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Listericidal and nonlistericidal mouse macrophages differ in complement receptor type 3-mediated phagocytosis of L. monocytogenes and in preventing escape of the bacteria into the cytoplasm.

Listeria monocytogenes is a facultative intracellular bacterium that escapes phagocytic vesicles and replicates in the cytoplasm, where it becomes coated with F-actin. Macrophages, important anti-Listeria effector cells, are heterogeneous in their ability to kill Listeria. Complement receptor type 3 (CR3) mediates most phagocytosis of Listeria by listericidal macrophages. Experiments described here tested whether nonlistericidal macrophages also phagocytosed Listeria through CR3 and whether the ability of Listeria to escape into the cytoplasm correlated with lack of listericidal activity. We show here that CR3 mediated an average of 66% of the phagocytosis of serum-opsonized Listeria by listericidal peptone-elicited macrophages but only 35% by nonlistericidal thioglycolate-elicited macrophages. In thioglycolate-elicited macrophages, most Listeria were cytoplasmic and actin coated, whereas in peptone-elicited macrophages most were retained in the phagosome. These results indicate that listericidal and nonlistericidal macrophages phagocytose Listeria through different receptors and that nonlistericidal macrophages allow Listeria to escape into the cytoplasm.

Actins↗

Extracellular chitinase production by Trichoderma harzianum in submerged fermentation.

Extra-cellular chitinase production by a chitinolytic fungus Trichoderma harzianum TUBF 966 using submerged fermentation was studied. Colloidal chitin (1.5% w/v) was used as sole carbon source. Maximum chitinase production (14.7 U/ml) was obtained when fermentation was carried out at 30 degrees C for 96 h using 72 h old mycelium in a medium containing colloidal chitin 1.5% (w/v) as carbon source and 0.42 (% w/v) peptone as nitrogen source (pH 5.5). Supplementation of additional carbon sources (0.75% w/v) showed no further enhancement in chitinase production while supplementation of nitrogen sources (0.42% w/v) such as peptone and tryptone in the fermentation medium showed a marked increase in production. The process parameters that controlled chitinase production by the fungus were studied and presented here.

Carbon↗

Effect of medium treatment on microbial degradation of sterols by Nocardia.

The rate of transformation of cholesterol by Nocardia sp. is affected by the culture medium composition as well as by the way it has been treated before inoculation. When 1% of peptone was sterilized separately and afterwards added to the basic medium, the transformation of cholesterol to bisnorchola-1,4-dienic acid, as well as further aerobic degradation, was accelerated by a factor of 2. If this enriched medium was kept at rest overnight, the rate of transformation was lowered to half. Heating of additional peptone together with a basic medium resulted only in a slight diminution of the transformation of the preceding 4-ene metabolite to the final 1,4-diene when compared with a normal basic medium without any additive.

Aerobiosis↗

Observations on the effect of the diluent used for diluting challenge toxin in the Clostridium botulinum potency test.

Clostridium botulinum vaccines were assayed for potency according to the method described in the European Pharmacopoeia. This method uses 20 white mice which are vaccinated with a dilution of vaccine in 0.9% saline. Twenty-one days later the vaccinated mice and ten unvaccinated control mice are challenged with C. botulinum toxin and observed for 7 days. In this study the challenge toxin was diluted in two separate diluents, 0.9% saline and peptone broth. More reproducible results were achieved with peptone broth as the diluent.

Animals↗

Growth of Nitrobacter in the presence of organic matter. I. Mixotrophic growth.

1. Culture filtrates of heterotrophic bacteria were tested for their stimulatory effect on nitrification of three strains of Nitrobacter. 2. Yeast extract-peptone solution, in which Pseudomonas fluorescens had grown, after removal of the cells was added to autotrophically growing cultures of Nitrobacter agilis; it caused a stimulated nitrite oxidation and growth of Nitrobacter agilis. 3. The degree of stimulation depended on: a) the proportion of the culture filtrate to the autotrophic medium; b) the composition of the complex medium in which Pseudomonas fluorescens had been grown; c) the time the heterotrophic bacterium had been grown in the complex medium. 4. The stimulatory effect was highest with Nitrobacter agilis, less with Nitrobacter winogradskyi and negligible with Nitrobacter K4. 5. It was possible to adapt nutrifying cells of Nitrobacter agilis to higher concentrations of yeast extract and peptone. After the nitrite had been completely oxidized the cell-N still increased up to 30% before growth stopped.

Culture Media↗

Comparison of vasoactive intestinal peptide (VIP) and secretin in gastric secretion and mucosal blood flow.

