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Stomach loss has occurred independently multiple times during gnathostome evolution with notable frequency within the Teleostei. Significantly, this loss of acid-peptic digestion has been found to correlate with the secondary genomic loss of the gastric proton pump subunits (atp4a, atp4b) and pepsinogens/pepsins (pga, pgc). Gastric glands produce gastric juice containing the acid and pepsin and thus their presence is a hallmark feature of a digestive system capable of acid-peptic digestion. However, in gobiid fishes although oesogaster and gastric glands have been identified histologically, their functional significance has been questioned. In the present study we address whether the gastric proton pump is present and expressed in gastric glands of the goby Neogobius species (Gobiidae) and in members of the family Oxudercidae, a group of amphibious gobiid fishes commonly known as mudskippers (genera: Periophthalmus, Boleophthalmus, Periophthalmodon and Scartelaos). We confirmed the presence of gastric glands and have immunohistochemically localized gastric proton pump expression to these glands in Neogobius fluviatilis and Periophthalmus novemradiatus, Periophthalmus barbarus and Boleophthalmus boddarti. Genome analysis in Neogobius melanostomus, Periophthalmus magnuspinnatus, Scartelaos histophorus, Boleophthalmus pectinirostris, and Periophthalmodon schlosseri revealed the presence of both atp4a and atp4b subunit orthologues in all species in a conserved genomic loci organization. Moreover, it was possible to deduce that the complete open reading frame and the key functional amino acid residues are present. The conserved expression of the gastric proton pump provides clear evidence of the potential for gastric acid secretion indicating that acid digestion is retained in these gobiid fishes and not lost.

One major pepsinogen, PG-I, and two minor pepsinogens, PG-II and PG-III were purified from lizardfish stomach by ammonium sulfate precipitation and two chromatographic columns. The three purified PGs migrated as single bands in native-PAGE gels with molecular weights (MW) ranging from 36 to 38 kDa. Each PG was converted to pepsin (P) at pH 2.0, and the MW were determined as 32 kDa (for P-I), 31 kDa (for P-II) and 30 kDa (for P-III). The optimum pH and temperature of pepsins were 2.0–3.5, and 40–50 °C. All 3 pepsins were strongly inhibited by pepstatin A. Divalent cations slightly stimulated the pepsin activities, but ATP had no effect on the pepsins. Purified pepsins were effective in the hydrolysis of various proteins. Km and kcat of the three pepsins for hemoglobin hydrolysis were 107.64–276.61 µM and 18.30–32.68 s−1, respectively. The new pepsins have potential for use in protein food procession and modification.

The study investigated the protective effect of walnut oligopeptides (WOPs) against ethanol-induced gastric injury using Sprague-Dawley (SD) rats. Rats were randomly divided into seven groups based on body weight (10/group), normal group, ethanol group, whey protein group (220 mg/kg body weight), omeprazole group (20 mg/kg body weight), and three WOPs groups (220, 440, 880 mg/kg body weight). After 30 days of treatment with WOPs, rats were given 5 mL/kg absolute ethanol by gavage to induce gastric mucosal injury. Gastric ulcer index (GUI) were determined and the following measured; gastric content pH, gastric mucin, endogenous pepsinogens (PG), prostaglandin E2 (PGE2), inflammatory cytokines, oxidative stress indicators, and the expression of apoptosis-related proteins were measured to evaluate the gastroprotective effect of WOPs. The results showed that the administration with WOPs markedly mitigated the hemorrhagic gastric lesions caused by ethanol in rats, and decreased the GUI, the gastric content pH, PG1, PG2, and NO levels, enhanced mucin and PGE2. Also, WOPs repressed gastric inflammation through the reduction of TNF-α, IL-6, IL-1β and increase IL-10 levels, and revealed antioxidant properties with the enhancement of superoxide dismutase, glutathione, and catalase activity, while reduction of malondialdehyde. Moreover, WOPs treatment significantly down-regulated Bax, caspase-3 and nuclear factor-κB p65 (NF-κB p65) expression, while up-regulating the expression of Bcl-2 and inhibitor kappa Bα (IκBα) protein. These results indicated that WOPs have protective effects against ethanol-induced gastric mucosal injury in rats through anti-inflammatory, anti-oxidation, and anti-apoptosis mechanisms.

The nucleotide sequences of largemouth bass pepsinogens (PG1, 2 and 3) were determined after molecular cloning of the respective cDNAs. Encoded PG1, 2 and 3 were classified as fish pepsinogens A1, A2 and C, respectively. Molecular evolutionary analyses show that vertebrate pepsinogens are classified into seven monophyletic groups, i.e. pepsinogens A, F, Y (prochymosins), C, B, and fish pepsinogens A and C. Regarding the primary structures, extensive deletion was obvious in S'1 loop residues in fish pepsin A as well as tetrapod pepsin Y. This deletion resulted in a decrease in hydrophobic residues in the S'1 site. Hydrolytic specificities of bass pepsins A1 and A2 were investigated with a pepsin substrate and its variants. Bass pepsins preferred both hydrophobic/aromatic residues and charged residues at the P'1 sites of substrates, showing the dual character of S'1 sites. Thermodynamic analyses of bass pepsin A2 showed that its activation Gibbs energy change (∆G(‡)) was lower than that of porcine pepsin A. Several sites of bass pepsin A2 moiety were found to be under positive selection, and most of them are located on the surface of the molecule, where they are involved in conformational flexibility. The broad S'1 specificity and flexible structure of bass pepsin A2 are thought to cause its high proteolytic activity.

Six pepsinogens were purified from the gastric mucosa of largemouth bass (Micropterus salmoides) by DEAE-Sephacel chromatography, Sephadex G-100 gel filtration, and Mono Q FPLC. The potential specific activities of two major pepsinogens, PG1-1 and PG2-2, against hemoglobin were 51 and 118 units/mg protein, respectively. The activity of pepsin 2-2 was the highest among the pepsins reported to date; this might be linked to the strongly carnivorous diet of the largemouth bass. The molecular masses of PG1-1 and PG2-2 were 39.0 and 41.0 kDa, respectively. The N-terminal amino acid sequences of PG1-1 and PG2-2 were LVQVPLEVGQTAREYLE- and LVRLPLIVGKTARQALLE-, respectively, showing similarities with those of fish type-A pepsinogens. The optimal pHs for hemoglobin-digestive activity of pepsins 1-1 and 2-2 were around 1.5 and 2.0, respectively, though both pepsins retained considerable activity at pHs over 3.5. They showed maximal activity around 50 and 40 °C, respectively. They were inhibited by pepstatin similarly to porcine pepsin A. The cleavage specificities clarified with oxidized insulin B chain were shown to be restricted to a few bonds consisting of hydrophobic/aromatic residues, such as the Leu(15)-Tyr(16), Phe(24)-Phe(25) and Phe(25)-Tyr(26) bonds. When hemoglobin was used as a substrate, the kcat/Km value of bass pepsin 2-2 was 4.6- to 36.8-fold larger than those of other fish pepsins. In the case of substance P, an ideal pepsin substrate mimic, the kcat/Km values were about 200-fold larger than those of porcine pepsin A, supporting the high activity of the bass pepsin.

