Helicobacter pylori (Hp) IgG ELISA of the New-Generation GastroPanel® Is Highly Accurate in Diagnosis of Hp-Infection in Gastroscopy Referral Patients.

BACKGROUND/AIM: Helicobacter pylori (Hp) infection affects a substantial proportion of the world population and is a major risk factor of gastric cancer (GC). The caveats of common Hp-tests can be evaded by a serological biomarker test (GastroPanel®, Biohit Oyj, Helsinki), the most comprehensive Hp-test on the market. The clinical validation of Helicobacter pylori IgG ELISA of the new-generation GastroPanel® test is reported. The aim of the study is to validate the clinical performance of the Helicobacter pylori IgG ELISA test in diagnosis of biopsy-confirmed Hp-infection in gastroscopy referral patients. PATIENTS AND METHODS: A cohort of 101 patients (mean age=50.1 years) referred for gastroscopy at the outpatient Department of Gastroenterology (SM Clinic, St. Petersburg) were examined by two test versions to validate the new-generation GastroPanel®. All patients were examined by gastroscopy and biopsies, which were stained with Giemsa for specific identification of Hp in the antrum (A) and corpus (C). RESULTS: Biopsy-confirmed Hp-infection was found in 64% of patients, most often confined to antrum. The overall agreement between Hp IgG ELISA and gastric biopsies in Hp-detection was 91% (95%CI=84.1-95.8%). Hp IgG ELISA diagnosed biopsy-confirmed Hp (A&C) with sensitivity (SE) of 92.3%, specificity (SP) of 88.6%, positive predictive value (PPV) of 93.8% and negative predictive value (NPV) of 86.1%, with AUC=0.904 (95%CI=0.842-0.967). In ROC analysis for Hp detection (A&C), Hp IgG ELISA shows AUC=0.978 (95%CI=0.956-1.000). CONCLUSION: The Hp IgG ELISA test successfully concludes the clinical validation process of the new-generation GastroPanel® test, which retains the unrivalled diagnostic performance of all its four biomarkers, extensively documented for the first-generation test in different clinical settings.

Purification and partial characterization of feline pepsinogen.

OBJECTIVE: To purify and partially characterize feline pepsinogen (fPG) from the gastric mucosa and compare fPG with PGs of other species. SAMPLE POPULATION: Stomachs of 6 cats. PROCEDURE: A crude protein extract was prepared from the gastric mucosa of feline stomachs. Feline PG A was purified by ammonium sulfate precipitation, weak-anion-exchange chromatography, size-exclusion chromatography, and strong-anion exchange chromatography. Partial characterization consisted of estimation of molecular weights (MWs) and isoelectric points, N-terminal amino acid sequencing, and investigation of susceptibility to pepstatin inhibition. RESULTS: Several fPG A-group isoforms were identified. The MWs of the isoforms ranged from 37,000 to 44,820. Isoelectric points were all < pH 3.0. The proteolytic activity of the activated PGs was inhibited completely by pepstatin in a range of equimolar to 10-fold molar excess. The specific absorbance of fPG A was 1.29. The N-terminal amino acid sequence of the first 25 residues of the predominant fPG A7 had 75%, 72%, 64%, and 56% homology with PG A of dogs, rabbits, cattle, and humans, respectively. Sequences of 4 other fPG A-group isoforms were similar to fPG A7. All isoforms were immunologically cross-reactive with sheep anti-fPG A7 antiserum. CONCLUSIONS AND CLINICAL RELEVANCE: PG A is the only identified type of PG in cats and, similar to pg in other species, comprises multiple isoforms. The availability of fPG A may be used to facilitate the development of an immunoassay to quantify serum fPG A as a potential marker for gastric disorders in cats.

N-terminal modifications increase the neutral-pH stability of pepsin.

