Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

Therapy Consortium EU-FP7. Composition and function of macroencapsulated human embryonic stem cell-derived implants: comparison with clinical human islet cell grafts.

High-level expression of human trypsinogens as inclusion bodies in Escherichia coli requires deletion of the secretory signal sequence and placement of an initiator methionine at the N terminus. Trypsinogen preparations obtained this way contain a mixture of abnormal N termini, as a result of processing by cytoplasmic aminopeptidases. Here, we describe an expression system that produces recombinant human cationic trypsinogen with a native, intact N terminus, using intein-mediated protein splicing and an aminopeptidase P (pepP) deficient E. coli strain. As a first application of this system, the effect of the pancreatitis-associated mutation A16V on the autoactivation of human cationic trypsinogen was characterized. The use of the novel pepP knock-out E. coli strain should be generally applicable to the expression of recombinant proteins, which undergo unwanted N-terminal trimming by aminopeptidase P.

Background: The archetypal mammalian elastase (ELA1) is not expressed in the human pancreas, because evolutionary mutations suppressed transcription of the ELA1 gene. Aims: In this study, we tested the theory that the unique duplication of the ELA2 gene in humans might compensate for the loss of ELA1. Methods: Recombinant ELA2A and ELA2B were expressed in Escherichia coli, and their activity was tested on Glt-Ala-Ala-Pro-Leu-p-nitroanilide, DQ elastin and bovine milk protein. Results: Surprisingly, recombinant ELA2B was completely devoid of proteolytic activity, while ELA2A readily hydrolyzed all three test substrates. Furthermore, ELA2A formed an SDS-resistant complex with α₁-antitrypsin, whereas ELA2B did not bind covalently to the inhibitor. Finally, chimeras and point mutations engineered between ELA2A and ELA2B revealed that multiple evolutionary mutations inactivated ELA2B. Conclusions: The results indicate that ELA2B is not an elastase enzyme and confirm that ELA2A is the major elastase in the human pancreas.

Mesotrypsin, an inhibitor‐resistant human trypsin isoform, does not activate or degrade pancreatic protease zymogens at a significant rate. These observations led to the proposal that mesotrypsin is a defective digestive protease on protein substrates. Surprisingly, the studies reported here with α1‐antitrypsin (α1AT) revealed that, even though mesotrypsin was completely resistant to this serpin‐type inhibitor, it selectively cleaved the Lys10–Thr11 peptide bond at the N‐terminus. Analyzing a library of α1AT mutants in which Thr11 was mutated to various amino acids, we found that mesotrypsin hydrolyzed lysyl peptide bonds containing Thr or Ser at the P1′ position with relatively high specificity (kcat/KM∼105 m−1·s−1). Compared with Thr or Ser, P1′ Gly or Met inhibited cleavage 13‐ and 25‐fold, respectively, whereas P1′ Asn, Asp, Ile, Phe or Tyr resulted in 100–200‐fold diminished rates of proteolysis, and Pro abolished cleavage completely. Consistent with the Ser/Thr P1′ preference, mesotrypsin cleaved the Arg358–Ser359 reactive‐site peptide bond of α1AT Pittsburgh and was rapidly inactivated by the serpin mechanism (ka∼106 m−1 s−1). Taken together, the results indicate that mesotrypsin is not a defective protease on polypeptide substrates in general, but exhibits a relatively high specificity for Lys/Arg–Ser/Thr peptide bonds. This restricted, thrombin‐like subsite specificity explains why mesotrypsin cannot activate pancreatic zymogens, but might activate certain proteinase‐activated receptors. The observations also identify α1AT Pittsburgh as an effective mesotrypsin inhibitor and the serpin mechanism as a viable stratagem to overcome the inhibitor‐resistance of mesotrypsin.

Repair and replicative DNA synthesis were measured at different stages of the cell cycle in control and cadmiumtreated Chinese hamster ovary (CHO-K1) cells. Cells were synchronized by counterflow centrifugal elutriation. Elutriation resulted in five repair and four replication subphases. On Cd treatment, repair synthesis was elevated in certain subphases. Replicative subphases were suppressed by Cd treatment, with some of the peaks almost invisible. The number of spontaneous strand breaks measured by random oligonucleotide primed synthesis assay showed a cell-cycle-dependent fluctuation in control cells and was greatly increased after Cd treatment throughout the S phase. Elevated levels of the oxidative DNA damage product, 8-oxodeoxyguanosine, were observed after Cd treatment, with the highest level in early S phase, which gradually declined as damaged cells progressed through the cell cycle.Keywords: 8-oxodeoxyguanosine; elutriation; permeable cells; strand breaks; synchronization. [6,7,10±12]. Heavy metals generate oxidizing radicals through Fenton chemistry and by the Haber±Weiss reaction leading to the hypothesis that metal carcinogenesis is mediated primarily by the elevated level of free radicals (reviewed by Kasprzak [13]). According to this view, heavy-metal-induced oxidative stress can lead to different types of DNA damage as a consequence of consumption of molecular oxygen in multiple steps of incomplete O 2 reduction, ultimately producing water.Common types of oxidative damage cause changes in DNA structure, the long-term effect of which can lead to malignant transformation. Structural changes involve alterations in nucleotide bases, cross-links, strand breaks, formation of bulky DNA adducts, etc. DNA damage suppresses DNA replication at checkpoints to avoid mutagenic changes being perpetuated in the genome of the next generation of cells [14±16]. A comparison of cell cycle profiles of replicative repair synthesis in permeable cells showed opposite trends. The rates of repair synthesis and replication are inversely correlated [17].The detection of oxidative DNA damage leading to degenerative diseases associated with aging involves chromatographic immunochemical approaches, measurement of oxidized bases such as 8-hydroxyguanine, 8-hydroxyadenine, and thymine glycol as well as oxidized DNA adducts such as 8-hydroxy-2 H -deoxyguanosine and thymidine glycol. The aim of this paper is to describe how oxidative damage of Cd treatment reduces replicative, and increases repair DNA synthesis during cell cycle. Changes have been expressed as the ratio of replication/repair synthesis. Generation of DNA strand breaks and the carcinogen indicator 8-deoxyguanosine were also measured in a cell-cycle-dependent manner. M A T E R I A L S A N D M E T H O D S Growth conditionsChinese hamster ovary cells (CHO-K1, ATCC No. CCL61) were kept in suspension culture in spinner flasks using F12 medium supplemented with 10% fetal bovine serum at 37 8C and 5% CO 2 . Cadmium treatmentCHO cells were treated with CdCl 2 (0.2±1 mm) 9...

Exponentially growing Drosophila S2 cells in suspension culture were synchronized at low- and high-resolution centrifugal elutriation, and DNA synthesis was measured by [(3)H]-thymidine incorporation throughout the S phase. At low resolution, one repair peak at the G(1)/G(0) border and two replication peaks known as early and late S subphases were observed. At high resolution, six chronologic compartments were distinguished. The distribution of these peaks indicated one repair peak at 2.05 C value, one minor replication peak at 2.43C, and four major subphases of replication corresponding to 2.64C, 2.89C, 3.32C, and 3.60C, representing 6.7%, 3.4%, 15.3%, 20.4%, 32.1%, and 22.0% of the synthetic activity, respectively. The five major peaks of cell growth with 2.32C, 2.56C, 2.85C, 3.18C, and 3.58C values consistently preceded those of replication subphases.

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