Chitinases are enzymes that hydrolyze chitin, a polymer of β-1, 4-linked N-acetyl-D-glucosamine (GlcNAc). Chitin has long been considered as a source of dietary fiber that is not digested in the mammalian digestive system. Here, we provide evidence that acidic mammalian chitinase (AMCase) can function as a major digestive enzyme that constitutively degrades chitin substrates and produces (GlcNAc)2 fragments in the mouse gastrointestinal environment. AMCase was resistant to endogenous pepsin C digestion and remained active in the mouse stomach extract at pH 2.0. The AMCase mRNA levels were much higher than those of four major gastric proteins and two housekeeping genes and comparable to the level of pepsinogen C in the mouse stomach tissues. Furthermore, AMCase was expressed in the gastric pepsinogen-synthesizing chief cells. The enzyme was also stable and active in the presence of trypsin and chymotrypsin at pH 7.6, where pepsin C was completely degraded. Mouse AMCase degraded polymeric colloidal and crystalline chitin substrates in the gastrointestinal environments in presence of the proteolytic enzymes. Thus, AMCase can function as a protease-resistant major glycosidase under the conditions of stomach and intestine and degrade chitin substrates to produce (GlcNAc)2, a source of carbon, nitrogen and energy.

Development of resistance to anthelmintic drugs has motivated the search for diagnostic methods to identify animals for targeted selective treatments. We compared three methods for the diagnosis of nematode infection in relation to milk production in a fully grazing dairy herd of 150 cows in the humid Pampa (Argentina). Animals had feces, blood and milk sampled during the first postpartum month for EPG, pepsinogen and anti-Ostertagia antibody determination, respectively. With the results obtained two groups of cows, divided in high and low parasite burden, were conformed for each method, and milk production was then compared between groups. When cows were separated by the EPG method (EPG=0 (N=106) vs. EPG0 (N=44)) a difference of nearly 800 l of milk per cow per lactation was found (P0.05). On the other hand, milk production between groups separated by Pepsinogen (mUtyr ≤ 1000 vs. mUtyr1000) or by anti-Ostertagia (ODR ≤ 0.5 vs. ODR0.5) results did not differ. Interestingly, proportion of cows in each group differed between methods (P0.0001), and the anti-Ostertagia method yielded significantly more cows in the high index group compared to results using the EPG or Pepsinogen method. No correlations were found between parasite indexes determined by the different methods. High parasite burden estimation found may be ascribed to the production system, fully grazing all year round, and to the sampling time, at the beginning of lactation with cows in negative energy balance and depressed immunity. The fact that the cows were born and reared outside, on pasture with continuous nematode larvae exposure, may also account for the results obtained. In conclusion, EPG counting during the first postpartum month may be a useful tool for the diagnosis of production impairment induced by high nematode burden in adult grazing dairy cows. The anthelmintic treatment of only the EPG-positive recently calved cows would improve milk production, while reducing selective pressure on nematode population for the development of resistance.

Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37-38 kDa and 31-34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians.

To summarize the literature over the past year on the regulation of gastric exocrine and endocrine secretion.Gastric acid secretion by parietal cells is precisely regulated by overlapping neural, hormonal, and paracrine pathways, both centrally and peripherally. Too much acid can induce gastroduodenal injury. Too little acid can interfere with the absorption of iron, calcium, vitamin B12, and certain drugs as well as predispose the patient to enteric infection. A number of peptides implicated in the central control of food intake such as ghrelin, orexin, and leptin are present in the stomach and are capable of modulating acid secretion. The precise mechanisms whereby Helicobacter pylori produces perturbations in acid secretion are not precisely known but appear to involve changes in somatostatin and perhaps ghrelin secretion. Both gastrin and gastrin-receptor knockout mice as well as gastrin-overexpressing and cAMP-overexpressing mice develop gastric atrophy; gastric atrophy is associated with antiparietal cell antibodies and may be a model for autoimmune gastritis.A better understanding of the pathways and mechanisms regulating acid secretion as well as the development of genetically engineered mouse models should lead to new strategies to prevent and treat a variety of gastric disorders, including peptic ulcer disease, neoplasia, and autoimmune gastritis.

A study was made of the effect of body fat stored by ewes in early pregnancy on the subsequent immune response to gastrointestinal parasites around parturition. Pregnant ewes were given access to a lucerne pelleted diet either ad libitum (H) or at approximately 0.70 of their maintenance requirements (L) from the 42nd day of pregnancy in order to achieve a clearly differentiated level of body reserves by the 90th day of pregnancy. Then, all animals were put on the same plane of nutrition till 3 weeks after parturition. All ewes in both groups received 7,000 infective larvae of Haemonchus contortus per week for 7 weeks prior to lambing. The dietary treatments led to large differences between both groups of ewes in back-fat thickness that had a high correlation with mean plasma concentrations of leptin. In spite of the fact that animals were on the same plane of nutrition at infection time, host resistance, as measured by faecal egg counts, was significantly affected by the nutritional treatment established during early pregnancy. This effect produced noticeable differences in worm size and in worm burden at lambing. The response was accompanied by a marked increase in circulating eosinophils in better-fed ewes than in those maintained on a restricted diet in early pregnancy. Serum pepsinogen concentration, however, was inversely affected by the nutritional treatment till lambing, showing a maximum difference as early as 2 weeks after infection. The results support the view that higher levels of nutrition during early pregnancy enhance the expression of immunity against gastrointestinal parasites around parturition. Furthermore, the differences in the immune response appeared associated with serum leptin levels suggesting that leptin may be a key link between nutritional status and the protective immune reactivity against GI nematode infection.

Twenty-four calves unexposed to pasture were allocated to four groups and inoculated with either two doses of 5 million Eimeria alabamensis oocysts at turn-out (E), 90,000 L3 of Ostertagia ostertagi and Cooperia oncophora divided on six occasions (N) or both oocysts and larvae as above (E + N). A control group was left uninoculated (C). For 10 weeks, the groups grazed in separate uniform paddocks not previously grazed by cattle. By day 5, most calves in groups E and E + N developed clinical coccidiosis that resulted in reduced weight gain compared to C and N. Mean trichostrongylid faecal egg counts in groups N and E + N never exceeded 300 eggs per gram of faeces, and average serum pepsinogen levels were less than 3.8 U tyrosine. This experiment demonstrates the potential impact of E. alabamensis on the performance of previously unexposed calves, whereas no aggravated effects were observed due to concurrent infections with gastrointestinal nematodes.

Aim: To develop conditionally immortalized gastric mucosal cell lines that show distinct types of cell differentiation from transgenic mice harboring temperature-sensitive simian virus 40 (tsSV40) large T antigen. Methods: Gastric mucosal cells from the transgenic mice were cultured at a permissive temperature (33°C), and proliferative cells were then cloned by colony formation. Results: Eight gastric cell lines showed epithelial-like morphology and grew at 33°C. Three different types of the cell lines have been established: (1) MGE12-1, MGE3-2, and MGE509 cells expressing mRNAs for pit cell markers (gastric mucin and cathepsin E); (2) MGE02, MGE503, and MGE511 cells expressing mRNAs for pit and zymogenic (pepsinogen F) cell markers, and (3) MGE507 and MGE727 cells expressing mRNAs for pit, zymogenic, and parietal (H,K-ATPase α-subunit) cell markers. Moreover, the TaqMan assay showed that mRNA levels of mucin, H,K-ATPase α-subunit, and pepsinogen F were influenced by nonpermissive temperature (39°C) in MGE503 and MGE727 cells. Conclusion: These gastric epithelial cell lines seem to reflect different stages of development of gastric mucosal cells.

