Measurement of Chitinase Activity in Biological Samples

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body ...

Yale University School of Medicine

Progressive familial intrahepatic cholestasis 1 (PFIC1) results from mutations in ATP8B1, a putative aminophospholipid flippase. Conflicting hypotheses have been proposed for the pathogenesis of PFIC1. The aim of this study was to determine whether ATP8B1 deficiency produces cholestasis by altering the activity of the farnesoid X receptor (FXR) or by impairing the structure of the canalicular membrane.

ATP8B1 deficiency disrupts the bile canalicular membrane bilayer structure in hepatocytes, but FXR expression and activity are maintained.

The premature activation of digestive proenzymes, specifically proteases, within the pancreatic acinar cell is an early and critical event during acute pancreatitis. Our previous studies demonstrate that this activation requires a distinct pathological rise in cytosolic Ca(2+). Furthermore, we have shown that a target of aberrant Ca(2+) in acinar cells is the Ca(2+)/calmodulin-dependent phosphatase calcineurin (PP2B). In this study, we hypothesized that PP2B mediates in vivo protease activation and pancreatitis severity. To test this, pancreatitis was induced in mice over 8 h by administering hourly intraperitoneal injections of the cholecystokinin analog caerulein (50 microg/kg). Treatment with the PP2B inhibitor FK506 at 1 and 8 h after pancreatitis induction reduced trypsin activities by greater than 50% (P < 0.005). Serum amylase and IL-6 was reduced by 86 and 84% relative to baseline (P < 0.0005) at 8 h, respectively. Histological severity of pancreatitis, graded on the basis of pancreatic edema, acinar cell vacuolization, inflammation, and apoptosis, was reduced early in the course of pancreatitis. Myeloperoxidase activity from both pancreas and lung was reduced by 93 and 83% relative to baseline, respectively (P < 0.05). These data suggest that PP2B is an important target of the aberrant acinar cell Ca(2+) rise associated with pathological protease activation and pancreatitis.

Protease activation during in vivo pancreatitis is dependent on calcineurin activation.

L&#39;activation pr&eacute;matur&eacute;e des proenzymes digestifs, plus pr&eacute;cis&eacute;ment des prot&eacute;ases, dans les cellules acineuses du pancr&eacute;as est un &eacute;v&eacute;nement pr&eacute;coce et critique au cours de la pancr&eacute;atite aigu&euml;. Nos &eacute;tudes pr&eacute;c&eacute;dentes montrent que cette activation n&eacute;cessite une augmentation pathologique dans Ca(2+) cytosolique. En outre, nous avons montr&eacute; qu&#39;une cible de Ca(2+) aberrant dans les cellules acineuses est la calcineurine phosphatase de Ca(2+)/calmoduline-d&eacute;pendante (PP2B). Dans cette &eacute;tude, nous avons &eacute;mis l&#39;hypoth&egrave;se que PP2B intervient dans la s&eacute;v&eacute;rit&eacute; de l&#39;activation et la pancr&eacute;atite prot&eacute;ase in vivo. Pour tester ceci, pancr&eacute;atite a &eacute;t&eacute; induite chez les souris sur 8 h en administrant des injections intrap&eacute;riton&eacute;ales horaires de la chol&eacute;cystokinine analogique c&eacute;rul&eacute;ine (50 microg/kg). Traitement avec l&#39;inhibiteur PP2B FK506, 1 et 8 h apr&egrave;s l&#39;induction de la pancr&eacute;atite r&eacute;duit les activit&eacute;s de la trypsine par plus de 50 % (P &lt; 0,005). IL-6 et l&#39;amylase s&eacute;rique a &eacute;t&eacute; r&eacute;duit de 86 et 84 % par rapport &agrave; la ligne de base (P &lt; 0,0005) &agrave; 8 h, respectivement. Gravit&eacute; histologique de pancr&eacute;atite, class&eacute;e sur la base d&#39;un oed&egrave;me du pancr&eacute;as, une vacuolisation des cellules acineuses, l&#39;inflammation et l&#39;apoptose, a &eacute;t&eacute; r&eacute;duite au d&eacute;but, au cours de la pancr&eacute;atite. Activit&eacute; my&eacute;loperoxydase du pancr&eacute;as et du poumon a &eacute;t&eacute; r&eacute;duite respectivement de 93 et 83 % par rapport &agrave; la ligne de base, (P 0,05 &lt;). Ces donn&eacute;es sugg&egrave;rent que PP2B est une cible importante de la cellule acineuse aberrante Ca(2+) &eacute;l&eacute;vation associ&eacute;e &agrave; la pancr&eacute;atite et l&#39;activation de la prot&eacute;ase pathologique.

