Response of enteric luminous bacteria to environmental conditions in the gut of the fish

The response of luminous bacterial cultures to conditions encountered in the fish gut such as neutral pH, the presence of bile salts, gastric juice and lysozyme was examined. The organisms preferred neutral pH. Bile salts did not inhibit their growth. Neither lysozyme nor gastric juice affected their growth and viability to any extent. In the light of these findings, the adaptability of luminous bacteria to conditions existing in the gut of fish was discussed.     

Response of enteric luminous bacteria to environmental conditions in the gut of the fish

The response of luminous bacterial cultures to conditions encountered in the fish gut such as neutral pH, the presence of bile salts, gastric juice and lysozyme was examined. The organisms preferred neutral pH. Bile salts did not inhibit their growth. Neither lysozyme nor gastric juice affected their growth and viability to any extent. In the light of these findings, the adaptability of luminous bacteria to conditions existing in the gut of fish was discussed.     

Fenvalerate-induced chromosome aberrations and sister chromatid exchanges in the bone marrow cells of mice in vivo

To investigate whether subjects with low-acid states are exposed to increased genetic risk with respect to controls, we evaluated mutagenicity and presence of clastogenic factors (CF) in the gastric juice of chronic atrophic gastritis and omeprazole-treated patients. Mutagenic gastric juice was found in 8/15 (53%) chronic atrophic gastritis patients, 8/11 (73%) omeprazole-treated patients, and 2/13 (15%) healthy control subjects. The mean mutagenicity ratio of omeprazole-treated patients (1.52+/-0.48/0.1 ml gastric juice) was significantly higher than those of either controls (1.07+/-0.15; P<0.01) or chronic atrophic gastritis patients (1.16+/-0.21; P<0.05). Only chronic atrophic gastritis patients showed an increased clastogenic index with respect to healthy controls (2.67+/-2.13 versus 0.38+/-0.51; P<0.001). These findings expand our knowledge of gastric disease risk factors, and indicate that there may well be a risk of mucosal DNA damage arising from the presence of mutagenic and CF in the gastric juice.     

Rate constants studies of the dye-sensitized photoinactivation of lysozyme

Summary The rate constants for the photodynamic inactivation of hen egg-white lysozyme at different temperatures were studied. Arrhenius plots of the methylene blue sensitized photo-inactivation of lysozyme gave an experimental activation energy of 7.5 kcal/mol. The rate constants for the photodynamic inactivation of lysozyme in the presence of riboflavin decreased almost linearly in the temperature range 4–38° C. The photosensitized oxidation of lysozyme at –20° C in freezing and non-freezing solvents was possible only in the presence of riboflavin. The effect of dye concentration on the quantum yield and rate constant for the photodynamic inactivation of lysozyme was examined. The quantum yields were lower when the concentrations of methylene blue used were low, and increased on increasing dye concentration, getting to a maximum and then declined at higher dye concentrations. It was found that in the case of riboflavin sensitized photo-inactivation of lysozyme both the rate constant and the quantum yield increased as the dye concentration increased. No maximum was observed over the range of dye concentrations studied. A new mechanism is postulated for the photodynamic action of lysozyme in the presence of riboflavin.     

Characterisation of the barrier caused by luminally secreted gastro-intestinal proteolytic enzymes for two novel cystine-knot microproteins