Pepsin is initially produced as the zymogen pepsinogen, containing a 44 residue prosegment (PS) domain. When folded without the PS, pepsin forms a thermodynamically stable denatured state (refolded pepsin, Rp). To guide native folding, the PS binds to Rp, stabilizes the folding transition state, and binds tightly to native pepsin (Np), thereby driving the folding equilibrium to favor the native state. It is unknown whether these functionalities of the PS are encoded within the entire sequence or within discrete segments. PS residues 1p-29p correspond to a highly conserved region in pepsin-like aspartic proteases and we hypothesized that this segment is critical to PS-catalyzed folding. This notion was tested in the present study by characterizing the ability of various truncated PS peptides to bind Rp, catalyze folding from Rp to Np, and to inhibit Np. Four PS truncations were examined, corresponding to PS residues 1p-16p (PS1-16), 1p-29p (PS1-29), 17p-44p (PS17-44) and 30p-44p (PS30-44). The three PS functionalities could be ascribed primarily to discrete regions within the highly conserved motif: 1p-16p dictated Rp binding, 17p-29p dictated Np binding/inhibition, while the entire 1p-29p dictated transition state binding/catalyzing folding. Conversely, PS30-44 played no obvious role in PS-catalyzed folding; it is hypothesized that this more variable region may serve as a linker between PS1-29 and the mature domain. The high sequence conservation of PS1-29 and its role in catalyzing pepsin folding strongly suggest that there is a conserved PS-catalyzed folding mechanism shared by pepsin-like aspartic proteases with this motif.

Background.Helicobacter pylori lipopolysaccharide (LPS) affects pepsinogen release by a nontoxic mechanism. We hypothesized that this effect was characteristic of the organism and related to the clinical status of the strain. Materials and methods. LPS was isolated from 11 H. pylori strains whose pathogenic profile was known and four other nongastric bacteria. The effects of luminal LPS on guinea pig gastric mucosal pepsinogen release was evaluated using the Ussing chamber technique. CCK-8 (10−9M) was used as a positive control. Results.H. pylori LPS dose-dependently stimulated pepsinogen release with a maximal stimulation at 250 µg/ml (~4500%; p

The appearance and development of prochymosin- and pepsinogen-producing cells were investigated in the ovine abomasum from fetus to adult using immunohistochemistry. Prochymosin immunoreactivity appeared first in the proper gastric glands of the 100-day-old fetus. The intensity and distribution of prochymosin-immunoreactive cells increased gradually with the progress of gestation, and their most intense immunoreactivities and widest distribution were observed in 3-day-old lambs. They were subsequently reduced throughout postnatal growth. A few prochymosin-immunoreactive cells were scattered in the glands of adult sheep. Pepsinogen immunoreactivity appeared at first in a small number of cells in the base of some proper gastric glands of 120-day-old fetuses. After 130 days, pepsinogen-immunoreactive cells increased their staining intensities and distribution. They reached a peak in area at 21 days, which is comparable to adult sheep. In the pyloric glands, prochymosin- and pepsinogen-immunoreactive cells appeared from 100 and 130 days, respectively. Numbers were reduced in comparison to gastric glands and their occurrence was capricious. The results demonstrated that the ontogeny of prochymosin- and pepsinogen-immunoreactive cells in the abomasum of sheep is more similar to that in cattle than to that in the goat. The present data will contribute to the overall understanding of the development of ruminant gastric proteases.

Background/Aims: Early weaning has been shown to induce intestinal ornithine decarboxylase (ODC) activities and cell proliferation in rats. No information is available about the effect of early weaning on ODC activity in the stomach. Methods: Suckling rats were prematurely weaned on postnatal day 15 and followed through day 21. Oxyntic gland mucosa of stomach was obtained on postnatal days 15, 16, 18 and 21 (days 0, 1, 3 and 6 after early weaning) and assayed for ODC activity, DNA, protein and pepsinogen activity. α-Difluoromethyl ornithine (DFMO), a specific ODC inhibitor, was given orally to early-weaned pups and its resultant effects were assessed on days 1 and 6 after early weaning. Results: Stomach mucosal wet weight, DNA, protein and pepsinogen activities significantly increased on day 6 after early weaning. ODC activity increased on days 1, 3, and 6 after early weaning, with the highest increase (3-fold) on day 1 when compared to controls. The increases of ODC activity, DNA and protein contents as induced by early weaning were significantly suppressed when pups were exposed to DFMO. However, no suppression of pepsinogen activity was observed. Conclusions: Our study shows that early weaning induces ODC activity and functional growth in the stomach. Gastric ODC activity is essential in gastric mucosal growth processes but not in differentiation. The induction of stomach ODC may act as an early marker in the growth of stomach mucosa induced by early weaning in rats.

Pepsinogen levels in ape stomachs were comparable to those in macaques and significantly higher than those in the stomachs of other mammals, including carnivores and ruminants. The occurrence of multiple forms of pepsinogens was remarkable. Nine, sixteen, eight, and fourteen pepsinogens were purified or partially purified from the gastric mucosa of a gibbon, orang-utan, gorilla, and chimpanzee, respectively. Most of these were type-A pepsinogens, and only one type-C pepsinogen was identified in each ape. The two types could be readily distinguished by staining for proteolytic activity on polyacrylamide gel electrophoresis (PAGE) in the presence/absence of pepstatin. Type-A pepsinogens were further divided into two subtypes. One subtype, constituting a major group of pepsinogens in apes, exhibited high hemoglobin-digestive activity. The other subtype was specified by a relatively high content of Lys and low hemoglobin-digestive activity. It is likely that pepsinogen-A genes have been duplicated several times as hominoids, including humans, evolved in the primate lineage. The presence of multiple pepsinogens in apes might be advantageous in the efficient digestion of a wide variety of foods.

The consequences for lambs of infection over the winter with Teladorsagia circumcincta were quantified by deliberate, trickle infection of selected animals at 7 months of age. Infected and control uninfected animals were each allocated into four groups, relatively resistant animals on a normal diet, relatively resistant animals on an isocaloric diet supplemented with urea, and relatively susceptible animals on the same two diets. Resistance and susceptibility was assessed by faecal egg counts following natural infection during the summer preceding the deliberate infection. During the deliberate infection egg counts remained low and most parasites recovered at necropsy were inhibited larvae. Nonetheless, infection reduced weight gain, decreased albumin and fructosamine concentrations and provoked a noticeable pepsinogen and eosinophil response. As most larvae were inhibited these responses may have been largely a consequence of immuno-inflammatory responses in the host rather than the direct action of parasites themselves. Relatively resistant animals on the supplemented diet allowed fewer larvae to establish and had higher fructosamine concentrations, higher albumin concentrations and decreased pepsinogen responses. Therefore, a combination of relatively resistant sheep and nutritional supplementation appears most efficient at controlling infection.

Nematode larvae developing within the glands cause local loss of parietal cells and mucous cell hyperplasia whereas reduced acid secretion, increased serum gastrin and pepsinogen concentrations and generalized histological changes are associated with parasites in the abomasal lumen. Parietal cells with dilated canaliculi and/or degenerative changes typical of necrosis are present soon after the transplantation of adult worms, and abomasal secretion is also affected. Anaerobic bacteria survive in greater numbers as the pH rises, with bacterial densities becoming similar to ruminal populations at an abomasal pH of 4 and above. Failure to lyse bacteria may affect adversely the nutrition of the host. The parasites may initiate the pathophysiology through the release of excretory/secretory (ES) products which either act directly on parietal cells or indirectly through enterochromaffin-like (ECL) cells by provoking inflammation or by disrupting the protective mucosal defence system. Parietal cell dysfunction is proposed as a key event which leads to loss of mature chief cells and mucous cell hyperplasia, as well as hypergastrinaemia. Inflammation increases circulating pepsinogen concentrations and may also contribute to increased gastrin secretion. Stimulation of mucosal proliferation and differentiation of parietal cells in the isthmus by the raised serum gastrin levels will be beneficial by generating a new population of active parietal cells and adequate acid secretion.

