CCK-JMV-180, an analog of cholecystokinin, releases intracellular calcium from an inositol trisphosphate-independent pool in rat pancreatic acini.

In pancreatic acinar cells cholecystokinin and its analogs, caerulein and CCK-JMV-180, stimulate an increase in intracellular free [Ca2+] by releasing Ca2+ from non-mitochondrial intracellular pools. It is generally believed that the caerulein-induced release of Ca2+ is mediated by phospholipase C-catalyzed production of 1,4,5-inositol triphosphate (IP3). In this study we have investigated the source and mechanism of Ca2+ release induced by CCK-JMV-180 using streptolysin O-permeabilized pancreatic acinar cells. Caerulein-stimulated release of Ca2+ was completely blocked by either neomycin, an inhibitor of phospholipase C, or by heparin, an IP3 receptor antagonist. These observations are compatible with the conclusion that caerulein releases Ca2+ from an IP3-sensitive pool. In contrast to caerulein, however, CCK-JMV-180-stimulated release of Ca2+ was not blocked by either neomycin or by heparin, indicating that CCK-JMV-180 releases Ca2+ by mechanisms which do not involve the generation or action of IP3. CCK-JMV-180 stimulated the release of Ca2+ even after the IP3-sensitive pool had been completely emptied by prior exposure to a supramaximally stimulating concentration of IP3 (40 microM). Prestimulation of permeabilized acini with 20 mM caffeine did not abolish the CCK-JMV-180-induced Ca2+ release. These results indicate that CCK-JMV-180 stimulates release of Ca2+ from a hitherto uncharacterized intracellular storage pool which is insensitive to either IP3 or caffeine.     

Biotransformation of lantadene A (22β-angeloyloxy-3-oxoolean-12-en-28-oic acid), the pentacyclic triterpenoid, by Alcaligenes faecalis

A bacterial strain capable of biotransformation oflantadene A(22-angeloyloxy-3-oxo-olean-12-en-28-oic acid),the pentacyclic hepatotoxin of lantana (Lantanacamara var. aculeata) has been isolated fromsoil using lantadene A as the sole carbon source. Theorganism is Gram negative, rod shaped, motile,catalase positive and has been identified as Alcaligenes faecalis. The isolate has been found tobe specific for lantadene A and did not utilizelantadene B. In studies using sucrose as an additionalcarbon source, A. faecalis elicitedbiotransformation of lantadene A to its trans isomer22-tigloyloxy-3-oxoolean-12-en-28-oic acid,designated as lantadene X and two other minormetabolites which could not be isolated in pure state.     

Development of a new mouse model of acute pancreatitis induced by administration of L-arginine

The pathogenesis of acute pancreatitis is not fully understood. Experimental animal models that mimic human disease are essential to better understand the pathophysiology of the disease and to evaluate potential therapeutic agents. Given that the mouse genome is known completely and that a large number of strains with various genetic deletions are available, it is advantageous to have multiple reliable mouse models of acute pancreatitis. Presently, there is only one predominant model of acute pancreatitis in mice, in which hyperstimulatory doses of cholecystokinin or its analog caerulein are administered. Therefore, the aim of this study was to develop another mouse model of acute pancreatitis. In this study, C57BL/6 mice were injected intraperitoneally with L-arginine in two doses of 4 g/kg each, 1 h apart. Serum amylase, myeloperoxidase, and histopathology were examined at varying time points after injection to assess injury to the pancreas and lung. We found that injection of L-arginine was followed by significant increases in plasma amylase and pancreatic myeloperoxidase accompanied by marked histopathological changes. The injury to the pancreas was slow to develop and peaked at 72 h. Subsequent to peak injury, the damaged areas contained collagen fibers as assessed by increased Sirius red staining. In contrast, D-arginine or other amino acids did not cause injury to the pancreas. In addition, acute inflammation in the pancreas was associated with lung injury. Our results indicate that administration of L-arginine to mice results in severe acute pancreatitis. This model should help in elucidating the pathophysiology of pancreatitis.     

