Relationship between carbachol hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen or NF-kappaB activation in rats in vitro / 华中科技大学学报（医学）（英德文版）

The relationship between M3 cholinergic receptor agonist (carbachol) hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen activation or NF-kappaB activation in rats was studied in vitro. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc), and NF-kappaB inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The cellular injury was evaluated by measuring the leakage of LDH from pancreatic acinar cells. The results showed that as compared with control group, 10(-3) mol/L carbachol induced a significant increase of the intracellular trypsin activity and the leakage of LDH from pancreatic acinar cells. Pretreatment with 2 mmol/L pefabloc could significantly decrease the activity of trypsin and the leakage of LDH from pancreatic acinar cells (P 0.05). It was concluded that intracellular trypsinogen activation is likely involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro. NF-kappaB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro.

Partially folded intermediates during trypsinogen denaturation

The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was delta GH2O = 6.99 + ou - 1.40 kcal/mol for guanidine hydrochloride and delta GH2O = 6.37 + ou - 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 + ou - 0.4 angstron to 26.0 + ou - 0.3 angstron 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 + ou - 0.3 angstron to 25.7 + ou - 0.6 angstron for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

Influência do regime alimentar na evoluçäo tardia da pancreatite aguda. Estudo experimental em ratos / Influence of diet composition on the evolution of acute pancreatitis. Experimental study in rats

Com a finalidade de estudar o efeito de regimes dietéticos diversos sobre a recuperaçäo das funçöes do pâncreas exócrino pós pancreatite aguda (PA), dois lotes de ratos submetidos, durante três semanas, a dietas isoprotéicas, diferindo apenas no teor de gordura (normo e hiperlipídica), foram distribuídos em grupos controles e com pancreatite aguda moderada, induzida com taurocolato de sódio a 1%. Amostras de sangue para dosagem de amilase sérica e fragmentos de tecido pancreático, para determinaçäo de enzimas triptolíticos, amilase, proenzimas e nucleotídeos foram colhidos nos grupos controles e nos com pancreatite após um, quatro, sete e 15 dias da induçäo da doença. Os resultados foram comparados estatisticamente por ANOVA, entre grupoos sob o mesmo regime alimentar e pelo teste "t" de Student, entre grupos correspondentes, sob regimes alimentares diversos (p <0.05). Os autores verificaram que, nas condiçöes experimentais utilizadas, a pancreatite agravou-se progressivamente até o quarto dia pós-PA, independentemente do regime alimentar prévio. A recuperaçäo funcional no órgäo manifestou-se a partir do sétimo dia pelo aumento de RNA/DNA, mas foi incompleta durante o período deste estudo. No décimo quinto dia pós-PA, ocorreu a normalizaçäo de parâmetros histopatológicos e bioquímicos, para os grupos com ambas as dietas. Os autores concluíram que, nas condiçöes experimentais deste estudo, o agravamento da doença, traduzido pela capacidade de síntese do pâncreas exócrino, foi progressivo até o quarto dia e independeu do teor de gordura na dieta

A plausible identification of secondary binding site in trypsin and trypsinogen

1. The determination of the binding of 4,4'diazoamino-bis-benzamidine (DABB) to alfa-trypsin by equilibrium measurements in columns indicated a stoichiometry of 2 mol ligan/mol enzyme. One molecule binds to the secondy binding site, sith Ki2=mMat pH8,0, 25-C. 2. Bovine pancreatic trypsin inhibitor (BPTI) prevented binding of DABB to both sites, indicating that they are topographically close and within the interface of the trypsin-BPTI complex. 3. On the basis of data from the interface of the trypsin-BPTI complex, we concluded that the secondary binding site of trypsin is plausibly identified as the same site in trypsin that binds the Arg-17 reside of BPTI, i.e., Tyr-39 and Tyr-151 in bovine trypsin. This site would then correspond to subsite S'2 on the enzyme surface