The present study investigated (1) the pharmacological profile of cholecystokinin (CCK) receptor subtypes involved in the regulation of gastric pepsinogen secretion, (2) the influence of gastric acidity on peptic responses induced by CCK-8-sulfate (CCK-8S) or gastrin-I; and (3) the mechanisms accounting for the effects of CCK-like peptides on pepsinogen secretion. In anaesthetized rats, i.v. injection of CCK-8S or gastrin-I increased both pepsinogen and acid secretion. The pepsigogue effect of CCK-8S was higher than that of gastrin-I, whereas acid hypersecretion after CCK-8S was lower than that induced by gastrin-I. Peptic output following CCK-8S was partly blocked by i.v. injection of the CCK1 receptor antagonist, devazepide (-75.3%), or the CCK2 receptor antagonist, L-365,260 [3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3 yl)-N'-(3-methyl-phenyl)urea; -27.9%], but was fully prevented by combined administration of devazepide and L-365,260. The gastric acid hypersecretory effect of CCK-8S was enhanced by devazepide (+84.5%) and blocked by L-365,260. In contrast, the gastric secretory actions of gastrin-I were insensitive to devazepide, but abolished by L-365,260. Excitatory effects of CCK-8S and gastrin-I were not modified by vagotomy or atropine, whereas cimetidine or alpha-fluoromethylhistidine (irreversible blocker of histidine decarboxylase) partly prevented acid hypersecretion induced by both peptides without affecting their pepsigogue effects. After pretreatment with omeprazole, both CCK-8S and gastrin-I failed to stimulate acid secretion, while they increased pepsinogen output. In rats with gastric perfusion of acid solutions, CCK-8S or gastrin-I increased peptic output in a pH-independent manner either with or without pretreatment with omeprazole. Ablation of capsaicin-sensitive sensory nerves as well as application of lidocaine to the gastric mucosa failed to modify the excitatory effects of CCK-8S or gastrin-I on pepsinogen and acid secretion. Blockade of the nitric oxide (NO) synthase pathway by N(G)-nitro-L-arginine-methyl ester prevented the pepsigogue actions of both CCK-8S and gastrin-I (-61.8% and -71.7%, respectively), without affecting the concomitant increase in acid output. In addition, both these peptides significantly increased the release of NO breakdown products into the gastric lumen. The present results suggest that: (1) both CCK1 and CCK2 receptors mediate the peptic secretory responses induced by CCK-like peptides; (2) the excitatory inputs of CCK-8S and gastrin-I to chief cells are not driven through acid-dependent mechanisms or capsaicin-sensitive afferent sensory nerves; and (3) under in vivo conditions, the stimulant actions of CCK-like peptides on pepsinogen secretion are mediated, at least in part, by an increase in NO generation.

Three groups of pepsinogens exist in vertebrates, namely, pepsinogen A, pepsinogen C, and prochymosin, which are produced at different developmental stages. In the chicken, prochymosin is expressed only in the embryonic stage, while pepsinogens A and C are secreted from adult chicken proventricular (glandular stomach) mucosa. In order to understand the mechanism of transcriptional regulation of these genes, we have cloned the genes encoding chicken pepsinogens A and C and analyzed the sequences possibly involved in their regulation. 5'-Upstream sequences of both genes contain putative binding motifs for transcription factors such as GATA, Sox, and HNF-3 beta, which are expressed in the chicken gut epithelium. Moreover, we found seven putative binding motifs for human MZF-1 in intron 8 of pepsinogen A gene. These transcription factors may act as regulators of expression of chicken pepsinogen genes.

Three antimicrobial peptides, which had strong antimicrobial activity against a broad spectrum of microorganisms, were isolated from the stomach of the bullfrog, Rana catesbeiana. Two of the antimicrobial peptides were found to be derived from the N-terminal sequences of pepsinogen A and C prosequences. The amino acid sequences of the new antimicrobial peptides, named bullfrog pepsinogen A-derived antimicrobial peptide (bPaAP) and bullfrog pepsinogen C-derived antimicrobial peptide (bPcAP), were Gly-Val-Val-Lys-Val-Ser-Arg-Leu-Lys-Gly-Glu-Ser-Leu-Arg-Ala-Arg-Leu (MW 1865.5) and Ile-Ile-Lys-Val-Pro-Leu-Lys-Lys-Phe-Lys-Ser-Met-Arg-Glu-Val-Met-Arg-A sp-His-Gly-Ile-Lys-Ala-Pro-Val-Val-Asp-Pro-Ala-Thr-Lys-Tyr (MW 3691.6), respectively. The bPaAP and bPcAP adopted 35% and 42% amphipathic alpha-helical structure in 50% trifluoroethanol, respectively, and were non-hemolytic up to a concentration of 200 microg/ml. Synthesized pepsinogen C prosequences of monkey and human, which had similar structural characteristics as bPaAP and bPcAP, also showed antimicrobial activity at concentrations of 10-200 microg/ml. The third peptide was buforin I, previously found in the stomach of the Asian toad, Bufo bufo gargarizans. These findings strongly suggest that peptides derived from the prosequences of pepsinogens, along with buforin I, may contribute to the antimicrobial function of the gastrointestinal mucosa of vertebrates, including human.

It is generally known that the anuran stomach begins to express pepsinogens (Pg) during metamorphosis. To clarify the mechanisms of differentiation of Pg-producing cells, we examined immunohistochemically the epithelial transformation from larval to adult form in Xenopus laevis stomach at the cellular level. At the beginning of metamorphic climax, concomitantly with the modification of the basement membrane, apoptotic cells labelled by TUNEL suddenly increased in number in the entire epithelium except for the primordia of adult epithelial cells in the basal region of larval glands. Subsequently, with the development of connective tissue, the adult epithelial cells actively proliferated and replaced the larval cells from the basal to the luminal region. Following the start of morphogenesis of adult glands, Pg-producing cells became differentiated in newly formed adult glands, but not in the adult surface epithelium. We then developed an organ culture system and examined effects of thyroid hormone (TH) on the differentiation of Pg-producing cells in X. laevis stomach in vitro. In the presence of TH, just as in spontaneous metamorphosis, Pg-producing cells differentiated from the adult epithelial primordia after the apoptosis of larval epithelial cells. In contrast, in the absence of TH, neither apoptotic larval cells no Pg-producing cells were detected. Therefore, we conclude that TH triggers organ-autonomously the entire process leading to the differentiation of Pg-producing cells in X. laevis stomach. In addition, the strict localization of Pg-producing cells in the adult glands both in vivo and in vitro suggests the correlation between the differentiation of Pg-producing cells and morphogenesis of the glands surrounded by the developed connective tissue.

Multiple isoforms of the Na+/H+ exchanger (NHE) are expressed at high levels in gastric epithelium, but the physiological role of individual isoforms is unclear. To study the function of NHE2, which is expressed in mucous, zymogenic, and parietal cells, we prepared mice with a null mutation in the NHE2 gene. Homozygous null mutants exhibit no overt disease phenotype, but the cellular composition of the oxyntic mucosa of the gastric corpus is altered, with parietal and zymogenic cells reduced markedly in number. Net acid secretion in null mutants is reduced slightly relative to wild-type levels just before weaning and is abolished in adult animals. Although mature parietal cells are observed, and appear morphologically to be engaged in active acid secretion, many of the parietal cells are in various stages of degeneration. These results indicate that NHE2 is not required for acid secretion by the parietal cell, but is essential for its long-term viability. This suggests that the unique sensitivity of NHE2 to inhibition by extracellular H+, which would allow upregulation of its activity by the increased interstitial alkalinity that accompanies acid secretion, might enable this isoform to play a specialized role in maintaining the long-term viability of the parietal cell.