It was the aim of this study to evaluate the stability of two novel cystine-knot microproteins (CKM) SE-ET-TP-020 and SE-MC-TR-020 with potential clinical relevance towards luminally secreted proteases of the gastrointestinal tract in order to gain information about their potential for oral administration. Therefore, the stability of the two CKM and the model-drug insulin towards collected porcine gastric and small intestinal juice as well as towards isolated proteolytic enzymes was evaluated under physiological conditions. No intact SE-ET-EP-020 was detected after few seconds of incubation with porcine small intestinal juice. SE-ET-TP-020 was also degraded in porcine gastric juice. Furthermore, SE-ET-TP-020 was extensively degraded by isolated chymotrypsin, trypsin and pepsin. Moreover, it was degraded by elastase. SE-MC-TR-020 was degraded entirely within approximately 2 h when incubated in porcine small intestinal juice, whereas no degradation was observed within a 3 h incubation period with porcine gastric juice. In presence of the isolated proteolytic enzymes, SE-MC-TR-020 was only slightly degraded by trypsin and pepsin, whereas elastase caused no degradation to SE-MC-TR-020 at all. Chymotrypsin was the protease that caused most degradation to SE-MC-TR-020. The model drug insulin was degraded extensively by chymotrypsin, elastase, pepsin and trypsin as well as by porcine gastric and porcine small intestinal juice. In conclusion, a precise characterisation of SE-ET-TP-020 and SE-MC-TR-020 degrading luminally secreted GI enzymes has been made, which is an important and substantial prerequisite for the further optimisation of these CKM.     

Effect of lysozyme and sodium EDTA on shrimp microflora

Summary The influence of lysozyme and salts on the growth of the microflora of shrimp was investigated. It was found that lysozyme at concentrations up to 150 g/ml could retard microbial growth in nutrient broth at 28°C. Growth of shrimp microflora was not affected much at low concentrations (0.05% and 0.1%) of EDTA but was totally inhibited in the presence of 0.5% Na₂EDTA. No growth was discernible using concentrations of 50 g/ml lysozyme and 0.02% Na₂EDTA, either in nutrient broth or in 2% shrimp homogenate.     

Bovine colostrum or milk as a serum substitute for the cultivation of a mouse hybridoma

A mouse-mouse hybridoma was grown in serum-free medium supplemented with bovine milk or colostrum. Bovine colostrum supported growth of the hybridoma whereas bovine milk alone did not support cellular proliferation. For growth in medium supplemented with colostrum, the maximum cell concentration achieved was 1.4 x 10(6) cells/mL in 2.2% colostrum, which is 44% of that obtained in 9% serum. When cells were grown in media containing milk and low amounts of serum (<1%) the maximum cell concentration in 2.2% milk with 0.4% serum was 2 x 10(6) cells/ml, whereas it was only 0.2 x 10(6) cells/ml and 1.3 x 10(6) cells/ml in 2.2% milk alone and 0.4% serum alone, respectively. Similar behavior was observed for growth in media containing colostrum and low amounts of serum. The monoclonal antibody production in media containing combinations of serum and milk or colostrum was comparable to that obtained in media with higher serum concentrations. Experiments performed with conditioned media suggest that the rapid decrease in viability, after the maximum cell concentration has been reached, is partially due to the presence of some inhibitory components generated during the cell culture rather than due to depletion of some serum components.     

Effect of inflammatory cytokines on human endothelial cell proliferation.

Neovascularization, a common occurrence in chronic inflammatory lesions, requires endothelial cell (EC) proliferation. Because this form of inflammation is often mediated by immunologically generated cytokines, the effects of such cytokines on human umbilical vein EC proliferation in vitro were investigated. Low concentrations of recombinant interferon gamma (rIFN-gamma) (10-100 U/ml), but not a higher concentration (1,000 U/ml), enhanced both basal and endothelial cell growth factor (ECGF)-stimulated EC proliferation. Recombinant interleukin 1 (rIL-1) and recombinant tumor necrosis factor-alpha (rTNF) had minor effects on basal EC proliferation, but significant inhibition was observed in the presence of ECGF. A combination of rIFN-gamma and rTNF induced marked suppression of EC proliferation, which appeared to be due to a cytotoxic effect on the EC, as demonstrated by 51Cr release. In contrast, the combination of rIFN-gamma and rIL-1 had only an additive effect on EC proliferation, with no evidence of cytotoxicity. These results suggest that cytokines have important regulatory roles in local vascular proliferation. These effects varied not only with the individual cytokine, but also with the combination of cytokines used. The most striking effects were 1) the stimulation of proliferation by IFN-gamma at a low concentration and 2) the inhibition by both rIL-1 and rTNF of ECGF-stimulated proliferation.     