VIP and secretin were compared in regard to their effects on gastric acid and pepsin secretion induced by pentagastrin histamine or a peptone meal as well as on gastric mucosal blood flow and meal induced serum gastrin level in conscious dogs provided with gastric fistulas and denervated fundic pouches. Both VIP and secretin caused a dose-related stimulation of basal pepsin outputs and inhibition of pentagastrin-induced acid secretion. VIP, like secretin, inhibited pentagastrin and meal-induced gastric acid secretion but in contrast to secretin it caused inhibition of acid response to histamine. Inhibition of acid secretion by VIP or secretin was accompanied by secondary reduction in gastric mucosal blood flow in tests with pentagastrin or histamine and by depression of the serum gastrin level in tests with a peptone meal. This study indicates that in comparison with secretin, VIP has a wider spectrum of inhibition of stimulated gastric secretion and may be considered as one of the enterogastrones released in the small intestine.

Aminopyrine↗

Effect of low-dose exogenous secretin on pentagastrin- and meal-stimulated gastric acid secretion in humans.

Intravenous infusion of secretin in a dose of 0.05 CU/kg/hr inhibited pentagastrin-stimulated (100 ng/kg/hr) acid secretion by 42% (P less than 0.05) abd meal-stimulated (10% peptone, pH 5.5) acid secretion by 33% (P less than 0.05) in 10 healthy subjects. Median serum gastrin concentration during peptone stimulation was reduced by 24% (P less than 0.05) during secretin infusion. Median plasma secretin concentrations were 6.0 and 5.2 pmol/liter, respectively. Since these secretin concentrations are of the same magnitude as those seen after duodenal acidification, it is concluded that secretin may participate in the physiological inhibition of gastric acid secretion.

Adult↗

Factors influencing the intestinal phase of pancreatic exocrine secretion in the turkey.

The present study was done to investigate the factors regulating the intestinal phase of exocrine pancreatic secretion in the turkey. The intestine of turkeys equipped with pancreatic fistulas was perfused with peptone solution, fat emulsion and hydrochloric acid (HCl), and pancreatic flow and protein output were measured. Neither peptone solution nor fat emulsion had any effects on pancreatic secretion. HCl enhanced the flow rate of pancreatic juice but not protein output. To clarify the neural mechanism of this phenomenon, the vagal postganglionic blocker atropine was continuously infused and pancreatic secretion in response to intestinal HCl was measured. Atropine completely suppressed both pancreatic flow and protein output. It is suggested that the avian intestinal phase of pancreatic secretion is mainly controlled by cholinergic action though HCl stimulation.

Animals↗

Effect of rice--glycerol complex medium on the production of Lovastatin by Monascus ruber.

Response surface methodology (RSM) was employed to study the effect of the composition of the rice-glycerol complex medium on the production of lovastatin (Lvs) by the ascomycete Monascus ruber in mixed solid-liquid (or submerged) cultures at 25 degrees C. Four components (rice powder, peptone, glycerol, glucose) were studied to evaluate the approximate polynomial for all dependent variables, explaining their effects on the production of Lvs. The best composition derived from RSM regression was (in g/L) rice powder 34.4, peptone 10.8,, glucose 129, KNO3 8.0, MgSO4.7H2O 4.0 and glycerol 36.4 mL/L. With this composition, the Lvs production was 157 mg/L after 10 d of cultivation. In comparison with glycerol and glucose, the rice powder becomes a more suitable carbon source and represents a great potential for the production of Lvs.

Glucose↗

Production and induction of manganese peroxidase isozymes in a white-rot fungus Pleurotus ostreatus.

The production of MnP by Pleurotus ostreatus in different liquid cultures was investigated. The highest level of activity was observed after 8 days of culture in peptone-glucose-yeast extract medium (PGY), whereas maximal activity was achieved after 30 days in glucose-yeast extract medium (GY). MnP was purified to homogeneity from PGY (designated MnP-PGY) and GY (MnP-GY). The isoelectric points of MnP-PGY and MnP-GY were 3.77 and 4.06, respectively. The molecular mass of both enzymes was 42 kDa. Analysis of the N-terminal amino acid sequence of purified MnPs and nucleotide sequence of cloned mnp indicated that MnP-GY has VTCATGQTTANE at the N-terminus, whereas MnP-PGY has ATCADGRTTANA. A putative exposed tryptophan residue (W170) was found in MnP-GY. Both isozymes oxidized veratryl alcohol, although the K(m) of MnP-GY was lower than that of MnP-PGY. Thus, the presence of peptone in the medium affected the production of MnP isozymes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that the synthesis of MnP isozymes is controlled by culture conditions at the transcriptional level.

Amino Acid Sequence↗

Optimization of alkaline protease production from Bacillus sp. by response surface methodology.