The effect of ivermectin delivered by an orally administered prototype sustained-release (IVM SR) bolus was evaluated over a period of 140 days using 12 steers aged approximately 8-10 months and ranging in weight from 150 to 175 kg. They were allocated to two groups of 6 steers each by restricted randomization based on their liveweight. Each of the treatment group received an IVM SR bolus designed to deliver 12 mg of the ivermectin/day for 135 days. The other group was designated as non-medicated controls. The groups were placed on adjacent 3 acre paddocks obtained by sub-dividing of 6.0 acre permanent pasture which had previously been grazed by young untreated cattle so exposing experimental steers to a similar challenge of a contaminated paddock. The faecal egg counts, herbage larval counts and worm burdens of the major gastro-intestinal nematodes of cattle were significantly reduced by the use of the IVM SR boluses. These parasitological effects were reflected in the increased liveweight gains in the IVM SR bolus-treated steers. The control steers required occasional salvage treatments over the trial period and the herbage on their paddock was heavily contaminated with infective larvae as reflected in the high worm burden in the control steers necropsied at trial termination and in tracer calves introduced into the paddocks during the initial (Day 31), interim (Day 69) and final (Day 100) stages of the experiment.

The efficacy of a move to aftermath in July combined with moxidectin or fenbendazole treatment for the control of parasitic gastroenteritis (PGE) in calves was evaluated in three field experiments in the Netherlands. In all five treated groups high gastrointestinal nematode infections and PGE were prevented by a dose and move in July. Cooperia infections increased to moderate levels in two groups treated with moxidectin and one group treated with fenbendazole. In both other groups and also for Ostertagia in these three groups, low to extremely low infections were acquired. In the first experiment high primary infections, resulting in high faecal egg counts and a moderate increase of blood pepsinogen values occurred before the dose and move. Nevertheless, these primary infections were not high enough to result in PGE. In both other experiments primary infection levels were low and faecal egg counts increased to 100–650 eggs/g faeces at the end of the grazing season. The blood pepsinogen values of non-treated control groups demonstrated that it took more than a month after their move to aftermath before substantial reinfection occurred on the new pasture. In the first and the last experiment only, high Ostertagia and Cooperia infections developed in the control group at the end of the grazing season, though it did not result in clinical PGE. The experiments demonstrate all theoretical risks of the dose and move system: (1) PGE early in the grazing season as a result of high overwintered pasture infectivity. (2) PGE just before the move as a result of an early midsummer increase in pasture infectivity. (3) PGE around housing as a result of insufficient suppression of pasture infectivity late in the grazing season. (4) Underexposure to nematode infections due to a high suppression of nematode infections. Nevertheless, it can be concluded that under normal conditions the dose and move system remains to be a valuable and easily applicable system for the control of PGE.

Sonic hedgehog (Shh) gene encodes a secreted protein that acts as an important mediator of cell-cell interactions. A detailed analysis of Shh expression in the digestive organs of the chicken embryo was carried out. Shh expression in the endoderm begins at stage 7, when the formation of the foregut commences, and is found as narrow bands in the midgut. Shh expression around the anterior intestinal portal at stage 15 is restricted to the columnar endoderm lined by the thick splanchnic mesoderm, suggesting that the existence of thick splanchnic mesoderm might be necessary for Shh expression in the columnar endoderm. After the gut is closed, Shh expression is found universally in digestive epithelia, including the cecal epithelium. However, its expression ceases in the epithelium of the proventricular glands, the ductus choledochus and ductus pancreaticus that protrude from the main digestive duct. When the gizzard epithelium differentiated into glands under the influence of the proventricular mesenchyme, the glandular epithelium lost the ability to express Shh. These findings suggest that Shh expression in the epithelium may be regulated by surrounding mesenchyme throughout organogenesis of the digestive organs and is closely involved in epithelial-mesenchymal interactions in developing digestive organs.

The influence of different sulfonylureas on the rate of acid and pepsinogen secretion was studied in isolated rabbit gastric glands. Neither tolbutamide (10-500 microM), chlorpropamide (10-500 microM), glibenclamide (1-50 microM) nor glipizide (1-50 microM) exerted a secretory effect. In contrast, gliquidone caused a marked and dose-dependent stimulation of acid production in gastric glands incubated under basal conditions and potentiated the stimulatory effect of both histamine and carbachol. Gliquidone also increased the rate of pepsinogen release in gastric glands incubated either under basal conditions or in the presence of cholecystokinin-octapeptide or isoproterenol. The secretory effects of gliquidone were associated with a significant increase in the glandular content of cyclic AMP, caused by a competitive inhibition of low-Km cyclic AMP phosphodiesterase. Our results indicate that, among the assayed sulfonylureas, only gliquidone, in the micromolar range, stimulates acid and pepsinogen secretion through a cyclic AMP-dependent mechanism.

The objective of this study was to evaluate the effect of different infection levels of Ostertagia ostertagi and Cooperia oncophora in a simulated `first grazing season' on the resistance of calves to an artificial challenge infection. The infection levels were determined by the infection schedules and the chemoprophylaxis used. Thirty six 7–11-month old Holstein–Friesian bull calves were randomly divided into four groups. The animals of group B received an ivermectin sustained release bolus (ISRB) on day 0. The calves of group D were treated on days 0 and 56 with a subcutaneous injection of doramectin (0.2 mg kg−1 BW). Group C was the untreated control group. The calves of group N were used as helminth-naive controls, while the animals of groups B, C and D were trickle infected for 24 weeks. The infection schedules were designed to simulate the expected infection pattern for each treatment group under set-stocked conditions in temperate climate areas. After the last infection, all animals were treated with oxfendazole. One week later, all animals received a challenge infection of 50,000 O. ostertagi L3 and 100,000 C. oncophora L3, spread over 10 consecutive days. During the primary infection period the faecal egg output and the serum pepsinogen and antibody levels reflected the different levels of host–parasite contact between the groups (group C>group D>group B>group N). After the challenge infection, faecal egg counts, total Ostertagia burden, size of the adult worms and abomasal globule leucocyte counts all indicated a positive relationship between the level of Ostertagia infection during the primary infection period and the level of acquired resistance. A reduction of host–parasite contact during the primary infection period, as a consequence of the infection schedule and the chemoprophylaxis used, resulted in a diminished level of resistance to the artificial challenge infection with O. ostertagi. Faecal cultures and small intestine worm counts indicated that all previously infected groups had acquired a high degree of resistance to the Cooperia challenge infection.

A total of 70 strongyle-free French Alpine dairy goats were exposed to a combination of sequential and challenge infections with Haemonchus contortus and Trichostrongylus colubriformis third-stage larvae. The sequential infection consisted of three inoculations at 50-day intervals, each infection being abbreviated by anthelmintic treatment at 40 days postinoculation. The challenge infection, composed of the same nematode strains, was undertaken 2 months later, when goats were at their 1st month of lactation. Fecal egg counts (FECs), packed cell volumes (PCVs), pepsinogen concentrations, inorganic phosphate concentrations, and peripheral eosinophil numbers were measured at 30-40 days after each inoculation. Goats were defined as being resistant or susceptible according to their level of nematode egg output following the first inoculation. Significant differences in FECs were recorded between the two groups throughout the further inoculations and the challenge infection. The reliability of FECs was supported by the high repeatability values found within and between infections. With regard to blood constituents, only PCVs related to H. contortus infection showed values that differed significantly between the two groups, resistant goats having higher PCVs after the first and the third inoculations than did susceptible animals. However, this difference was not detectable after the challenge infection. The milk production yield for the current lactation was significantly lower in the resistant goats. Moreover, resistant animals exhibited constantly greater body condition scores as compared with susceptible animals. These results indicate that the individual responsiveness of dairy goats to experimental nematode infection can be estimated on the basis of FECs and PCVs (for H. contortus) and is negatively related to the level of milk production of the animals.

