ABSTRACT
G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies.
Subject(s)
Receptor, Melanocortin, Type 4/genetics , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Alleles , Animals , Disease/genetics , Ethylnitrosourea/chemistry , Gene Expression , Gene Knockout Techniques/methods , Genetic Variation , Humans , Mutagenesis/genetics , Mutation , Mutation, Missense , Phenotype , Rats , Receptor, Melanocortin, Type 4/metabolism , Receptors, Lysophosphatidic Acid/genetics , Receptors, Lysophosphatidic Acid/metabolismABSTRACT
Complications attributable to direct pressure may result from the use of pneumatic tourniquets during surgical procedures. Traditional estimates have determined the pressures employed rather than the minimal pressure necessary to produce a bloodless field. To determine this pressure, pre-operative and post-operative systolic blood pressures and the tourniquet pressure at which capillary bleeding occurred were measured in a group of patients undergoing elective surgery of the upper and lower limbs. From these results two equations were derived, one for each of the upper and lower limbs, which give the minimum tourniquet pressures to produce bloodless fields. In an average sized, normotensive patient, 200mm Hg was found to be adequate for the upper limb and 250mm Hg for the lower limb.
