Studies of saturated absorption and measurements of optical frequency for lines in the nu1 + nu3 and nu1 + 2nu4 bands of ammonia at 1.5 microm.

A cavity-enhanced absorption spectrometer was used to saturate several lines of ammonia in the 1510 nm - 1560 nm region. Analysis of power broadening of the saturated absorption feature for one of the ammonia lines yielded a dipole moment value comparable to that of the lines in the nu(1)+nu(3) band in acetylene. Highly reproducible frequency measurements of four ammonia line centres were carried out using a frequency comb generated by a mode-locked Cr(4+):YAG laser. These results demonstrate the possible application of ammonia saturated absorption lines for frequency metrology and calibration in a spectral region lacking strong absorbers. To our knowledge, this is the first frequency measurement of saturated absorption lines in ammonia at near infrared frequencies and the first reported observation of saturated absorption lines in the nu(1)+2nu(4) band.

Ultrashort pulse Cr4+:YAG laser for high precision infrared frequency interval measurements.

A cavity stabilized, SESAM mode-locked Cr4+:YAG laser capable of generating sub-100 fs pulses has been developed. Locking the 130-MHz pulse repetition frequency to that of a hydrogen maser-referenced frequency synthesizer provides a 30-nm wide frequency comb for the 1530-nm wavelength region. In conjunction with a pair of acetylene stabilized, external cavity diode lasers, this laser provides a high precision measurement tool for the determination of acetylene transition frequencies.

Comparison of the efficacy and safety of ebrotidine in the treatment of duodenal ulcer. A multicentre, double-blind, placebo-controlled phase II study.

Ebrotidine (N-[(E)-[[2-[[[2-[(diaminomethylene)amino]-4 -thiazoly]methyl]thio]ethyl]amino]methylene]-4-bromo-benzenesulfon amide, CAS 100981-43-9, FI-3542) is a new H2-receptor antagonist characterized by its high receptor affinity and gastroprotective effect. This Phase II study has been undertaken to establish the efficacy and safety of ebrotidine, administered in four dosages as a single evening dose versus placebo in the treatment of duodenal ulcer. A total of 110 duodenal ulcer patients were studied in a randomized, double-blind, placebo-controlled, multicentre clinical trial. The patients were assigned to 5 groups: placebo, 200 mg, 400 mg, 600 mg and 800 mg of ebrotidine once daily. Controls were performed at baseline and every two weeks at four follow-up visits unless ulcer healed before. Endoscopic examination was the main parameter for the assessment of treatment efficacy and ulcer healing rate. Vital signs and blood/ urine analysis were used to establish safety. The three groups treated with higher dosages (400 to 800 mg of ebrotidine daily) showed an endoscopic ulcer healing rate of 90-95%, significantly higher than 55% achieved with placebo (p < 0.05), whilst the differences between these three dosages of ebrotidine were not statistically significant. Healing rate in the group treated with 200 mg of ebrotidine daily was not significantly different from that in the placebo group. The development of symptoms, number of episodes of ulcer-related pain, total ulcerated surface area or subjective ratings by the patients and investigators also differed significantly between ebrotidine (400, 600 and 800 mg daily) and placebo, and again, no marked differences were found between these three doses of ebrotidine. As far as tolerance is concerned, no clinically or statistically significant changes were observed in vital signs and analytical parameters. The incidence of side effects was less than that presented by the placebo group, possibly due to a greater consumption of antacids in this group. Results showed that a daily dose of 400 mg ebrotidine is effective and safe in the treatment of duodenal ulcers.

Enhancement in the protective qualities of gastric mucus by ebrotidine during duodenal ulcer healing.

1. Gastric mucus from duodenal ulcer patients before and following therapy with a new antiulcer agent, ebrotidine, at 400, 600 and 800 mg dose was examined for changes in the physicochemical qualities and anti-H. pylori activity. 2. The results of physical measurements revealed that successful therapy with ebrotidine was accompanied by a 25% increase in gastric mucus viscosity, and a 20% increase in H+ retardation capacity, while its hydrophobicity increased by 11%. 3. The enhancement in the physical properties of mucus with ebrotidine therapy were also reflected in a marked (2.6-2.9-fold) increase in the proportion of the high molecular weight form of mucin. Furthermore, following therapy with ebrotidine, the gastric mucins showed a 36% higher content of sulfate as compared to that before the therapy. 4. Assays on the H. pylori aggregating titer of gastric mucin revealed that ebrotidine therapy at all three doses evoked a 4-fold increase in mucin anti-H. pylori activity. 5. The data demonstrate that duodenal ulcer therapy with ebrotidine leads to a marked improvement in the protective qualities of gastric mucus essential for the maintenance of mucosal integrity and enhances the inherent mucosal defense against H. pylori infection.