Over a period of 14 months, faecal and serum samples were collected monthly from cattle of 1 to 30 months old on three ranches (20-25 animals ranch-1 sample-1) in Mindanao, Philippines. Individual faecal egg counts, serum pepsinogen levels and Mecistocirrus digitatus specific antibodies were determined, and infective larvae from pooled faecal samples were examined. Post mortem worm counts were carried out on tracer calves (n = 16) that had been kept for 30 days with the herds on two of the ranches. A number of gastrointestinal nematode species were present: M. digitatus, Haemonchus placei, H. similis, Trichostrongylus axei, Cooperia punctata, C. pectinata, Bunostomum phlebotomum and Oesophagostomum radiatum. Strongyle eggs first appeared in the faeces of calves of two months old, egg counts peaked when animals were 3-6 months old, after which they steadily and significantly declined. Faecal egg output of M. digitatus increased up to the age of 10 months, after which there was a steady but not significant decline until the age of 24 months. In faecal cultures the relative proportion of Cooperia larvae decreased from over 70% in 0-6 month old animals to 30% in 19-24 month old animals, and a corresponding 30% increase in the proportion of Mecistocirrus was observed. Both serum pepsinogen and M. digitatus specific antibodies levels increased during the first year of life, and then remained at the same level in older animals. Whereas the total faecal egg output did not significantly vary throughout the year, worm counts from tracer calves indicated a marked seasonality in transmission.

The native folding of certain zymogen-derived enzymes is completely dependent upon a prosegment domain to stabilize the folding transition state, thereby catalyzing the folding reaction. Generally little is known about how the prosegment accomplishes this task. It was previously shown that the prosegment catalyzes a late-stage folding transition between a stable misfolded state and the native state of pepsin. In this study, the contributions of specific prosegment residues to catalyzing pepsin folding were investigated by introducing individual Ala substitutions and measuring the effects on the bimolecular folding reaction between the prosegment peptide and pepsin. The effects of mutations on the free energies of the individual misfolded and native ground states and the transition state were compared using measurements of prosegment-pepsin binding and folding kinetics. Five out of the seven prosegment residues examined yielded relatively large kinetic effects and minimal ground state perturbations upon mutation, findings which indicate that these residues form strengthened and/or non-native contacts in the transition state. These five residues are semi- to strictly conserved, while only a non-conserved residue had no kinetic effect. One conserved residue was shown to form native structure in the transition state. These results indicated that the prosegment, which is only 44 residues long, has evolved a high density of contacts that preferentially stabilize the folding transition state over the ground states. It is postulated that the prosegment forms extensive non-native contacts during the process of catalyzing correct inter- and intra-domain contacts during the final stages of folding. These results have implications for understanding the folding of multi-domain proteins and for the evolution of prosegment-catalyzed folding.

The purpose of this study was to characterize time-dependent changes in pepsinogen (PG) synthesis of porcine gastric chief cells during long-term monolayer culture. Porcine chief cells were isolated by pronase/collagenase treatment of fundic mucosa and enriched by density gradient and counterflow centrifugation. PG isoenzymes were identified in [L-35S]methionine-labelled cultured chief cells by native polyacrylamide gel electrophoresis followed by phosphor imager analysis, protease detection and immunoblots with specific PG A and C antibodies. The obtained results suggest that porcine chief cell cultures, after an initial settling period, reached an approximate steady state in total protein content and synthesis as well as in PG content and isoenzyme pattern from days 3 to 9 of culture. The latter was characterized by the presence of at least two PG A and two PG C isoenzymes. During the supposed steady-state total PG synthesis averaged out at 34 +/- 2% of total protein synthesis, as detected by [L-35S]methionine incorporation, due to the synthesis of, mainly, PG A2 and, to a much lesser extent, PG C and A1. In line with an active secretion, PG A2 proportion was on average significantly higher in released (44 +/- 3%) than in intracellular labelled proteins (19 +/- 2%). In addition, PG release from chief cells cultured for 6 and 9 days could be stimulated by cholecystokinin-octapeptide. These data suggest that porcine chief cells in monolayer culture are a model well suited for the quantitative and qualitative characterization of PG isoenzyme synthesis and release during long-term investigations, for which an establishment of a culture steady state appears to be a useful prerequisite.

CdxA, a chicken homeobox-containing gene related to caudal in Drosophila, has been implicated in the regionalization of endoderm. It is reported here that, in the development of the chicken embryo, CdxA expression appears in the endoderm at day 1.5 of development as bilateral bands on either side of the splanchnopleure which later contribute to intestinal epithelium. The CdxA-expressing area extends medially and caudally as formation of the gut tube progresses. It is also shown that the rostral limit of CdxA expression demarcates the boundary between stomach and duodenum after day 3 of development. CdxA is not expressed in digestive tract appendages which open into the intestine, such as pancreas, liver and allantois. Early restriction of CdxA expression in intestinal lineage suggests that the intestinal specification involving CdxA expression commences before the gut tube is formed. The expression of CdxA in epithelial-mesenchymal tissue recombinants suggests that mesenchymal influence regulating CdxA expression plays an important role in confirming the boundary between the stomach and intestine. Chronological change in the spatial distribution of CdxA transcripts and the results of tissue recombination experiments, together with precise fate maps of early endoderm and splanchnic mesoderm, lead to a model of mechanisms by which intestinal specification is brought about.

Undifferentiated glandular stomach tissue fragments from 16.5-day fetal rats were transplanted under the kidney capsule of syngeneic adult rats, and the proliferation, differentiation and morphogenesis of the transplanted tissues were investigated. Gastric epithelial cells began to invaginate 3-4 days after the transplantation and immature glands were formed after 1 week. During the period, there was a gradual increase in the expression of pepsinogen and cathepsin E, markers of cytodifferentiation of the stomach epithelia, both at protein and mRNA levels. Cathepsin E was weakly expressed in undifferentiated gastric epithelial cells at 16.5 days of gestation, and a higher level of the expression was observed in differentiated epithelia of the transplants. In contrast, the pepsinogen-producing cells first appeared around days 3-4 after transplantation and gradually increased in number to about 30% of the epithelial cells and became localized at the bottom of the gland. During the period of the experiment up to 1 month, the pepsinogen-producing cells were all positive for class III mucin and cathepsin E, indicating the immature character of these cells. In addition, no parietal cells were observed. When the tissue fragments were transplanted into adrenalectomized animals, the epithelial differentiation and morphogenesis was suppressed, but its proliferation was enhanced. The observed changes were reversed by hydrocortisone replacement. These results suggest that the development of the 16.5-day fetal stomach is regulated intrinsically to a certain extent by the genetic program of the cells involved and various gastric functions develop in the absence of luminal stimulation, stage-specific systemic hormonal change, neuronal regulation or other systemic influences, and that glucocorticoids modulate the developmental program of the fetal stomach tissues.