Microflora of gastric juice and its sensitivity to antibiotics in post-gastrectomy gastritis

Fifty four patients with postresection gastritis and 13 healthy adults were examined. 141 microbial cultures belonging to 33 species were isolated from the gastric juice of the patients. In the healthy persons 27 cultures belonging to 7 species were isolated from the gastric juice. In the gastric juice of the patients there predominated enteric bacteria and enterococci (64.8 +/- 6.5 and 57.4 +/- 6.7 per cent respectively). Among the enteric bacteria Escherichia coli and Proteus spp. were the most frequent. Contamination of the gastric juice by such microbes amounted to 10(5)-10(9) microbial bodies per 1 ml. The isolates were mainly sensitive to gentamicin (84.8 +/- 2.9 per cent).     

The effect of grapefruit juice and seville orange juice on the pharmacokinetics of dextromethorphan: the role of gut CYP3A and P-glycoprotein

The objective of this study was to determine the effects of grapefruit juice and seville orange juice on dextromethorphan (DM) pharmacokinetics. Eleven healthy volunteers were studied over a 3-week period consisting of 5 study days each separated by a three-day washout. All subjects refrained from drinking caffeine containing beverages (coffee, soda, etc.) 8 h before orally taking DM (30 mg) with 200 ml water, 200 ml grapefruit juice, 200 ml water, 200 ml seville orange juice, and 200 ml water on Study Days 1 to 5. Aliquots of urine samples were assayed and analysed for DM, and the DM metabolites dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan using a validated HPLC method employing a phenyl column and a fluorescence detection. Results suggests that DM could provide some useful information on P-glycoprotein or related membrane efflux protein activity in the human gastro-intestinal tract. Bioavailability (F) of DM increased significantly with grapefruit and seville orange juice, but only returned to half the baseline value after three days of washout. This confirms that grapefruit and seville orange juice are long-lasting and perhaps irreversible inhibitors of gut CYP3A/P-glycoprotein. Grapefruit and seville orange juice appeared to have the same overall effect on DM pharmacokinetics. In addition, this paper presents a novel method of phenotyping for CYP2D6, CYP3A and P-glycoprotein using DM as a probe.     

Study on the repeated oral administration method in infant rats

The purpose of this study was to establish a new technique for repeated oral administration to infant rats. To determine the maximum volume which could be administered to infant rats the following amounts of the Chinese ink were given by metal gastric zonde for mice: 0.01, 0.04 and 0.08ml/g B. W. General conditions and the arrival distance of the Chinese ink in the gastro-intestinal tract were also observed in infant rats. The best way of administration to infant rats was decided as follows: infant rats were isolated from the dums for one hour before administration and held tenderly by their neck to sustain their mouth upward, then a metal gastric zonde for mice 2 cm long was inserted to their mouth and drug solution was injected slowly. From the observation of general conditions and pathological examination, the maximum volumes for single or repeated administration were considered to be 0.04ml/g B. W. and 0.01 ml/g B. W. respectively. Daily oral administration of 0.1ml/g B. W. of distilled water, 1% CMC solution or 1% tragacantha gum suspension for 44 days caused no effects in infant rats when administration was begun 4 days after birth. These results show that the new method for administration to infant rats is useful to evaluate the toxicity or pharmacological activity of drugs.     