High yields (1939 U/ml) of an alkaline protease were obtained in batch fermentation of a Bacillus sp. using a response surface methodology. The interaction of four variables, viz., starch, peptone, incubation time, and inoculum density, suggested inoculum density to be an insignificant variable. However, incubation time had a profound effect on protease yields at all the concentrations of carbon and nitrogen used. The response surface raised and flattened with increase in time of incubation, and maximum protease production up to 1939 U/ml was obtained after 96 h of incubation. The model equation obtained was validated experimentally at maximum starch (15 mg/ml) and peptone (7.5 mg/ml) concentration with increased incubation time up to 144 h in the presence of minimum inoculum density (1%). An overall 2.6-fold increase in protease production was obtained as compared with mean observed response (750 U/ml) at zero level of all variables.

Analysis of Variance↗

Isolation and identification of hyper-ammonia producing bacteria from swine manure storage pits.

Storage of swine manure is associated with the microbiological production of a variety of odorous compounds, including ammonia, organic acids, and alcohols, phenolics, and sulfides. Until recently, little was known about the microorganisms responsible for their production. Results from our laboratory have demonstrated that the predominant microbial populations of stored swine manure are anaerobic, low (G + C), Gram-positive bacteria. However, studies on pure cultures isolated from manure have found few microorganisms that produce appreciable ammonia concentrations. Therefore, selective and enrichment techniques were employed to isolate ammonia-producing bacteria from stored swine manure by using media containing peptone and amino acids as carbon and energy sources. We now report on the isolation of 40 bacterial cultures, a number of which are capable of producing at least 40 mM ammonia in peptone-amino acid medium, concentrations similar to those produced by hyper-ammonia producing (HAP) bacteria isolated from the rumen of cattle. The manure HAP isolates are phylogenetically distinct from the ruminal isolates and may prove to be intimately involved in the production of ammonia during storage of swine manure.

Actinobacteria↗

Proteases production by two Vibrio species on residuals marine media.

A comparative study was carried out on the growth and production of alkaline proteases by two Vibrio species using different marine peptones from fish viscera residues. The bacteria tested, Vibrio anguillarum and Vibrio splendidus, are producers of high levels of proteolytic enzymes which act as factors of virulence in fish cultures, causing high mortality rates. The kinetic assays and subsequent comparison with the parameters obtained from the adjustment to various mathematical models, highlighted the potential interest of the media formulated, for their possible production on an industrial scale, particularly the production of proteases by V. anguillarum growing in rainbow trout and squid peptones.

Alkalies↗

Gastric acidification inhibits meal-stimulated gastric acid secretion after prostaglandin synthesis inhibition by indomethacin in humans.

The effects of cyclooxygenase inhibition by indomethacin on gastric acid secretion were studied in 8 healthy men. Oral doses of indomethacin (200 mg), administered 15 and 2 h before testing, were known to inhibit prostaglandin synthesis by 90% in 3 of the subjects as determined by prostaglandin E2 generation assay on endoscopically obtained gastric mucosal biopsy specimens. Acid-induced inhibition of gastric acid secretion was evaluated in a randomized and blinded study in which acid output was measured for 2 h during basal conditions by aspiration, for the next 2 h by intragastric titration during distention with isotonic glucose, and for the following 2 h by intragastric titration during meal stimulation with peptone. The studies were done on separate days, and intragastric pH was maintained at either 2.5 or 5.5 after administration of indomethacin or placebo. Basal acid output was not altered by indomethacin treatment. Distention of the stomach stimulated acid output significantly to a similar degree in all groups, without affecting plasma gastrin. Meal stimulation increased plasma gastrin and acid output significantly more at pH 5.5 (47 +/- 12 pM, 13 +/- 2 mmol/30 min) than at pH 2.5 (30 +/- 8 pM, 6 +/- 2 mmol/30 min). No effect of indomethacin treatment was observed. It is concluded that the participation of cyclooxygenase products in the mechanisms by which acid inhibits the gastric phase of acid secretion in humans is likely to be minor. These results also cast doubt on an important physiologic role for cyclooxygenase products in the regulation of basal acid secretion or of acid secretion stimulated by distention or a peptone meal.

Adult↗

Gastrin is a major mediator of the gastric phase of acid secretion in dogs: proof by monoclonal antibody neutralization.

