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Tumors and cancers of the gastrointestinal tract and pancreas are commonly derived from precursor lesions so that understanding the physiological, cellular, and molecular mechanisms underlying the pathogenesis of precursor lesions is critical for the prevention and treatment of those neoplasms. Pancreatic neuroendocrine tumors (PNETs) can also be derived from precursor lesions. Pancreatic α cell hyperplasia (ACH), a specific and overwhelming increase in the number of α cells, is a precursor lesion leading to PNET pathogenesis. One of the 3 subtypes of ACH, reactive ACH is caused by glucagon signaling disruption and invariably evolves into PNETs. In this article, the existing work on the mechanisms underlying reactive ACH pathogenesis is reviewed. It is clear that the liver secretes a humoral factor regulating α cell numbers but the identity of the liver factor remains elusive. Potential approaches to identify the liver factor are discussed.

Aim: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells., Methods: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition. After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells. The bacmids DNA was verified to replicate in insect cells and packaged into baculovirus particles via PCR and electronic microscopic analysis. The insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-lauryl sarcosine sodium (NLS) and purified by metal-chelated-affinity chromatography, then the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbant assay and its enzymatic activity was detected., Results: The HCV NS3 protease domain was expressed in insect cells at high level and it was partially solved in NLS. Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5 x 10(7) cells, and both antigenicity and specificity of the protein were evaluated to be high when used as antigen to detect hepatitis C patients' sera in indirect ELISA format. In vitro cleavage assay corroborated its enzymatic activity., Conclusion: The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.

Aim: To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine protease based on the recombinant protease and substrate, and to evaluate its feasibility in screening the enzyme inhibitors., Methods: Based on the crystallographic structure of hepatitis C virus (HCV) serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with three primers and then cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing. The single-chain recombinant protease was expressed when the E.coli was induced with IPTG and the expression conditions were optimized to produce large amount of soluble protease. The recombinant substrate NS5ab that covers the cleavage point NS5A/B was also expressed in E.coli. Both of the protease and substrate were purified by using Ni-NTA agarose metal affinity resin, then they were mixed together in a specific buffer, and the mixture was analyzed by SDS-PAGE. The cleavage system was used to evaluate some compounds for their inhibitory activity on serine protease., Results: The single-chain recombinant protease was over-expressed as soluble protein when the E.coli was induced at a low dosage of IPTG (0.2 mM) and cultured at a low temperature (15 degrees ). The protease was purified by using Ni-NTA agarose metal affinity resin (the purity is over 95 %). The recombinant substrate NS5ab was expressed in an insoluble form and could refold successfully after purification and dialysis. A simple and convenient assay in vitro was established, in which the purified single-chain serine protease could cleave the recombinant substrate NS5ab into two fragments that were visualized by SDS-PAGE. PMSF had an effect on inhibiting activity of serine protease, while EDTA had not., Conclusion: A simple and convenient assay in vitro for hepatitis C virus NS3 serine protease is based on recombinant substrate NS5ab and single-chain serine protease. This assay can be used in screening of enzyme inhibitors.