Gastric juice neopterin in Helicobacter pylori infection

Abstract Neopterin, a pteridine compound produced by macrophages activated by interferon-gamma, is widely used to assess the activation of cellular immunity. An elevation in serum or urinary neopterin reflects immune activation in many different disorders, including viral infections, cancer, autoimmune diseases or acute myocardial infarction, but less attention has been paid to neopterin concentration in other biological fluids. The aim of the present study was to examine neopterin concentration in gastric juice. An association with the presence of Helicobacter pylori , a bacterium linked to the most common disorders of upper digestive tract, was also investigated. Gastric juice was obtained at endoscopy from 61 patients. Neopterin was determined by a radioimmunoassay and the presence of H. pylori was examined by urease test. The macroscopic finding of bile in gastric juice was associated with significantly higher neopterin levels compared to patients where no bile was noted (15.5 ± 15.6 vs. 2.1 ± 3.0 nmol/l, P < 0.001). However, similar concentrations were observed in the H. pylori positive and H. pylori negative patients (7.6 ± 12.0 vs. 11.1 ± 14.9 nmol/l). Even in the absence of macroscopic bile contamination, no significant difference could be found between the infected and uninfected patients (2.3 ± 3.2 vs. 1.3 ± 1.9 nmol/l), and the patients with duodenal ulcer and normal findings (3.8 ± 4.6 vs 1.6 ± 1.9 nmol/l). The contamination of gastric juice with bile represents the limitation for the use of neopterin as a marker of immune activation in the gastric mucosa. Rather than an index of immune activation, gastric juice neopterin concentration represents a marker of duodenogastric reflux.     

Hydrolysis of soybean trypsin inhibitor by the gamma subunit of 7SNGF and EGF-BP

Although soybean trypsin inhibitor (STI) does not inhibit the esterase activity of either epidermal growth factor binding protein (EGF BP) or the gamma subunit of 7SNGF, it does behave as a substrate for proteolysis. Cleavage of the active site peptide bond of STI does occur when incubated in the presence of either EGF-BP or the gamma subunit of 7SNGF. The hydrolysis id pH dependent with maximum proteolysis at pH 6.0-7.0. the newly formed C-terminal arginine residue in modified STI can be released by carboxypeptidase B digestion. Both enzymes are inhibited by low concentrations (2-4 microgram/ml) of the microbial protease inhibitors leupeptin and antipain. These inhibitors are specific for trypsin-like proteases. Since both enzymes can be found as part of high molecular weight complexes with growth factors these results confirm the hypothesis that they are involved during a postranslational modification event.     

Antimicrobial actions of degraded and native chitosan against spoilage organisms in laboratory media and foods

The objective of this study was to determine whether chitosan (poly-beta-1,4-glucosamine) and hydrolysates of chitosan can be used as novel preservatives in foods. Chitosan was hydrolyzed by using oxidative-reductive degradation, crude papaya latex, and lysozyme. Mild hydrolysis of chitosan resulted in improved microbial inactivation in saline and greater inhibition of growth of several spoilage yeasts in laboratory media, but highly degraded products of chitosan exhibited no antimicrobial activity. In pasteurized apple-elderflower juice stored at 7 degrees C, addition of 0.3 g of chitosan per liter eliminated yeasts entirely for the duration of the experiment (13 days), while the total counts and the lactic acid bacterial counts increased at a slower rate than they increased in the control. Addition of 0.3 or 1.0 g of chitosan per kg had no effect on the microbial flora of hummus, a chickpea dip; in the presence of 5.0 g of chitosan per kg, bacterial growth but not yeast growth was substantially reduced compared with growth in control dip stored at 7 degrees C for 6 days. Improved antimicrobial potency of chitosan hydrolysates like that observed in the saline and laboratory medium experiments was not observed in juice and dip experiments. We concluded that native chitosan has potential for use as a preservative in certain types of food but that the increase in antimicrobial activity that occurs following partial hydrolysis is too small to justify the extra processing involved.     

The development of the peripheral autonomic nervous system in relation to the gastro-intestinal tract

In this study the development of vagus nerves and the development of sympathetic nerves related to the development of the upper gastro-intestinal tract was studied in the rat from 12.5 days p.c. until birth by means of enzyme histochemical methods applied to sections and toto preparations. A striking time relation between the ingrowth of the vagus nerves as well as the sympathetic nerves and the appearance of acetylcholinesterase (AChE)-positive cells in the wall of the upper gastro-intestinal tract is established. The maturation of the upper enteric ganglion cells is dependent on the presence of a vagal influence. The vagal ingrowth of the stomach starts at about day 12.5 p.c. In AChE toto preparations it is established that the basic distribution pattern as described in the adult rat is complete at day 15 p.c. However, in contrast with the adult state, gastric branches can be traced up to the pylorus and the greater curvature until day 18 p.c. During the 18th day p.c. there is a tremendous increase in the surface area of the stomach. This increase in surface area of the gastric wall and relative decrease in vagal outgrowth leads to the adult configuration of vagal ramifications in relation to the wall of the stomach which can already be observed on the 19th day p.c.     