We developed a monoclonal antibody, 28.2, that binds specifically to the amidated carboxyl terminal region common to gastrin and cholecystokinin. This immunoglobulin G1 antibody has high affinity (ID50 = 30-70 pM for gastrin and cholecystokinin peptides), binds labeled gastrin similarly at 37 degrees C and 4 degrees C, and shows minimal inhibition of binding in the presence of 40% canine serum. Antibody 28.2 was used to carry out in vivo immunoneutralization studies in 8 dogs previously prepared with chronic gastric fistulas. Preliminary studies revealed that a single intravenous dose of 0.75 mg of partially purified immunoglobulin G of monoclonal antibody 28.2 completely inhibited the acid stimulatory effect of exogenous gastrin-17 given intravenously at 200 pmol/kg.h, a physiologic dose, and inhibited by 70% the acid response to a supraphysiologic dose, 800 pmol/kg.h. The same dose of antibody decreased the acid secretory response obtained during distention of the stomach with 300 ml of 5.8% glucose solution by 98% and decreased the response to distention with 300 ml of 8% peptone solution by 68%. A 10-fold higher dose of antibody decreased the acid response to peptone by 96%. The gastrin antibody had no effect on the acid response to exogenous histamine. A control antibody, specific for the biologically inactive glycine-extended gastrin/cholecystokinin peptapeptide region, had no significant effect on gastric acid secretion stimulated by gastrin or by gastric distention with nutrients. These studies indicate that circulating gastrin is of major importance in the gastric phase of gastric acid stimulation caused by distention of the stomach with nutrients.

Animals↗

Intestinal fat does not inhibit gastric function through a hormonal somatostatin mechanism in dogs.

In awake dogs with chronic gastric, duodenal, and jejunal fistulas, F(ab)1 fragments of somatostatin monoclonal antibody (mAb S607) were administered intravenously (IV) to test the hypothesis that intraintestinal lipid inhibits peptone-stimulated gastric acid secretion and emptying by a hormonal somatostatin mechanism. Plasma somatostatin was increased significantly by duodenal and jejunal perfusion with 20% lipid. Somatostatin administered IV caused dose-dependent inhibition of meal-stimulated gastric acid secretion and gastric emptying similar to that seen after intestinal perfusion with lipid. Administration of mAb S607 F(ab)1 fragments significantly reversed somatostatin (400 pmol.kg-1.h-1, IV)-induced inhibition of peptone-stimulated acid output and gastric emptying. Acid output inhibited by intraduodenal lipid was reversed partially after F(ab)1 administration, but the inhibitory effect of intrajejunal lipid was not altered. Inhibition of acid secretion by IV somatostatin and by intraintestinal fat was not caused by a decrease in circulating gastrin concentrations. Gastric emptying delayed by intraintestinal lipid was unaffected by antibody administration. Somatostatin does not appear to be a major hormonal mediator of intestinal fat-induced inhibition of gastric acid secretion or delayed gastric emptying in dogs.

Animals↗

Cholecystokinin receptor antagonist MK-329 blocks intestinal fat-induced inhibition of meal-stimulated gastric acid secretion.

MK-329, a selective type A cholecystokinin (CCK) receptor antagonist, was given to dogs to test the hypothesis that CCK is one of the principal physiological enterogastrones mediating fat-induced decreases in gastric acid secretion. Gastric acid secretion in response to 300 mL 8% peptone meals was measured by intragastric titration to pH 5.5 in six awake dogs with chronic gastric, duodenal, and jejunal fistulas. Gastric emptying was measured by a dye-dilution technique. During the last hour of peptone stimulation, the intestine was perfused with either control solution or 20% lipid (Intralipid; Kabi Vitrum, Alamedo, CA) intraduodenally or intrajejunally. Compared with control perfusions, mean gastric acid outputs were decreased significantly after lipid perfusion of the duodenum (47% of control) and jejunum (24% of control). Similarly, mean gastric emptying rates were significantly less after lipid perfusion of the duodenum (56%) and jejunum (26%). Oral pretreatment with MK-329 (1 mg/kg) significantly reversed the inhibition of gastric acid output caused by lipid perfusion of the duodenum and jejunum, but fat-induced inhibition of gastric emptying was not significantly affected. These studies provide evidence for an important inhibitory role for CCK as an enterogastrone but do not implicate CCK as being important in fat-induced delayed gastric emptying of a liquid meal in dogs.

Animals↗

Validation of the antral mucosal/submucosal sleeve preparation: studies of gastrin and acetylcholine release in response to luminal stimulation.

In the present study we developed an experimental model for direct assessment of antral endocrine cell and cholinergic neural responses to luminal stimulation. A sleeve of antral mucosal/submucosal tissue was prepared from rat antrum, mounted in perfusion chamber, and perfused in both luminal and submucosal compartments. Morphological and functional integrity of the antral sleeve were confirmed by histological examination and measurement of protein synthesis. Antral gastrin release was assessed in response to luminal stimulation with acid, peptone and distension. Luminal acid (pH3) inhibited basal gastrin release by -70.4% and luminal peptone stimulated gastrin release to 210% above control (p < 0.02). Distention of the antral sleeve by hydrostatic pressure (3-25cm H2O) caused stepwise and significant increase in gastrin release that was reversible. 3H-acetylcholine was stimulated significantly by KCl (56mM) to values twice control. In summary, these results establish the integrity and responsiveness of the antral sleeve to pharmacological and luminal stimulation. The antral sleeve may be a useful model in assessing antral function in response to luminal stimulation.

Acetylcholine↗