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1

L-alanine:2-oxoglutarate aminotransferase isoenzymes from Arabidopsis thaliana leaves

100%

Wisniewski P. , Szklarczyk J. , Maciaga M. , Paszkowski A.

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tom 28

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nr 6

EN

The occurrence of four L-alanine:2-oxoglutarate aminotransferase (AOAT) isoenzymes (AOAT-like proyeins): alanine aminotransferase 1 and 2 (AlaATl and AlaAT2, EC 2.6.1.2) and L-glutamate:glyoxylate aminotransferase 1 and 2 (GGAT1 and GGAT2, EC 2.6.1.4) was demonstrated in Arabidopsis thaliana leaves. These enzymes differed in their substrate specificity, susceptibility to pyridoxal phosphate inhibitors and behaviour during molecular sieving on Zorbax SE-250 column. A difference was observed in the electrostatic charge values at pH 9.1 between GGAT1 and GGAT2 as well as between AlaAT1 and AlaAT2, despite high levels of amino acid sequence identity (93 % and 85 %, respectively). The unprecedented evidence for the monomeric structure of both AlaAT1 and AlaAT2 is presented. The molecular mass of each enzyme estimated by molecular sieving on Sephadex G-150 and Zorbax SE-250 columns and SDS/PAGE was approx-mately 60 kDa. The kinetic parameters: Km (Ala) = 1.53 mM, Km (2-oxoglutarate) = 0.18 mM, kcat = 124.6 s⁻¹, kcat/Km = 8.1 x 10⁴ M⁻¹-s⁻¹ of AlaAT1 were comparable to those determined for other AlaATs iso-ated from different sources. The two studied GGATs also consisted of a single subunit with molecular mass of 47.3-70 kDa. The estimated Km values for L-glutamate (1.2 mM) and glyoxylate (0.42 mM) in the transamination catalyzed by putative GGAT1 contributed to indentification of the enzyme. Based on these results we concluded that each of four AOAT genes in Arabidopsis thaliana leaves expresses different AOAT isoenzyme, functioning in a native state as a monomer.

2

Leptin is able to stimulate pancreatic enzyme secretion via activation of duodeno-pancreatic reflex and CCK release

76%

Nawrot-Porabka K. , Jaworek J. , Leja-Szpak A. , Palonek M. , Szklarczyk J. , Konturek S. J. , Pawlik W. W.

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nr 2

47-57

EN

Leptin, 16- kDa protein produced and secreted from white adipocytes is known to regulate food intake and energy expenditure. Leptin receptors have been detected in the pancreas and it has been shown that systemic application of this protein diminished postprandial pancreatic secretion. Leptin is also produced in the stomach and released into the gastrointestinal lumen but the implication of luminal leptin in the regulation of pancreatic enzyme secretion has not been elucidated. The aim of our study was to evaluate the effects of intraduodenal (i.d.) leptin administration on pancreatic enzyme secretion and to assess the involvent of afferent nerves and CCK in above effects. The secretory studies were carried out on anaesthetized Wistar rats with acute pancreatic fistulae. Leptin was administered to the animals at doses of 0.1 1.0 or 10.0 µg/kg i.d. Tarazepide (2.5 mg/kg i.d.), a CCK1 receptor antagonist, was given to the rats prior to the application of leptin. Rats with capsaicin deactivated sensory nerves were used in part of the study. Samples of pancreatic juice were taken at 15 min intervals to measure the volume flow and protein and amylase concentrations. CCK plasma level was measured by radioimmunoassay (RIA) following administration of leptin to the rats. Intraduodenal administration of leptin (1.0 or 10.0 µg/kg) to the fasted rats significantly and dose-dependently increased pancreatic protein and amylase outputs. Pancreatic secretory responses to leptin were totally abolished by prior capsaicin deactivation of sensory nerves or by pretreatment of the rats with tarazepide. Under basal conditions plasma CCK level averaged about 15.46 ± 1,4 pg/ml. Exogenous leptin, given i.d. at doses of 0.1 1.0 or 10.0 µg/kg i.d. to the rats with intact or capsaicin-deactivated sensory nerves resulted in dose-dependent rise of plasma CCK level, reaching the highest value at the dose of 10.0 µg/kg i.d. We conclude that leptin given i.d. stimulates pancreatic enzyme secretion and this effect could be related to the stimulation of CCK release and activation of duodeno-pancreatic reflexes.

3

Effect of neonatal endotoxemia on the pancreas of adult rats

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Jaworek J. , Leja-Szpak A. , Nawrot-Porabka K. , Bonior J. , Szklarczyk J. , Kot M. , Konturek S. J. , Tomaszewska R. , Pawlik W. W.

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nr 4

87-102

4

Endotoxemia in newborn rats attenuates acute pancreatitis at adult age

51%

Jaworek J. , Konturek S. J. , Macko M. , Kot M. , Szklarczyk J. , Leja-Szpak A. , Nawrot-Porabka K. , Stachura J. , Tomaszewska R. , Siwicki A. , Pawlik W. W.

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nr 1

EN

Bacterial endotoxin (lipopolysaccharide, LPS), at high concentration is responsible for sepsis, and neonatal mortality, however low concentration of LPS protected the pancreas against acute damage. The aim of this study was to investigate the effect of exposition of suckling rats to LPS on the course of acute pancreatitis at adult age. Suckling rat (30-40g) received intraperitoneal (i.p.) injection of saline (control) or LPS from Escherichia coli or Salmonella typhi (5, 10 or 15 mg/kg-day ) during 5 consecutive days. Two months later these rats have been subjected to i.p. caerulein infusion (25 µg/kg) to produce caerulein-induced pancreatitis (CIP). The following parameters were tested: pancreatic weight and morphology, plasma amylase and lipase activities, interleukin 1ß (IL-1 ß), interleukin 6 (IL-6), and interleukin 10 (IL-10) plasma concentrations. Pancreatic concentration of superoxide dysmutase (SOD) and lipid peroxidation products; malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) have been also measured. Caerulein infusion produced CIP in all animals tested, that was confirmed by histological examination. In the rats, which have been subjected in the neonatal period of life to LPS at doses 10 or 15 mg/kg-day x 5 days, all manifestations of CIP have been reduced. In these animals acute inflammatory infiltration of pancreatic tissue and pancreatic cell vacuolization have been significantly diminished. Also pancreatic weight, plasma lipase and a-amylase activities, as well as plasma concentrations of IL-1ß and IL-6 have been markedly decreased, whereas plasma anti-inflammatory IL-10 concentration was significantly increased in these animals as compared to the control rats, subjected in the infancy to saline injection instead of LPS. Caerulein-induced fall in pancreatic SOD concentration was reversed and accompanied by significant reduction of MDA + 4 HNE in the pancreatic tissue. The effects of LPS derived from E.coli or S.typhi were similar. Pretreatment of suckling rats with LPS at dose of 10 mg/kg-day x 5 days resulted in the most prominent attenuation of acute pancreatitis at adult age, whereas LPS at dose of 5 mg/kg-day x 5 days given to the neonatal rats failed to affect significantly acute pancreatitis induced in these animals 2 months later. We conclude that: 1/ Prolonged expositon of suckling rats to bacterial endotoxin attenuated acute pancreatitis induced in these animals at adult age. 2/ This effect could be related to the increased concentration of antioxidative enzyme SOD in the pancreatic tissue and to the modulation of cytokines production in these animals.