Antimicrobial Actions of Degraded and Native Chitosan against Spoilage Organisms in Laboratory Media and Foods

The objective of this study was to determine whether chitosan (poly-β-1,4-glucosamine) and hydrolysates of chitosan can be used as novel preservatives in foods. Chitosan was hydrolyzed by using oxidative-reductive degradation, crude papaya latex, and lysozyme. Mild hydrolysis of chitosan resulted in improved microbial inactivation in saline and greater inhibition of growth of several spoilage yeasts in laboratory media, but highly degraded products of chitosan exhibited no antimicrobial activity. In pasteurized apple-elderflower juice stored at 7°C, addition of 0.3 g of chitosan per liter eliminated yeasts entirely for the duration of the experiment (13 days), while the total counts and the lactic acid bacterial counts increased at a slower rate than they increased in the control. Addition of 0.3 or 1.0 g of chitosan per kg had no effect on the microbial flora of houmous, a chickpea dip; in the presence of 5.0 g of chitosan per kg, bacterial growth but not yeast growth was substantially reduced compared with growth in control dip stored at 7°C for 6 days. Improved antimicrobial potency of chitosan hydrolysates like that observed in the saline and laboratory medium experiments was not observed in juice and dip experiments. We concluded that native chitosan has potential for use as a preservative in certain types of food but that the increase in antimicrobial activity that occurs following partial hydrolysis is too small to justify the extra processing involved.     

Protein crystal growth. Growth kinetics for tetragonal lysozyme crystals

A method for immobilizing protein crystals has been devised for determining face growth rates, and used to investigate the growth kinetics of hen egg white lysozyme crystals. Growth rates were determined at 22 degrees C in 0.1 M sodium acetate, 5% NaCl, pH 4.0, on the visually identified (110) face of tetragonal lysozyme crystals. Protein concentrations ranged from 13 to 57 mg/ml (saturation concentration = 1.7 mg/ml). Growth rate data were fit to the equation R = kappa sigma ri, where R = rate in cm/s; kappa = constant; sigma i = solute growth interface supersaturation; and r = rate dependence upon super-saturation, with the result that kappa = 0.146 X 10(-8) cm/s and r = 2.0. A model of the growth process was developed and the experimental data were used to determine the relative roles of transport and interfacial kinetics in the growth of this crystal. Values for the width of the boundary layer delta, the interfacial concentration Ci, and growth rate R were determined. The model may be used to extrapolate to other growth conditions. The relative role of transport and interfacial kinetics can be expressed by the coefficient gamma = (CB - Ci)/(CB - Cs), when CB is the bulk concentration and Cs the saturation. Values for gamma were found to range from much less than 0.1 for submicron-size crystals to approximately 0.15 for cm sizes. The results indicate that attachment or surface effects are rate-limiting in lysozyme crystal growth in Earth's gravity because solutal convection always provides more transport of solute than can be accommodated by the interface. In order to grow such crystals under transport limiting conditions, it would be necessary to suppress this solutal convection.     

Intragastric ascorbic but not uric acid is depleted in relation with the increased pH in patients with atrophic body gastritis and H. pylori gastritis