Inhibition of gastric mucosal mucin receptor by Helicobacter pylori lipopolysaccharide: effect of sulglycotide.

1. A receptor for mucin was isolated from the solubilized gastric epithelial cell membrane by affinity chromatography on Sepharose-bound wheat germ agglutinin. 2. The receptor protein displayed a molecular weight of 97 kDa and exhibited specific affinity towards mucin-coated surfaces. The optimum for mucin binding occurred at 60-100 micrograms/ml, while the values for the receptor were 2.0-3.1 micrograms/ml. 3. The mucin binding to the receptor was susceptible to Helicobacter pylori lipopolysaccharide which caused maximum inhibition of 91% at 30 mu/ml. This inhibitory effect of the lipopolysaccharide was abolished by a gastroprotective agent, sulglycotide. 4. The effect of sulglycotide was dose dependent and at 50 micrograms/ml produced a 94% restoration in receptor-mucin binding. Furthermore, sulglycotide was also capable of enhancing (97%) the mucin binding to its receptor in the absence of the lipopolysaccharide. 5. The results demonstrate that H. pylori through its lipopolysaccharide interferes in the interaction of mucin with gastric epithelial surfaces and that a gastroprotective agent, sulglycotide, counteracts this effect, and hence is capable of preventing the loss of mucin coat continuity occurring with H. pylori infection.

Gastric mucosal EGF and PDGF receptor expression with ulcer healing by ebrotidine.

OBJECTIVES: Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) play important roles in the process of mucosal repair and restitution, and their biological effects are mediated by receptors located on the target cell surfaces. The purpose of this study was to assess the effect of the antiulcer agent, ebrotidine, on the expression of EGF and PDGF receptors with chronic ulcer healing. METHODS: Chronic gastric ulcers were developed in the rat by acetic acid technique. The animal were divided into two groups and were treated twice daily for 14 consecutive days, either with ebrotidine at 100 mg/kg, or placebo. At different stages of treatment, the animals were sacrificed and used for the isolation and quantification of gastric mucosal EGF and PDGF receptors. RESULTS: The binding assays revealed that ulcer healing was accompanied by an increase in mucosal expression of both types of receptors. A 1.7-1.8-fold increase in PDGF and EGF receptors occurred by the 4th day after the development of ulcer and reached a maximum of 3-fold increase by the 14th day, when the ulcer was essentially healed. Treatment with ebrotidine caused accelerated ulcer healing (7 days) which was accompanied by a significant enhancement in receptor expression. Compared to the controls, a 1.5-fold increase in EGF and 1.7-fold increase in PDGF receptor expression occurred by the 7th day of ebrotidine treatment, and a 1.4- to 1.5-fold increase was still observed at the 14th day of treatment. CONCLUSIONS: The results suggest that ebrotidine is capable of enhancement of gastric mucosal proliferative activities associated with ulcer healing through the stimulation of EGF and PDGF receptor expression.

Gastric mucosal laminin receptor expression with ulcer healing by ebrotidine.

1. The effect of antiulcer agent, ebrotidine, on the expression of gastric mucosal laminin receptor during ulcer healing was investigated. 2. Rats with acetic acid-induced chronic gastric ulcers were treated twice daily for 14 consecutive days either with ebrotidine at 100 mg/kg or placebo, and then at different stages of treatment used for the isolation and quantitation of gastric mucosal laminin receptor. 3. The binding assays revealed that the ulcer healing was accompanied by an increase in mucosal expression of laminin receptor. A 2.7-fold increase in the receptor expression occurred by 4th day following the development of ulcer and reached a maximum of 8.6-fold increase by the 14th day when the ulcer was essentially healed. 4. Treatment with ebrotidine caused accelerated ulcer healing (7 days), which was accompanied by a remarkable enhancement in the laminin receptor expression. A 2.5-fold increase in the receptor expression over that of controls occurred by the 4th day of ebrotidine treatment, and a 1.7-fold increase was still observed at the 14th day of treatment. 5. The results suggest that ebrotidine, by evoking enhanced mucosal cell laminin receptor expression, promotes reepithelization and, hence, hastens the ulcer healing.

Enhancement in gastric mucosal epidermal growth factor receptor expression by sulglycotide.

The effect of intragastric administration of sulglycotide, a cytoprotective sulfated glycopeptide, on the expression of gastric mucosal epidermal growth factor receptor was investigated. The experiments were conducted with groups of rats, one receiving twice daily for 5 consecutive days a dose of 200mg/kg sulglycotide, and the other only vehicle. Mucosal cell membranes were isolated from the stomachs at 16, 40 and 88h after the last dose, and used for EGF receptor assays. The binding assays revealed a marked increase in mucosal EGF receptor expression with sulglycotide. Compared to the controls, the sulglycotide-treated group showed a 4-fold increase in the EGF receptor expression at 16h after the last dose of sulglycotide, a 4.7-fold increase in the EGF receptor was observed by the 40h, and a 4.2-fold increase was still evident at 88h following the treatment. The results demonstrate that sulglycotide exhibits remarkable ability to enhance the gastric mucosal expression of EGF receptor.