Activation de la prot&eacute;ase au cours de la pancr&eacute;atite in vivo d&eacute;pend de l&#39;activation de la calcineurine.

L&#39;attivazione precoce di proenzymes digestivi, in particolare le proteasi, all&#39;interno delle cellule acinose del pancreas &egrave; un evento precoce e critico durante la pancreatite acuta. I nostri precedenti studi dimostrano che questa attivazione richiede un aumento patologico distinto in Ca(2+) citosolico. Inoltre, abbiamo dimostrato che una destinazione di aberrante Ca(2+) nelle cellule acinose &egrave; il Ca(2+)/calmodulina-dipendente fosfatasi calcineurina (PP2B). In questo studio, abbiamo ipotizzato che la PP2B media gravit&agrave; attivazione e pancreatite proteasi in vivo. Per fare una prova, pancreatite &egrave; stata indotta nei topi oltre 8 h dalla somministrazione orarie iniezioni intraperitoneali della colecistochinina analogico caerulein (50 microg/kg). Il trattamento con l&#39;inibitore di PP2B FK506 a 1 e 8 ore dopo l&#39;induzione di pancreatite ridotta attivit&agrave; della tripsina di superiore al 50% (P &lt; 0,005). Amilasi sierica e IL-6 &egrave; stato ridotto da 86 e 84% rispetto al basale (P &lt; 0,0005) a 8 h, rispettivamente. Gravit&agrave; istologica di pancreatite, classificata sulla base di edema del pancreas, vacuolizzazione delle cellule acinose, infiammazione e apoptosi, &egrave; stata ridotta precocemente nel corso della pancreatite. Attivit&agrave; di mieloperossidasi da polmone e pancreas &egrave; stato ridotto da 93 e 83% rispetto al basale, rispettivamente (P &lt; 0,05). Questi dati suggeriscono che la PP2B &egrave; un obiettivo importante della cellula aberrante acinose Ca(2+) aumento associato con pancreatite e attivazione di proteasi patologica.

Attivazione di proteasi durante la pancreatite in vivo &egrave; dipendente dall&#39;attivazione della calcineurina.

Bile salt secretion is mediated primarily by the bile salt export pump (Bsep), a transporter on the canalicular membrane of the hepatocyte. However, little is known about the short-term regulation of Bsep activity. Ca(2+) regulates targeting and insertion of transporters in many cell systems, and Ca(2+) release near the canalicular membrane is mediated by the type II inositol 1,4,5-trisphosphate receptor (InsP3R2), so we investigated the possible role of InsP3R2 in modulating Bsep activity. The kinetics of Bsep activity were monitored by following secretion of the fluorescent Bsep substrate cholylglycylamido-fluorescein (CGamF) in rat hepatocytes in collagen sandwich culture, an isolated cell system in which structural and functional polarity is preserved. CGamF secretion was nearly eliminated in cells treated with Bsep small interfering RNA (siRNA), demonstrating specificity of this substrate for Bsep. Secretion was also reduced after chelating intracellular calcium, inducing redistribution of InsP3R2 by depleting the cell membrane of cholesterol, or reducing InsP3R function by either knocking down InsP3R2 expression using siRNA or pharmacologic inhibition using xestospongin C. Confocal immunofluorescence showed that InsP3R2 and Bsep are in close proximity in the canalicular region, both in rat liver and in hepatocytes in sandwich culture. However, after knocking down InsP3R2 or inducing its dysfunction with cholesterol depletion, Bsep redistributed intracellularly. Finally, InsP3R2 was lost from the pericanalicular region in animal models of estrogen- and endotoxin-induced cholestasis.

Type 2 inositol 1,4,5-trisphosphate receptor modulates bile salt export pump activity in rat hepatocytes.

We report a case of a 31-year-old man who presented to the emergency department after four episodes of syncope within a 24 h time span. He was found to have symptomatic complete heart block associated with episodes of ventricular asystole lasting 5-6 s. He underwent emergent permanent pacemaker insertion during which he was found to have no underlying rhythm. He was later found to have positive serologies for Lyme disease despite no known exposure to ticks and neither signs nor symptoms of the disease. The pacemaker was ultimately removed due to resolution of his heart block with antibiotic therapy. 

Samir Gautam

Pulmonary,
Critical Care and Sleep Medicine,
Pulmonary, Critical Care and Sleep Medicine

Yale University School of Medicine

Measurement of Chitinase Activity in Biological Samples

Samir Gautam

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Pulmonary,Critical Care and Sleep Medicine,Pulmonary, Critical Care and Sleep Medicine

Yale University School of Medicine

Measurement of Chitinase Activity in Biological Samples

Pulmonary,
Critical Care and Sleep Medicine,
Pulmonary, Critical Care and Sleep Medicine