The efficacy of morantel sustained release trilaminate (MSRT) bolus against gastrointestinal nematodes was evaluated under field conditions over a 10-month period. Twenty weaner calves were randomly divided into 2 groups of 10 calves each and grazed from March to December on adjacent, similarly contaminated paddocks. Group 1 calves (T-1) served as untreated controls while group 2 calves (T-2) were dosed at turnout with MSRT bolus designed to release morantel tartrate continuously for 90 days. The efficacy of MSRT was assessed by comparison of parasitological data (faecal worm egg counts, herbage larval counts, worm counts from tracer calves and set-stocked trial calves, determination of haematological parameters and pepsinogen levels), weight gains and clinical status of the animals. Faecal egg counts from the treated group (T-2) were reduced by 100% (P < 0.001) following treatment and remained significantly (P < 0.05) lower than counts from T-1 calves up to trial termination. The use of MSRT bolus resulted in a reduction of 92% (P < 0.001) in the number of gastrointestinal nematodes in set-stocked calves at the end of the study and a 55 to 85.7% reduction in herbage larval infectivity as reflected in lowered parasite burdens in tracer calves. At the trial termination, the control calves had gained on average (+/- s.d.) 59.4 +/- 4.8 kg (200.0 +/- 7.4 g day-1) and the treated ones on an average 128.6 +/- 10.5 kg (530.0 +/- 13.1 g day-1).

Objective and Design: The effects of sex, sex hormones and gonads were studied in dogs because men secrete more acid than woman and duodenal ulcer is commoner in men and remits in pregnancy and after stilboestrol.¶Material: Four male and 4 female greyhounds with chronic gastric fistulas were tested.¶Methods: Histamine was infused i.v. 1–16 μg/min and plateau acid output measured in the last 30 min of each 60 min infusion.¶Treatment: The gonads were then removed and secretion retested. The orchidectomised dogs were then given stilboestrol 2 mg daily (±progesterone 25 mg i.m. three times a week) and the oophorectomised bitches given testosterone propionate 25 mg twice weekly i.m. and secretion again retested.¶Results: Submaximal and maximal acid outputs (and serum pepsinogen) of dogs and bitches were similar and correlated with body weight. Gonadectomy did not alter these functions nor did hormones of the opposite sex given for 1–12 months.¶Conclusions: In the dog, as in the rat, and probably in man, histamine-stimulated gastric acid output is a function of body size and not of sex.

The development of immunity to Ostertagia ostertagi and Cooperia oncophora and interactions between these species were investigated in experimentally infected calves. Parasitological, serological and histological parameters were used for assessing immune responses. No conclusive evidence of an effect of C. oncophora on the course of an O. ostertagi infection in calves could be shown. Following a challenge with C. oncophora and O. ostertagi of C. oncophora primed calves, no significant reductions in establishment rate, faecal egg counts, worm length or the percentage of early fourth stage larvae could be demonstrated. Results also confirmed earlier work showing the very different degrees of immunity conferred following immunisation with either C. oncophora or O. ostertagi. While a protective immunity was generated in the case of C. oncophora, continuous infection of calves with 420 000 L3 of O. ostertagi during almost 5 months induced immune reactions which affected growth and fecundity of the worms but not the establishment rate.

1. Infection with the bovine abomasal nematode Ostertagia ostertagi results in a loss of acid-secreting parietal cells and an increase in gastric pH. The effects of an experimental infection on gastrin mRNA expression, blood and tissue gastrin concentrations, the different molecular forms of gastrin in each, and pyloric mucosal chromogranin A-derived peptides were investigated in the calf. 2. An increase in blood gastrin concentrations in the infected group reached a peak by day 28 postinfection (635 pg ml-1; P < 0.01). Gel chromatography analysis of blood samples revealed that the hypergastrinaemia comprised largely gastrin-34 (G-34) in parasitized calves while gastrin-17 (G-17) predominated in control animals. 3. An 11-fold increase in gastrin mRNA expression was recorded in the parasitized animals which was accompanied by a 23.8% reduction in pyloric mucosal gastrin content and an apparent drop of 24.7% in the number of gastrin-producing G cells detected. There was no major change in the relative abundance of G-17 and G-34 in the pyloric mucosa of infected calves. No significant differences in the concentration of pyloric mucosal chromogranin A-derived peptides were recorded between infected and control groups. 4. These data suggest that the hypergastrinaemia seen in parasitized calves results largely from an increase in gastrin synthesis and that depletion of previously stored peptide makes virtually no contribution to elevated blood gastrin concentrations.

The dose-response relation for the appearance of pepsinogen isozyme 1 (Pg 1)-altered pyloric glands (PAPG) and the related induction of adenocarcinomas were examined in male C3H mice given N-methyl-N-nitrosourea (MNU) in their drinking water at the concentration of 120 ppm (group 1), 60 ppm (group 2), 30 ppm (group 3) or 0 ppm (group 4) for 30 weeks and then normal tap water. Animals were killed at weeks 10, 30 and 42. Adenomatous hyperplasias and adenocarcinomas were noted from week 30 and their induction was dose-dependent at week 42. Almost all cells of pyloric gland cell type in those lesions had little or no immunohistochemically demonstratable Pg 1 content, as was also the case for the cells in PAPG, whose numbers per 100 normal-appearing pyloric glands were found to be MNU dose-dependent at all experimental time points. The numbers of PAPG at week 10 significantly correlated with the incidences of adenomatous hyperplasias and adenocarcinomas at week 42. Investigation of proliferation by immunohistochemical detection of bromodeoxyuridine (BrdU) labeling in the PAPG at week 10 demonstrated elevation (P < 0.05) as compared to normal pyloric glands. Intestinal metaplasia was not a feature in the present experiment and the results suggest that in mice, PAPG might be a preneoplastic lesion involved in gastric chemical carcinogenesis.

The modifying effects of Chelidonium majis L. (Papaveraceae) herb extract (CH), an analgesic traditionally prescribed for gastric and duodenal ulcer patients, on gastric tumor development were studied in rats given N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Sixty-four male 6-week-old Wistar rats were used. Group 1 rats were initially given MNNG (200 mg/kg b.w.) by gavage at days 0 and 14 as well as saturated sodium chloride solution (S-NaCl, 1 ml per rat) every 3 days during weeks 0-3 (six times), and then placed on basal diet containing 0.1 or 0.2% CH for 16 weeks from week 4. Rats of Group 2 and 3 were treated with MNNG together with S-NaCl or saline (0.9% NaCl, 1 ml per rat), respectively, timed as in Group 1 but without further treatment. All surviving animals were killed at week 20 and histopathologically investigated. In the glandular stomach, the number of preneoplastic pepsinogen 1 altered pyloric glands (PAPGs) in the MNNG + S-NaCl-->CH (0.1%) group (Group 1) was significantly smaller than in the MNNG + S-NaCl group (Group 2) (P < 0.02). The incidences of forestomach neoplastic lesions (papillomas and squamous cell carcinomas) also showed a tendency to decrease with the CH treatment. The results thus indicate that CH exerts inhibitory effects on glandular stomach carcinogenesis in the rat, so that it may have potential as a chemopreventive agent for stomach cancer in man.

Proton pump inhibitors were shown to affect the sensitivity of the gastric mucosa to chemical agents. This effect is associated with inhibition of proton back-diffusion and increase in the permeability of the gastric epithelium. We studied the effect of omeprazole on gastric secretion of bicarbonates and pepsinogen induced by irritation of the gastric mucosa in narcotized rats with a hypertonic solution of high acidity (500 mM NaCl, pH 2.0). Irritation of the gastric mucosa increased the basal secretion of bicarbonates and potentiated the secretion of $$ \mathrm{HCO}_3^{-} $$ and pepsinogen induced by electrostimulation of the vagus nerve. Omeprazole stimulated the prostaglandin-induced increase in the basal secretion of $$ \mathrm{HCO}_3^{-} $$ and pepsinogen. By contrast, bicarbonate production in response to vagal stimulation was suppressed in the presence of omeprazole. Our results indicate that proton pump blockade has a modulatory effect on gastric secretion of bicarbonates and pepsinogen induced by chemical stimulation of the gastric mucosa.

