabicyclo[2.2.1]-heptan-3-one oxime agonists examined, arylacetylene oximes showed little alteration in binding in either the D103E or D103N mutants, while the geometric isomers of several bicyclic aryl-ene-yne oximes showed significant changes in affinity, especially in the D103N mutant. Thus, overall size of the agonist and/or spatial orientation of the molecule within the binding pocket contribute to changes measured in binding.     

Hydrodynamic thickening of lubricating fluid layer beneath sliding mesothelial tissues

The delicate mesothelial surfaces of the pleural space and other serosal cavities slide relative to each, lubricated by pleural fluid. In the absence of breathing motion, differences between lung and chest wall shape could eventually cause the lungs and chest wall to come into contact. Whether sliding motion keeps lungs and chest wall separated by a continuous liquid layer is not known. To explore the effects of hydrodynamic pressures generated by mesothelial sliding, we measured the thickness of the liquid layer beneath the peritoneal surface of a 3-cm disk of rat abdominal wall under a normal stress of 2 cm H2O sliding against a glass plate rotating at 0-1 rev/s. Thickness of the lubricating layer was determined microscopically from the appearance of fluorescent microspheres adherent to the tissue and glass. Usually, fluid thickness near the center of the tissue disk increased with the onset of glass rotation, increasing to 50-200 microm at higher rotation rates, suggesting hydrodynamic pumping. However, thickness changes often differed substantially among tissue samples and between clockwise and counter-clockwise rotation, and sometimes thickness decreased with rotation, suggesting that topographic features of the tissue are important in determining global hydrodynamic effects. We conclude that mesothelial sliding induces local hydrodynamic pressure gradients and global hydrodynamic pumping that typically increases the thickness of the lubricating fluid layer, moving fluid against the global pressure gradient. A similar phenomenon could maintain fluid continuity in the pleural space, reducing frictional force and shear stress during breathing.     

Identification of residues in the monocyte chemotactic protein-1 that contact the MCP-1 receptor, CCR2.

The CC chemokine, MCP-1, has been identified as a major chemoattractant for T cells and monocytes, and plays a significant role in the pathology of inflammatory diseases. To identify the regions of MCP-1 that contact its receptor, CCR2, we substituted all surface-exposed residues with alanine. Some residues were also mutated to other amino acids to identify the importance of charge, hydrophobicity, or aromaticity at specific positions. The binding affinity of each mutant for CCR2 was assayed with THP-1 and CCR2-transfected CHL cells. The majority of point mutations had no effect. Residues at the N-terminus of the protein, known to be crucial for signaling, contribute less than a factor of 10 to the binding affinity. However, two clusters of primarily basic residues (R24, K35, K38, K49, and Y13), separated by a 35 A hydrophobic groove, reduced the level of binding by 15-100-fold. A peptide fragment encompassing residues 13-35 recapitulated some of the mutational data derived from the intact protein. It exhibited modest binding as a linear peptide and dramatically improved affinity when the region which adopts a single turn of a 3(10)-helix in the protein, which includes R24, was constrained by a disulfide bond. Additional constraints at the ends of the peptide, corresponding to the disulfide between the first and third cysteines in MCP-1, yielded further improvements in affinity. Together, these data suggest a model in which a large surface area of MCP-1 contacts the receptor, and the accumulation of a number of weak interactions results in the 35 pM affinity observed for the wild-type (WT) protein. The receptor binding site of MCP-1 also is significantly different from the binding sites of RANTES and IL-8, providing insight into the issue of receptor specificity. It was previously shown that the N-terminus of CCR2 is critical for binding MCP-1 [Monteclaro, F. S., and Charo, I. F. (1996) J. Biol. Chem. 271, 19084-92; Monteclaro, F. S., and Charo, I. F. (1997) J. Biol. Chem. 272, 23186-90]. Point mutations of six acidic residues in this region of the receptor were made to test their role in ligand binding. This identified D25 and D27 of the DYDY motif as being important. On the basis of our data, we propose a model in which the receptor N-terminus lies along the hydrophobic groove in an extended fashion, placing the DYDY motif near the basic cluster involving R24 and K49 of MCP-1. This in turn orients the signaling residues (Y13 and the N-terminus) for productive interaction with the receptor.     