Background. Helicobacter pylori gastritis induces reversible lowering of Ascorbic Acid (AA) intragastric concentrations. No studies have been aimed at determining the gastric juice AA concentration of atrophic body gastritis (ABG) patients. Uric Acid (UA), is another potent hydro‐soluble scavenger of ROS and its possible modification in the gastric juice of patients with H. pylori gastritis have never been investigated. This study was aimed at investigating the levels of AA and UA in the plasma and gastric juice of ABG patients, compared with H. pylori positive patients without corporal atrophy, and with healthy individuals. Materials and Methods. Thirteen ABG patients (Group 1): 32 Chronic non‐atrophic H. pylori gastritis patients (Group 2); and 13 healthy stomach controls (Group 3) attending gastroscopy with gastric biopsies (antrum = 3, corpus = 3) had plasma and intragastric levels of AA and UA measured. Results. Intragastric AA concentration was significantly lower in group 1 (median 0.21 µg/ml, range 0.1–24) compared both with groups 2 (median 5.5 µg/ml, range 0.1–33.2) (p = 0.043) and 3 (median 14.9 µg/ml, range 0.34–44.8) (p = 0.0028). Intragastric UA was not different between the three groups. Intragastric AA concentration resulted negatively correlated with the intragastric pH (Spearman r = ?0.47, p = 0.0003). In patients with gastritis (groups 1 and 2) there was a significant negative correlation between the sum of the Sydney Score variables in the body mucosa, and AA in the gastric juice (Spearman r = ?0.55; p = 0.0001). Conclusion. The study shows that intragastric pH is the key factor for the depletion of gastric juice AA observed in patients with corporal atrophy and to a lower extent with nonatrophic H. pylori gastritis.     

Motogenic effect of recombinant HGF on airway epithelial cells during the in vitro wound repair of the respiratory epithelium

Cell migration is the earliest mechanism involved in the wound repair process of the respiratory epithelium and could be potentially enhanced by growth factors. In the present work, we investigated the localisation of the hepatocyte growth factor (HGF) receptor (c-Met) during wound repair and evaluated the effect of recombinant HGF (rHGF) on cell migration by using an in vitro model of airway epithelial wound repair. By using immunohistochemical methods, we observed that the immunoreactivity of the c-Met proto-oncogene was increased in epithelial cells engaged in the process of tissue repair. The incubation of wounded cultures with increasing concentrations of rHGF (0.2, 2, 20, and 200 ng/ml) induced a significant (P < 0.02) dose-dependent effect on the wound repair index, with a maximum effect produced at 20 ng/ml (+31.3%). The cell migration speed reached 50.2 micrometer/h at this concentration, compared to 20.4 micrometer/h in the absence of rHGF. No significant effect on cell proliferation was observed in the repairing area in the presence of rHGF. These results suggest that rHGF is able to improve the wound repair process of the airway epithelium by increasing cell migration.     

Enterocin 012, a bacteriocin produced by Enterococcus gallinarum isolated from the intestinal tract of ostrich

Enterococcus gallinarum strain 012, isolated from the duodenum of ostrich, produced enterocin 012 which is active against Ent. faecalis, Lactobacillus acidophilus, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp., Clostridium perfringens, Pseudomonas aeruginosa and Salmonella typhimurium. One of the four pathogenic strains of Escherichia coli isolated from the intestinal tract of ostrich was inhibited by enterocin 012. No antimicrobial activity was recorded against Bacillus cereus, Cl. sporogenes, Cl. tyrobutyricum, Leuconostoc cremoris, Pediococcus pentosaceus, Staphylococcus carnosus and Streptococcus thermophilus. Enterocin 012 was resistant to treatment with lysozyme, catalase, lipase and papain, but sensitive to Proteinase K, alpha-chymotrypsin, trypsin and pepsin. Treatment of enterocin 012 with gastric juice from the duodenum resulted in a 50% loss of antibacterial activity. Half of the activity was lost when incubated at 80 degrees C for 30 min, or when kept overnight at a pH of 1.0-5.0 and pH 11.0 and 12.0, respectively. Enterocin 012 production started in mid-logarithmic growth and reached a maximum of 800 AU ml-1, but increased further to 1600 AU ml-1 in the stationary growth phase. The peptide is approximately 3.4 kDa in size, as determined after partial purification with Amberlite XAD-1180 and ammonium sulphate precipitation, followed by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mechanism of antimicrobial activity against Lact. sake LMG 13558 is bactericidal and caused cell lysis of active growing cells.