We have used three distinct bioassay systems (rat aorta (RA) relaxation; rat gastric longitudinal muscle (LM) contraction; human embryonic kidney 293 (HEK293) cell calcium signal) to evaluate the activity and receptor selectivity of analogues of the receptor-activating peptides derived either from the thrombin receptor (TRAPs, based on the human receptor sequence, SFLLRNPNDK...) or the proteinase-activated receptor 2 (PAR2APs, based on the rat receptor sequence SLIGRL...). Our main focus was on the activation of PAR2 by PAR2APs and the cross-activation of PAR2 by the TRAPs. In the RA and LM assay systems, PAR2APs that were either N-acetylated (N-acetyl-SLIGRL-NH2) or had a reverse N-terminal sequence (LSIGRL-NH2) were inactive, either as agonists or antagonists. An alanine substitution at position 3 of the PAR2AP (SLAGRL-NH2) led to a dramatic reduction of biological activity, as did substitution of threonine for serine at position 1 (TLIGRL-NH2). However, alanine substitution at PAR2AP position 4 caused only a modest reduction in activity, resulting in a peptide (SLIARL-NH2) with a potency equivalent to that of the human PAR2AP, SLIGKV-NH2. The order of potency of the PAR2APs in the RA, LM, and HEK assay systems was SLIGRL-NH2 > SLIARL-NH2 > SLIGKV-NH2 > TLIGRL-NH2 > SLAGRL-NH2. In HEK cells, none of the PAR2APs activated the thrombin receptor (PAR1). However, in the HEK cell assay, the TRAP, SFLLR-NH2, activated or desensitized both PAR1 and PAR2 receptors, whereas the xenopus TRAP, TFRIFD-NH2, activated or desensitized selectively PAR1 but not PAR2. By constructing human-xenopus hybrid peptides, we found that the TRAPs, TFLLR-NH2, and SFLLFD-NH2 selectively activated the thrombin receptor in HEK cells without activating or desensitizing PAR2. In contrast, the TRAPs SFLLRD-NH2 and AFLLR-NH2 activated or desensitized both PAR1 and PAR2. The order of potency for the TRAPs in all bioassay systems was SFLLR-NH2 approximately equal to SFLLRD-NH2 approximately equal to TFLLR-NH2 > SFLLFD-NH2 > TFRIFD-NH2. We conclude that the N-terminal domain of the PAR2AP as well as positon 3 plays important roles for PAR2 activation. In contrast, the first and fifth amino acids in the TRAP motif, SFLLR-NH2, do not play a unique role in activating the thrombin receptor, but if appropriately modified can abrogate the ability of this peptide to cross-desensitize or activate PAR2, so as to be selective for PAR1. The PAR1- and PAR2-selective peptides that we have synthesized will be of use for the evaluation of the roles of the PAR1 and PAR2 receptor systems in vivo.

1 The biological activities of the proteinase-activated receptor number 2 (PAR-2)-derived peptides, SLIGRL (PP6) SLIGRL-NH2 (PP6-NH2) and were measured in mouse and rat gastric longitudinal muscle (LM) tissue and in a rat aortic ring preparation and the actions of the PAR-2-derived peptides were compared with trypsin and with the actions of the thrombin receptor activating peptide, SFLLR-NH2 (TP5-NH2). 2 From a neonatal rat intestinal cDNA library, and from intestinal and kidney-derived cDNA, the coding region of the rat PAR-2 receptor was cloned and sequenced, thereby establishing its close sequence identity with the previously described mouse PAR-2 receptor; and this information, along with a reverse-transcriptase (RT) polymerase chain reaction (PCR) analysis of cDNA derived from gastric and aortic tissue was used to establish the concurrent presence of PAR-2 and thrombin receptor mRNA in both tissues. 3 In the mouse and rat gastric preparations, the PAR-2-derived polypeptides, PP6, PP6-HN2 and PP5-NH2 caused contractile responses that mimicked the contractile actions of low concentrations of trypsin (5 u/ml-'; 10 nM) and that were equivalent to contractions caused by TP5-NH2. 4 The cumulative exposure of the rat LM tissue to PP6-NH2 led to a desensitization of the contractile response to this polypeptide, but not to TP5-NH2 and vice versa, so as to indicate a lack of crossdesensitization between the receptors responsive to the PAR-2 and thrombin receptor-derived peptides. 5 In the rat gastric preparation, the potencies of the PAR-2-activating peptides were lower than the potency of TP5-NH2 (potency order: TP5-NH2 >>PP6-NH2k PP6 > PP5-NH2); PP6 was a partial agonist in this preparation. 6 The contractile actions of PP6 and PP6-NH2 in the rat gastric preparation required the presence of extracellular calcium, were inhibited by nifedipine and were blocked by the cyclo-oxygenase inhibitor, indomethacin and by the tyrosine kinase inhibitor, genistein, but not by the kinase C inhibitor, GF109203X. The contractile responses were not blocked by atropine, chlorpheniramine, phenoxybenzamine, propranolol, ritanserin or tetrodotoxin. 7 In a precontracted rat aortic ring preparation, with an intact endothelium, all of the PAR-2-derived peptides caused a prompt relaxation response that was blocked by the nitric oxide synthase inhibitor, NwO-nitro-L-arginine-methyl ester (L-NAME) but not by D-NAME; in an endothelium-free preparation, which possessed mRNA for both the PAR-2 and thrombin receptors, the PAR-2-activating peptides caused neither a relaxation nor a contraction, in contrast with the contractile action of TP5-NH2. The relaxation response to PP6-NH2 was not blocked by atropine, chlorpheniramine, genistein, indomethacin, propranolol or ritanserin. 8 In the rat aortic preparation, the potencies of PP6, PP6-NH2 and PP5-NH2 were greater than those of the thrombin receptor activating peptide, TP5-NH2 (potency order: PP6-NH2 > PP6 > PP5-NH2 > TP5-NH2).9 In the rat aortic preparation, the relaxant actions of the PAR-2-...