Determinants of friction in soft elastohydrodynamic lubrication

Elastohydrodynamic lubrication (EHL) protects soft tissues from damage and wear in many biological systems (e.g. synovial joints, cornea of the eye, and pleural surfaces of the lung and chest wall). Among studies of lubrication of deformable solids, few have examined the effects of external loads, geometry, and material properties on EHL of soft tissues. To examine these effects, we studied the tribology of soft tissues in a two-dimensional finite element simulation of a thin layer of fluid separating a sliding rigid surface from a soft asperity or bump with an initial sinusoidal shape. We computed the frictional force, deformation of the solid, and change in fluid thickness as functions of independent variables: sliding velocity, normal load, material properties, and bump amplitude and length. Double-logarithmic regression was used to determine the exponents of the scaling relationships of friction coefficient and minimum fluid thickness to the independent variables. The analysis showed that frictional shear force is strongly dependent on velocity, viscosity, and load, moderately dependent on bump length and elasticity, and only weakly dependent on the bump amplitude. The minimum fluid thickness is strongly dependent on velocity and viscosity, and changes moderately with load, elasticity, amplitude, and length. The shape of the bump has little effect. The results confirm that the shear-induced deformation of an initially symmetrical shape, including generalizations to other symmetrical geometries such as quadratic or piecewise linear bumps, leads to load-supporting behavior.     

Novel SCN5A variants identified in a group of Iranian Brugada syndrome patients

Brugada syndrome (BrS) is a rare hereditary arrhythmia syndrome that increases an individual’s risk for sudden cardiac death (SCD) due to ventricular fibrillation. This disorder is regarded as a notable cause of death in individuals aged less than 40 years, responsible for up to 40% of sudden deaths in cases without structural heart disease, and is reported to be an endemic in Asian countries. Mutations in SCN5A are found in approximately 30% of patients with Brugada syndrome. This study aimed to investigate mutations in the SCN5A gene in a group of Iranian Brugada syndrome patients. Nine probands (n = 9, male, mean age = 39) diagnosed with Brugada syndrome were enrolled in this study. Exon 2 to 29 were amplified by PCR and subjected to direct sequencing. Eight in silico prediction tools were used to anticipate the effects of non-synonymous variants. Seven known polymorphisms and 2 previously reported disease-causing mutations, including H558R and G1406R, were found in the studied cases. Twenty novel variants were identified: 15 missense, 2 frameshift, 2 synonymous, and one nonsense variants. In silico tools predicted 11 non-synonymous variants to have damaging effects, whereas frameshift and nonsense variants were considered inherently pathogenic. The novel variants identified in this study, alongside previously reported mutations, are highly likely to be the cause of the Brugada syndrome phenotype observed in the patient group. Further analysis is required to understand the physiological effects caused by these variants.

     

Child Overweight,Associated Psychopathology,and Social Functioning: A French School‐based Survey in 6‐ to 11‐year‐old Children

The aim of this study was to estimate the prevalence of child overweight in a regional sample of primary school‐aged children, and to examine the relationships among child overweight, psychopathology, and social functioning. A cross‐sectional survey was conducted in 2004 in 100 primary schools of a large French region, with 2,341 children aged 6–11 randomly selected. Child weight and height, lifestyle variables (leisure‐time physical activity (LTPA), watching television (TV), playing video games), and socioeconomic characteristics were collected in parent‐administered questionnaires. Child psychopathology outcomes were assessed using child‐ and parent‐reported instruments (Dominic Interactive (DI) and Strengths and Difficulties Questionnaire (SDQ)). Overweight and obesity were estimated according to the International Obesity Task Force (IOTF) definition. Response rates to the parent questionnaire and DI were 57.4 and 95.1%, respectively. Final sample size was 1,030 children. According to the IOTF, 17.3% of the children were overweight, of whom 3.3% were obese. In univariate analysis, correlates of overweight were low parental education, low monthly income, Disadvantaged School Areas (DSAs), self‐reported generalized anxiety, parent‐reported conduct disorders, emotional problems, and peer difficulties. High monthly income was less frequently associated with overweight. In multivariate analysis, parent‐reported peer difficulties (odds ratio (OR) = 2.06; 95% confidence interval = 1.27–3.35) and DSAs (1.88; 1.03–3.44) were independent factors significantly associated with child overweight. There was a trend of being overweight with elevated TV times (P for trend = 0.02). The psychosocial burden of excess weight appears to be significant even in young children. Findings should be considered for preventing strategies and public health interventions. School‐based overweight prevention programs should be implemented first in disadvantaged areas together with information about weight stigmatization and discrimination.