Use of hGluc/tdTomato pair for sensitive BRET sensing of protease with high solution media tolerance.

Due to the complicated media, monitoring proteases in real physiological environments is still a big challenge. Bioluminescence resonance energy transfer (BRET) is one of the promising techniques but its application is limited by the susceptibility to buffer composition, which might cause serious errors for the assay. Herein we report a novel combination of BRET pair with humanized Gaussia luciferase (hGluc) and highly bright red fluorescence protein tdTomato for sensitive and robust protease activity determination. As a result, the hGluc/tdTomato BRET pair showed much better tolerance to buffer composition, pH and sample matrices, and wide spectral separation (Δλ:~110 nm). With the protease sensor built with this pair, the detection limit for enterokinase reached 2.1 pM in pure buffer and 3.3 pM in 3% serum. The proposed pair would find broad use in both in vitro and in vivo assays, especially for samples with complicated matrix.

Buffer enhanced bioluminescence resonance energy transfer sensor based on Gaussia luciferase for in vitro detection of protease.

Bioluminescence resonance energy transfer (BRET) has gained favors in recent years as a detection technology for protease activity due to its extreme reliability, high sensitivity and low intrinsic backgrounds. Because of the sensitivity of the donors, substrates and the acceptors, it is expected that BRET systems are sensitive to buffer environments. However, no systematic study has been reported on how buffer components would affect the BRET ratio, and thus affect the determination of protease activity based on BRET. We present here that several environmental factors, including buffer agents, pH and divalent metal ions, influenced BRET ratio significantly, when humanized Gaussia luciferase (hGluc) was utilized as the donor and enhanced yellow fluorescence protein (EYFP) as the acceptor. Based on these findings, an enhancing solution was optimized to improve the performance of the BRET sensor for analysis of enterokinase activity in vitro, resulting in 10-fold and 7-fold improvement of the sensitivity and the detection limit, respectively. We anticipate the system will be applicable for improving performance of other in vitro BRET protease sensors, especially when the optimal conditions for protease activity would severely affect the BRET signal.

Roles of gastrointestinal and adipose tissue peptides in childhood obesity and changes after weight loss due to lifestyle intervention.

Childhood obesity is a global epidemic and associated with an increased risk of hypertension, diabetes mellitus, and coronary heart disease, in addition to psychological disorders. Interventions such as bariatric surgery are highly invasive and lifestyle modifications are often unsuccessful because of disturbed perceptions of satiety. New signaling peptides discovered in recent years that are produced in peripheral tissues such as the gut, adipose tissue, and pancreas communicate with brain centers of energy homeostasis, such as the hypothalamus and hindbrain. This review discusses the major known gut- and adipose tissue-derived hormones involved in the regulation of food intake and energy homeostasis and their serum levels in childhood obesity before and after weight loss as well as their relationship to consequences of obesity. Since most of the changes of gastrointestinal hormones and adipokines normalize in weight loss, pharmacological interventions based on these hormones will likely not solve the obesity epidemic in childhood. However, a better understanding of the pathways of body weight- and food intake-regulating gut- and adipose tissue-derived hormones will help to find new strategies to treat obesity and its consequences.

Intermittent sampling of portal venous blood and bile from guinea pigs.

A technique is described for intermittent collection of portal venous blood from guinea pigs through a catheter advanced from an ileal tributary of the cranio-mesenteric vein into the portal vein and for the collection of bile from a catheter in the gallbladder after ligature obstruction of the common bile duct.

Displacement of endogenous enterokinase into portal venous blood and bile following luminal perfusion of proximal small intestine in guinea pigs.

The displacement of endogenous enterokinase into portal venous blood or bile was studied in conscious guinea pigs both with the small intestine undisturbed and during gentle, intermittent luminal perfusion of a 25-cm segment of duodenum and proximal jejunum. Perfusates tested included water, 150 mM saline, 5% (v/v) ethanol, 0.2% (w/v) lysolecithin, and mixtures of ethanol and lysolecithin. Enterokinase activity was absent from portal venous blood of control guinea pigs with the intestine undisturbed but perfusion with luminal saline or water was consistently associated with substantial levels of active enterokinase in portal venous blood. Similar concentrations of enterokinase in portal blood were also detected in response to luminal ethanol and lysolecithin. The capacity of the normal liver rapidly to clear the enzyme from portal blood was demonstrated. Of the estimated total endogenous enterokinase displaced, 0.2-0.4% was recovered in catalytically active form from the pooled bile of luminally perfused but not control animals. The readiness with which enterokinase was displaced into the circulation in the absence of mucosal damage raises the unexpected possibility that the event may be physiological. Induced penetration of the mucosa and absorption of luminal components is clearly different from the release into portal venous blood of endogenous mucosal macromolecules.