Molecular structure, polymorphism, and toxicity of lantadene A, the pentacyclic triterpenoid from the hepatotoxic plant Lantana camara

Lantadene A (22 beta-angeloyloxy-3-oxo-olean-12-en-28-oic acid), a pentacyclic triterpenoid compound from lantana (Lantana camara) leaves has been obtained in two polymorphic forms I and II. Form I had white, fluffy, and rod-shaped uniform crystals. Form II particles were irregular, shining, and polyhedral. The two forms differed in melting behavior. The powder x-ray diffraction of form I showed sharp peaks whereas from II did not contain distinct peaks. From single-crystal three-dimensional x-ray structure determination, the molecular structure of form I has been established. A/B and B/C rings of the molecule are trans fused while D/E rings are cis fused. The packing of the molecule is stabilized by hydrogen bonding. Form I of lantadene A was non-toxic to guinea pigs on oral administration. Form II induced ictericity and toxicity associated with decrease in feed intake and fecal output, hepatomegaly, increase in plasma bilirubin, and acid phosphatase activity.     

Protein precipitation assay for quantitation of tannins: determination of protein in tannin-protein complex

A protein precipitation method for the determination of tannins has been developed. The protein in the tannin-protein complexes was measured using the ninhydrin assay of amino acids released by alkaline hydrolysis of the complex. Standard protein and the complex were hydrolyzed with 13.5 N NaOH at 120 degrees C for 20 min and the amino acids released were measured with ninhydrin. Tannins did not interfere in the determination of protein by ninhydrin assay. The bovine serum albumin (BSA) precipitated (y; mg) increased linearly with increase in tannic acid (x) from 0.2 to 0.9 mg (y = 2.598x - 0.258). The protein precipitation capacities (mg BSA precipitated/g dry wt) measured by the method for young and mature leaves of oaks were Quercus incana (young, 42.21; mature, 79.51), Q. ilex (young, 1.86; mature, 1.86), and Q. semecarpifolia (young, 733.54; mature, 304.32). The method can provide valuable information on the mechanisms of protein-tannin interactions and nutritional and physiological significances of tannins.     

Hepatotoxicity and cholestasis in rats induced by the sesquiterpene, 9-oxo-10,11-dehydroageraphorone, isolated from Eupatorium adenophorum

Eupatorium adenophorum leaves cause hepatotoxicity and cholestasis in rats. The hepatotoxicant has been characterized as 9-oxo-10,11-dehydroageraphorone (ODA), a cadinene sesquiterpene. Oral administration of ODA, mixed in feed to rats, caused jaundice in 24 h. The liver of the intoxicated animals had focal areas of hepatocellular necrosis, proliferation, and dilation of bile ducts with degenerative changes in the lining epithelium. There was marked increase in the conjugated form of plasma bilirubin and in the activities of the enzymes glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, alkaline phosphatase, lactate dehydrogenase, gamma-glutamyltranspeptidase, glutamate dehydrogenase, and 5'-nucleotidase. The histopathological lesions in liver and biochemical profile of marker enzymes show that ODA induced hepatotoxicity and cholestasis in rats. This is the first report on the toxicity of a cadinene sesquiterpene in rats.     

Protein-binding capacity of microquantities of tannins

The physiological effect of tannins is studied in terms of their protein-binding or precipitation capacity. A number of assays based on binding of hemoglobin or bovine serum albumin (BSA) and subsequent determination of unbound protein in supernatant or tannin in a protein-tannin complex are available but with various limitations. These methods are unable to estimate protein-binding capacity, if the quantity of tannin available is low. In the method reported here, tannins or other phenolics were applied on chromatography paper and reacted with BSA and unbound BSA was washed off. The protein in the tannin-protein complex was measured spectrophotometrically after staining with Ponceau S. It required microquantities of sample. Using this method the protein-binding capacity of total leaf extract and hydrolyzable and condensed tannins of Quercus incana, Q. semecarpofolia, and Q. dilatata was determined. The protein binding capacities of ellagic acid and quercetin (microgram BSA/mg) were 297.3 and 78.0, respectively.     

Isolation,purification and properties of tannase from Aspergillus niger van Tieghem

Tannase (tannin acyl hydrolase E.C. 3.1.1.20) has been isolated from Aspergillus niger van Tieghem and purified 29-fold. The enzyme had a temperature optimum of 60^°C, pH optimum of 6.0 with a second peak at pH 4.5, K_m of 0.20mM and V_max of 5.0mol min^–1 mg^–1protein.