A structure-function study was undertaken to determine the effects of N-terminal mutations in pepsin designed to introduce the Lys-X-Tyr motif and increase N-terminal flexibility. At pH 7.0, E7K/T12A/E13Q pepsin was inactivated more slowly compared to WT, whereas the mutants E7K and T12A/E13Q were not stabilized. Far-UV circular dichroism revealed that changes in secondary structure accompanied the inactivation process, and that the structural changes occurred at approximately the same rate as inactivation. All of the inactivated pepsin forms showed retention of substantial secondary structure, more than previously determined for pepsin denatured at pH 7.2 and 8.0, suggesting the presence of a structural intermediate at pH 7.0. The coupled mutations at positions 12 and 13 impacted the pH dependence of activity at pH 0.9, lowered affinity for a synthetic substrate, and lowered the turnover number. The introduction of Lys at position 7 apparently destabilized the interaction between prosegment-enzyme body as evidenced by activation at higher pH (>or= 4.0) compared to WT, but showed no change for pH dependence of activity, nor a statistically significant change in affinity for the synthetic substrate.

Purification and partial characterization of canine pepsinogen A and B.

OBJECTIVE: To purify and partially characterize various isoforms of canine pepsinogen (PG) from gastric mucosa. SAMPLE POPULATION: Stomachs obtained from 6 euthanatized dogs. PROCEDURE: Mucosa was scraped from canine stomachs, and a crude mucosal extract was prepared and further purified by use of weak anion-exchange chromatography, hydroxyapatite chromatography, size-exclusion chromatography, and strong anion-exchange chromatography. Pepsinogens were characterized by estimation of molecular weights, estimation of their isoelectric points (IEPs), and N-terminal amino acid sequencing. RESULTS: Two different groups of canine PG were identified after the final strong anion-exchange chromatography: PG A and PG B. Pepsinogens differed in their molecular weights and IER Pepsinogen B appeared to be a dimer with a molecular weight of approximately 34,100 and an IEP of 4.9. Pepsinogen A separated into several isoforms. Molecular weights for the various isoforms of PG A ranged from 34,200 to 42,100, and their IEPs ranged from 4.0 to < 3.0. The N-terminal amino acid sequence for the first 25 amino acid residues for PG A and B had good homology with the amino acid sequences for these proteins in other species. CONCLUSIONS AND CLINICAL RELEVANCE: Canine PG B and several isoforms of canine PG A have been purified. Availability of these PGs will facilitate development of immunoassays to measure PG in canine serum as a potential diagnostic marker for gastric disorders in dogs.

Soluble expression and purification of porcine pepsinogen from Pichia pastoris.

This paper presents a new system for the soluble expression and characterization of porcine pepsinogen from the methylotrophic yeast Pichia pastoris. The cDNA that encodes the zymogenic form of porcine pepsin (EC 3.4.23.1) was cloned into the EcoRI site of the vector pHIL-S1 downstream from the AOX1 alcohol oxidase promoter. After P. pastoris transformation, colonies were screened for expression of pepsinogen based on enzyme activity of the active form, pepsin. The recombinant enzyme was purified 138-fold by anion exchange and affinity column chromatography. Homogeneity was confirmed through SDS-PAGE, Western blot, and N-terminal sequencing. When compared to commercial pepsin, the recombinant pepsin had similar kinetic profiles, pH/temperature stability, and secondary/tertiary conformation. A glycosylated form was also isolated and found to exhibit kinetic and structural characteristics similar to those of the commercial and wild-type pepsin, but was slightly more thermal stable. The above results indicate that the P. pastoris expression system offers a convenient and efficient means to produce and purify a soluble form of pepsin(ogen).

Aromatic amino acids and their derivatives as ligands for the isolation of aspartic proteinases.

Affinity chromatography was used to study an interaction of aspartic proteinases with immobilized aromatic amino acids and their derivatives. The following ligands were used: L-tyrosine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, L-phenylalanine, p-iodo-L-phenylalanine and N-acetyl-L-phenylalanine. With the exception of the last one, ligands were coupled directly to divinyl sulfone activated Sepharose 4B. For the preparation of immobilized N-acetyl-L-phenylalanine, divinyl sulfone activated Sepharose 4-B with linked ethylene diamine was used. Porcine pepsin was used for the evaluation of the capacity of the prepared affinity carriers. The capacity of the immobilized amino acid derivatives significantly increased in comparison with the non-derivatized amino acids. The prepared immobilized ligands were further used for the separation of human pepsinogens.