Association of salivary bacterial aggregating activity with sulfomucin.

The requirements of human salivary mucins for aggregating potential towards the common cariogenic oral bacteria, S. mutans and S. sanguis, were investigated. Agglutination inhibition assays demonstrated that the aggregating capacity towards bacteria resides in the acidic mucin fraction. The inhibitory activity of the acidic mucin decreased only 2-4-fold following removal of sialic acid, whereas the desulfation caused a complete loss of the inhibitory potential against both bacteria. Furthermore, the aggregating capacity of mucin-derived sulfated oligosaccharide was found to be 16-fold higher than that of the sialic acid containing oligosaccharide. The results point towards the importance of salivary sulfomucins as a predominant factor in the defense of oral cavity against cariogenic bacteria.

Regulation of gastric mucosal calcium channel activity by an antiulcer agent, ebrotidine.

Ebrotidine is a new H2-receptor antagonist also known for its gastroprotective effect against ethanol-induced mucosal injury. In this study, we investigated the effect of ebrotidine on the activity of the gastric mucosal calcium channels. The channel complex was isolated from the solubilized gastric epithelial cell membranes by affinity chromatography on wheat germ agglutinin. The complex following labeling with [3H] PN200-110 was reconstituted into phosphatidylcholine vesicles which exhibited active 45Ca2+ uptake into intravesicular space and responded in a concentration-dependent manner to calcium channel activator, BAY K8644, as well as to calcium channel antagonist, PN200-100. The 45Ca2+ uptake was inhibited by ebrotidine which caused maximum inhibitory effect of 54.9% at 50 micrograms/ml. The gastric mucosal calcium channels on epidermal growth factor binding (EGF) in the presence of ATP responded by an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the vesicles containing the phosphorylated channels displayed a 48% greater 45Ca2+ uptake. This phosphorylation process was inhibited by ebrotidine which also interfered with the binding of EGF to calcium channel protein. The results point towards the importance of EGF in the maintenance of gastric mucosal calcium homeostasis, and suggest that ebrotidine has the ability to protect the cellular integrity from calcium imbalance by modulating the EGF-stimulated gastic mucosal calcium channel phosphorylation.

Sulglycotide effect on the proteolytic and lipolytic activities of Helicobacter pylori toward gastric mucus.

OBJECTIVES: Infection with Helicobacter pylori is now recognized as a major factor in the etiology of gastric disease, and among the detrimental effects this bacterium exerts on the mucosal integrity is the elaboration of extracellular protease and lipase enzymes capable of mucus protein and lipids degradation. We present here evidence that the activities of these enzymes are inhibited by an gastroprotective agent, sulglycotide. METHODS: The grown colonies of bacterium were washed with saline, filtered through sterilization filter, and the filtrate used as the enzyme source. RESULTS: In the absence of sulglycotide, the H. pylori protease caused extensive degradation of human gastric mucus, while free fatty acids, glycerol monooleate and lysophosphatidylcholine were produced by the action of H. pylori lipase and phospholipase A enzymes. Introduction of sulglycotide to the incubation systems led to the reduction in the rate of mucus protein and lipid degradation. The rate of proteolysis inhibition was proportional to sulglycotide concentration up to 45 micrograms/ml, at which point a 43% reduction in mucus degradation was attained, whereas the maximum inhibition of lipase (39%) and phospholipase A (98%) activities occurred at a sulglycotide concentration of 100 micrograms/ml. CONCLUSIONS: This study indicates that sulglycotide is capable of counteracting the mucolytic activity of H. pylori, and thus may be of value in the therapy of H. pylori-associated gastric diseases.

Inhibition of Helicobacter pylori glycosulfatase activity towards human gastric sulfomucin by a gastroprotective agent, sulglycotide.

1. A glycosulfatase activity towards human gastric sulfomucin was identified in the extracellular material elaborated by Helicobacter pylori, a pathogen implicated in the etiology of gastric disease. 2. The purified enzyme displayed an apparent molecular weight of 30 kDa, and exhibited maximum activity at pH 5.7 in the presence of 0.3% Triton X-100 and 100 mM CaCl2. 3. The H. pylori glycosulfatase activity towards human gastric sulfomucin was inhibited by a gastroprotective agent, sulglycotide. The inhibitory effect was proportional to the concentration of sulglycotide up to 20 micrograms/ml, at which a 98% decrease in mucin desulfation occurred. However, the drug lost the inhibitory effect following its chemical desulfation. 4. The results demonstrate that sulglycotide is a potent inhibitor of H. pylori glycosulfatase and, hence, may be of value in the treatment of gastric disease associated with this bacterial infection.