The biliary excretion of enterokinase in rats. Studies in normal, chronic ethanol-maintained and cirrhotic rats.

The excretion of catalytically active human or pig enterokinase in hepatic bile after intravenous administration to normal rats or rats that had been maintained on 20% (v/v) ethanol for 1 year showed similar kinetics to that described for other serum-derived bile proteins. The half-life in serum was 2.5 min or less, and most of the enzyme was excreted within 45 min of administration. This was maintained when up to six successive doses were given at 90 min intervals. The mean amount excreted per dose was independent of the dose number and varied from 0.8% to 2.1% in the normal animals and 1.2% to 2.0% in the chronic ethanol-maintained animals. When three doses of enzyme were given at 30 min intervals, the total amount of active enterokinase recovered in bile was dose-dependent and was consistently higher in the rats drinking 20% (v/v) ethanol. The serum half-life of enterokinase in rats made cirrhotic by inhalation of carbon tetrachloride vapour was extended to 6 min or more. The amount of active enzyme recovered in bile was at least 50% less than in weight-matched normal rats, and excretion was not complete 2h after intravenous administration. The possible significance of these findings in liver and pancreatic disease is discussed.

Identification of a defence mechanism in vivo against the leakage of enterokinase into the blood.

1. The serum proteinase inhibitors alpha 1-antitrypsin, alpha 2-macroglobulin, inter-alpha-trypsin inhibitor and C1-esterase inhibitor were found not to affect the catalytic activity of human enterokinase, whereas bovine trypsin activity was modified essentially as expected. Enterokinase was also not inhibited by Trasylol (trypsin inhibitor from bovine lung) or bovine pancreatic trypsin inhibitor. No other component in human or mouse serum complexing with enterokinase was identified. 2. Human enterokinase administered intravenously into mice was rapidly cleared from the circulation with a half-life of 2.5 min. This removal was not the result of the difference in species, since partially purified mouse enterokinase was cleared at the same rate as the human enzyme. Clearance was mediated by recognition of the carbohydrate portion of enterokinase and not through specific recognition of its catalytic site. Immunofluorescent staining showed that the enzyme accumulated in the liver. Attempts to block the clearance by the simultaneous infusion of competing glycoproteins suggested that enterokinase was taken up by hepatocytes. Of the glycoproteins tested only two, human lactoferrin (terminal fucosyl alpha 1 leads to 3 N-acetylglucosamine) and bovine asialo-fetuin (terminal galactosyl beta 1 leads to 4 N-acetylglucosamine) were weakly competitive. Two inhibitors of endocytosis, Intralipid and Triton WR1339, failed to delay the removal of enterokinase. It is proposed that enterokinase is cleared from the circulation by an as yet uncharacterized hepatocyte receptor.

Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum.

The activities of highly purified human enterokinase (enteropeptidase, EC 3.4.21.9) and bovine trypsin were tested against three synthetic substrates alpha-N-Benzoyl-L-arginine ethyl ester HCl, alpha-N-Benzoyl-DL-arginine-p-nitroanilide HCl and alpha-N-Benzoyl-DL-arginine-2-naphthylamide HCl. There was no detectable hydrolysis of these substrates by enterokinase whereas the kinetic parameters obtained for trypsin were in close agreement with those previously described by other workers. The values for Km and kcat were dependent on the Ca2+ concentration. Hydrolysis of glycine-tetra-L-aspartyl-L-lysyl-2-naphthylamide (Gly(Asp)4-Lys-Nap) by these protease was also studied. Enterokinase-catalysed hydrolysis obeyed simple steady-state kinetics and values for Km of 0.525 mM and 0.28 mM and for kcat of 21.5 s-1 and 28.3 s-1 were obtained in 0.1 mM and 10 mM Ca2+, respectively. Trypsin-catalysed hydrolysis was complex and the response to Ca2+ was sigmoidal partly due to the lability of trypsin at low Ca2+ concentrations. A sensitive specific assay for enterokinase was developed and applied to the measurement of the enzyme in serum; interference by nonspecific arylamidases was eliminated by the addition of Zn2+.