Reflux of gastric juice and glue ear in children.

Otitis media with effusion (glue ear) is the most frequent cause of deafness in children. We investigated the role of gastric juice reflux in this disease. We measured pepsin concentrations in middle ear effusions from children using ELISA and enzyme activity assays. 45 (83%) of 54 effusions contained pepsin/pepsinogen at concentrations of up to 1000-fold greater than those in serum. Our data suggest that reflux of gastric juice could be a major cause of glue ear in children.

Amphibian pepsinogens: purification and characterization of xenopus pepsinogens, and molecular cloning of Xenopus and bullfrog pepsinogens.

Two pepsinogens (Pg C and Pg A) were isolated from the stomach of adult Xenopus laevis by Q-Sepharose, Sephadex G-75, and Mono-Q column chromatographies. Autolytic conversion and activation of the purified Pgs into the pepsins were examined by acid treatment. We determined the amino acid sequences from the NH2-termini of Pg C, pepsin C, Pg A, and pepsin A. Based on the sequences, the cDNAs for Pg C and Pg A were cloned from adult stomach RNA, and the complete amino acid sequences of the Pg C and Pg A were predicted. In addition, a Pg A cDNA was cloned from the stomach of adult bullfrog Rana catesbeiana, and the primary structure of the Pg A was predicted. Molecular phylogenetic analysis showed that such anuran Pg C and Pg A belong to the Pg C group and the Pg A group in vertebrates, respectively. The molecular properties of Pg C and Pg A, such as size, sequences of the activation peptide and active site, profile of autolytic activation, and pH dependency of proteolytic activity of the activated forms, pepsin C and pepsin A, resemble those of Pgs found in other vertebrates. However, the hemoglobin-hydrolyzing activity of Xenopus pepsin C is completely inhibited in the presence of equimolar pepstatin, an inhibitor of aspartic proteinases. Thus, the Xenopus pepsin C differs significantly from other vertebrate pepsins C in its high susceptibility to pepstatin, and closely resembles A-type pepsins.

Purification and characterization of goat pepsinogens and pepsins.

Three type-A and two type-C pepsinogens, namely, pepsinogens A-1, A-2, A-3, C-1, and C-2, were purified from adult goat abomasum. Their relative levels in abomasal mucosa were 27, 19, 14, 25, and 15%, respectively. Amino acid compositions were quite similar between isozymogens of respective types, but different between the two types especially in the Glx/Asx and Leu/Ile ratios. NH2-terminal amino acid sequences of pepsinogens A-3 and C-2 were SFFKIPLVKKKSLRQNLIEN- and LVKIPLKKFKSIRETM-, respectively. Pepsins A and C showed maximal hemoglobin-digestive activity at around pH 2 and 3, respectively, and specific activities of pepsins C were higher than those of pepsins A. Two subtypes of pepsin A were obvious, namely pepsin A-2/3 which maintains its activity in the weakly acidic pH region over pH 3 and pepsin A-1, which does not. Hydrolysis of oxidized insulin B chain by goat pepsins A occurred primarily at Ala14-Leu15 and Leu15-Tyr16 bonds.

Purification and characterization of the main pepsinogen from the shark, Centroscymnus coelolepis.

The main pepsinogen from the mucosa of the shark, Centroscymnus coelolepis, has been purified and characterized. This zymogen, the most abundant protein in terms of quantity and activity (yield 72%), is a homogeneous monomer of molecular weight 42+/-0.7 kDa, as determined by electrophoresis. The aspartyl proteinase nature of this enzyme was confirmed by the considerable inhibition by pepstatin. Its specificity as to the oxidized B-chain of bovine insulin was determined using electrospray ionization mass spectrometry (ESI-MS) coupled with reversed phase high pressure liquid chromatography (RP-HPLC). The 15-16 Leu-Tyr bond was rapidly cleaved in this substrate, followed by the 24-25 Phe-Phe, 25-26 Phe-Tyr, and 11-12 Leu-Val bonds.