BACKGROUND & AIMS: Although epidermal growth factor (EGF) inhibits gastric acid secretion, the effects it exerts on gastric chief cells are unknown. The aim of this study was to investigate whether EGF modulates pepsinogen release and intracellular Ca2+ concentrations ([Ca2+]i) and whether the effect involves mitogen-activated protein (MAP) kinase, eicosanoid generation, and nitric oxide. METHODS: Chief cells were obtained by sequential digestion with collagenase and Ca2+ chelation. [Ca2+]i was measured in cells loaded with Fura-2 and NO generation by the NO coproduct citrulline. RESULTS: In situ hybridization, immunohistochemistry, and immunoblotting showed that EGF receptor and MAP kinases were constitutively expressed in chief cells. EGF caused a concentration-dependent stimulation of pepsinogen secretion and MAP kinase activity and determined a 2.5-7.0-fold increase in [Ca2+]i, inositol 1,4,5-tryphosphate, prostaglandin E2, and leukotriene B4. Tyrosine kinase inhibitors and cyclooxygenase and lipoxygenase inhibitors reduced pepsinogen secretion and eicosanoid generation induced by EGF. EGF increased citrulline generation and guanosine 3',5'-cyclic monophosphate accumulation sixfold; the effect was blocked by NG monomethyl-L-arginine, which is an NO synthase inhibitor. CONCLUSIONS: EGF stimulates pepsinogen secretion by activating eicosanoid generation, tyrosine kinases, MAP kinases, Ca2+, NO, and guanosine 3',5'-cyclic monophosphate. (Gastroenterology 1996 Oct;111(4):945-58)

A three-dimensional model of human cathepsin E, a possible endothelin-converting enzyme, is constructed using computer-aided molecular modeling techniques. The structure of porcine pepsin, another aspartic protease, was used as a template. The final structure, after all gaps and deletions were made, was optimized using the AMBER-4 package. A dipeptide (Trp-Val) representing the substrate was docked in the putative active site and the whole structure was optimized after several runs of minimization and dynamics calculations. The result of this modeling study showed that the structure of cathepsin E is similar to that of porcine pepsin and has three disulfide bonds that are conserved in both enzymes. There are two Asp-Thr-Gly sequences at the active site of enzyme. The active site cavity is large enough to accommodate its substrate.

Pepsinogen was purified from the gastric mucosa of soft-shelled turtle (Trionyx sinensis) by a series of chromatographies on DEAE-cellulose, Sephadex G-100, and Q-Sepharose. Upon chromatography on Q-Sepharose, it was separated into nine isoforms. These isoforms showed a relative molecular mass of approximately 43,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoforms 4 through 9 contained carbohydrate (approx. 2% each). Insofar as they were examined, their NH2-terminal sequences differed only in showing substitution at a few positions. At pH 2.0, they were rapidly activated to the corresponding isoforms of pepsin in a stepwise manner. The nine isoforms showed similar specific activity toward hemoglobin and hydrolyzed N-acetyl-L-phenylalanyl-L-diiodotyrosine, a good substrate for pepsin A, at somewhat different rates. They were inhibited by pepstatin to various extents, more strongly than human pepsin C but less strongly than human pepsin A. All isoforms appeared to have similar cleavage specificity toward oxidized insulin B chain, which resembled those of both human pepsins A and C. A cDNA clone for one of the zymogen isoforms was isolated and sequenced. The amino acid sequence thus deduced was more homologous with those of mammalian pepsinogens A than those of mammalian pepsinogens C or prochymosin.

Rab proteins are a family of ras-like proteins that are involved in intracellular membrane trafficking. Rab-GDP dissociation inhibitor prevents dissociation of GDP from Rab proteins and extracts Rab proteins from cell membranes in vitro. In the present study, we examined the effects of recombinant rab-GDI on Rab proteins in gastric chief cells. Rab-GDI extracted GTP-binding proteins, including Rab3 and Rab5, from chief cell membranes in a dose-dependent manner. Maximal concentrations of rab-GDI (or = 50 micrograms/ml) removed approx. 40% of membrane-associated Rab3. Moreover, preincubation of permeabilized chief cells with rab-GDI resulted in a 35% decrease in GTP gamma S(100 microM)-induced pepsinogen secretion, suggesting that membrane-associated Rab proteins are involved in the final stages of secretion. Immunostaining of chief cell cytosolic and membrane fractions with GDI-specific antisera revealed bands at 56 and 48 kDa, respectively, indicating that chief cells express two isoforms of rab-GDI. In gastric chief cells, regulation of Rab proteins by rab-GDI plays an important role in mediating exocytosis.

In order to determine whether tachykinins alter the function of chief cells and to characterize the receptors mediating the effect, we investigated the abilities of various substance P (SP)-related peptides to inhibit the binding of 125I-Bolton-Hunter labeled substance P (125I-BH-SP) and their abilities to alter cell function in dispersed chief cells from guinea pig stomach. Binding of 125I-BH-SP was saturable, reversible, time- and temperature-dependent and was inhibited by several SP-related peptides with relative potencies of SP = physalaemin (IC50: 0.19 nM) > SP methyl ester (SP-ME) (IC50: 3.3 nM) > eledoisin (IC50: 6.1 nM) > neurokinin A (NKA) (IC50: 65 nM) > neurokinin B (NKB) (IC50: 80 nM). Analyses of these binding data demonstrated that chief cells possess a high and low affinity class of binding sites. Neither 125I-NKA nor [phenylalanyl-3,4,5-3H]senktide demonstrated saturable binding to chief cells. Acid stripping experiments demonstrated rapid ligand internalization with 55% of the bound radioligand internalized by 10 min. Phospholipase C activating agents (carbachol, CCK-8), adenylate cyclase activating agents (secretin, VIP), TPA and the calcium ionophore, A23187, all inhibited the binding of 125I-BH-SP and it was due to inhibition of ligand internalization with no change in surface bound parameters. SP (0.1 μM) stimulated pepsinogen secretion but was 4-times less efficacious than CCK-8 (10 nM) or carbachol (1 mM). 10 nM SP stimulated a rapid increase in cytoplasmic free calcium concentration ([Ca2+]i) followed by a sustained elevation lasting 2 min. Single cell spectroscopy demonstrated SP (10 pM to 1 μM) did not cause calcium oscillations. The NK1 receptor antagonist, CP96,345 specifically inhibited the SP-stimulated changes in [Ca2+]i and pepsinogen secretion. The relative potencies of SP-related peptides to stimulate pepsinogen secretion and [Ca2+]i demonstrated a close agreement with their abilities to inhibit the binding of 125I-BH-SP, and comparison of the dose-response curves suggests occupation of the low affinity sites mediate changes in biologic activity. In conclusion, the present study demonstrates that chief cells possess a NK1 subtype of tachykinin receptor, occupation of the low affinity sites of this receptor cause calcium mobilization and pepsinogen secretion, and that binding to this receptor is regulated by agents that activate phospholipase C, adenylate cyclase, protein kinase C and calcium mobilization.

We report an efficient procedure for in situ hybridization with a multi-well format on Caenorhabditis elegans embryos for large scale screening of gene expression patterns in this organism. Each hybridization well contains embryos at various stages throughout embryogenesis. The validity of the method was confirmed through results with control genes whose expression patterns have been reported; glp-1 in very early embryos, myo-2 in pharyngeal muscle and unc-54 in body wall muscle. Several collagen genes and a pepsinogen gene were also examined to establish a set of lineage-specific markers. As a pilot project, we examined approximately 100 unique cDNA species classified by our cDNA project, finding that approximately 10% of the cDNA groups were expressed in specific cells and at specific stages.