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs).Although thrombin is a recognized physiological activator of PAR 1 and PAR 4 , the endogenous enzymes responsible for activating PAR 2 in settings other than the gastrointestinal system, where trypsin can activate PAR 2 , are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR 1 , PAR 2 , and PAR 4 ; 2) PAR-dependent calcium signaling responses in cells expressing PAR 1 , PAR 2 , and PAR 4 and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR 4 -dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR 1 , PAR 2 , and PAR 4 and to disarm/inhibit PAR 1 . Although hK5 and -6 were also able to activate PAR 2 , they failed to cause PAR 4 -dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR 2 -mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR 1 , PAR 2 , and PAR 4 . Proteinase-activated receptors (PAR 1-4 )3 compose a unique family of four G-protein-coupled cell surface receptors for certain proteinases (1-9). Proteolytic cleavage within the extracellular N terminus reveals a tethered ligand that binds to the extracellular receptor domains to initiate cell signaling (5, 6, 9, 10). Proteinases that activate PARs include coagulation factors, enzymes from inflammatory cells, and proteinases from epithelial cells and neurons. These enzymes, generated and released during injury and inflammation, can cleave and activate PARs on many cell types from a variety of species (humans, rats, and mice) to regulate the critically important processes of hemostasis, inflammation, pain, and tissue repair. Other proteinases that cleave PARs downstream of the N-terminal tethered ligand sequence disable the receptors for further proteolytic activation, thus abrogating PAR signaling. It is of considerable interest to identify the proteinases that activate and/or disable PARs, in view of the emerging role that these receptors can play in diseases such as asthma, arthritis, inflammatory bowel disease, and cancer (5-7).In some systems, the proteinases that activate PARs have been established. For example, in the circulatory system, PAR 1 , PAR 3 , and PAR 4 are recognized physiological targets for thrombin (4), which does not efficiently acti...

A study was conducted in a completely randomized design to evaluate the performance, excreta characteristics, and some blood nitrogen metabolite concentrations of 28-d-old male broilers fed 4 experimental diets in which CP was decreased in a stepwise manner from 23 to 17%. The other 4 diets were formulated to have 19 and 17% CP, in which 2 of them contained an additional 10% of particular essential amino acids (EAA) and 2 were supplemented with Gly and Glu. Ileal digestible quantities of all EAA were almost equal in the diets, and total amount of each EAA was maintained at or above NRC requirements. Decreasing dietary CP below 19% depressed performance and appetite and increased fat deposition in the whole body and abdominal cavity significantly. Adding the Gly and Glu mixtures to low-CP diets improved performance and decreased fat deposition. Uric acid, moisture, and acidity of excreta were decreased by reduction of dietary CP; excretory ammonia level was increased in 17% CP diets. Blood ammonia level was increased and plasma uric acid was decreased with reduction of CP to 17%. Supplementing Gly and Glu increased plasma and excretory uric acid level in spite of decreasing blood ammonia concentration. The aminostatic hypothesis cannot explain the sharp reduction in appetite in this experiment, because alteration of dietary CP had no significant influence on most plasma free amino acid levels. Therefore, reduction of CP to 19% not only does not impair performance but also decrease nitrogen, ammonia, and pH of excreta that may improve upon litter and air quality. Adding large amounts of crystalline EAA to diets with low intact CP increased blood and excretory ammonia concentration, which due to its negative effects on tissue metabolism may be the main cause of retarded growth and appetite in decreased CP diets below 19%.

SummaryBackgroundSurgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world.MethodsThis international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231.FindingsBetween Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p<0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p<0·001).InterpretationCountries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication.FundingDFID-MRC-Wellcome Trust Joint Global Health Trial Development Grant,...