Helicobacter pylori lipopolysaccharide inhibition of gastric mucosal laminin receptor: effect of sulglycotide.

1. The effect of cell-wall lipopolysaccharide from Helicobacter pylori, a bacterium implicated in the etiology of gastric disease, on the gastric mucosal laminin-receptor was investigated. 2. The receptor, isolated from gastric epithelial cell membrane by affinity chromatography on laminin-coupled Sepharose, was radioiodinated and incorporated into liposomes which exhibited specific affinity towards laminin-coated surface. 3. The binding of liposomal receptor to laminin-coated surface was inhibited by H. pylori lipopolysaccharide, which at 50 micrograms/ml caused a nearly complete (97%) inhibition in binding. 4. The inhibitory effect of the lipopolysaccharide was prevented by a cytoprotective agent, sulglycotide, that evoked a 92% restoration in binding at 40 micrograms/ml. 5. The results demonstrate that through its lipopolysaccharide H. pylori is capable of disrupting the gastric mucosal integrity and that this detrimental effect could be successfully countered by sulglycotide.

Characterization of gastric mucosal mucin receptor.

Mucin receptor was isolated from gastric epithelial cell membrane by a procedure involving membrane solubilization with octylglucoside followed by affinity chromatography on Sepharose-bound wheat germ agglutinin. The receptor protein yielded on SDS-PAGE a single 97kDa band and displayed specific affinity, in a concentration-dependent manner, towards the mucin-coated surface. The receptor showed requirement for carbohydrate chains in mucin for binding, as their removal caused a marked (87%) reduction in binding capacity. Scatchard analysis revealed a linear plot with a single class of high affinity binding (Kd = 43.8 nM; Bmax = 140 pmol/mg protein) sites. The results demonstrate for the first time the presence in gastric mucosa of a specific receptor for mucin.

Regulation of buccal mucosal calcium channel activity by salivary mucins.

1. The effect salivary mucins on the activity of calcium channel isolated from buccal mucosal cell membranes was investigated. The uptake of 45Ca2+ while only moderately (15%) affected by the intact low and high molecular weight mucin forms, was significantly inhibited, by the acidic low and high molecular weight salivary mucins which evoked 64 and 60% inhibition, respectively. 2. The inhibitory effect of salivary mucins was associated with the sialic acid and sulfate ester groups of the carbohydrate chains, as the removal of either group caused partial loss in the glycoproteins inhibition, and the complete loss in the inhibitory effect occurred following desialylation and desulfation. 3. The channel in the presence of epidermal growth factor (EGF) and ATP responded by an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the phosphorylated channels showed a 46% increase in 45Ca2+ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein. 4. The binding of EGF to calcium channel receptor protein was inhibited by the low and high molecular weight acidic mucins, causing 41.2 and 36.1% reduction, respectively. This reduction in binding was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoproteins' inhibitory capacity following removal of these groups. 5. The results for the first time demonstrate that salivary mucins actively participate in the modulation of the EGF-controlled buccal mucosal calcium channel activity expression, a process of importance to the preservation of oral tissue integrity.

Enhancement in gastric mucosal laminin receptor expression with ulcer healing.

The expression of gastric mucosal laminin receptor with chronic ulcer healing was investigated. The receptor protein was isolated from gastric epithelial cell membrane of rats at various stages of ulcer healing and following radioiodination incorporated into vesicles which exhibited specific affinity towards laminin-coated surface. The binding assays revealed that the ulcer healing was accompanied by an increase in laminin receptor expression. A significant increase (2.5-fold) in the receptor expression occurred by the third day following the development of ulcer, reached a maximum of 8.6-fold increase by the 14th day when the ulcer was virtually healed, and its high level remained for at least 20 days. The results demonstrate the importance of laminin receptor as an indice of gastric mucosal repair in ulcer healing.

Effect of ebrotidine on gastric mucosal EGF and PDGF receptor expression.

The effect of ebrotidine, a new H2-blocker with gastroprotective properties, on the expression of gastric mucosal epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors, was investigated. Mucosal cell membranes were isolated from rats receiving twice daily for 5 days a dose of 100mg/kg ebrotidine or 100mg/kg ranitidine or vehicle only. Assays for EGF and PDGF revealed the presence of both types of receptors, activation of which led to enhanced tyrosine kinase activity. The receptor binding values in the control group were 2.4 fmol for EGF and 1.45 fmol/mg protein for PDGF, whereas the values in the ebrotidine group increased for EGF by 65.7% and 38.6% for PDGF, but no such effect was observed with ranitidine. The results suggest that the gastroprotective properties of ebrotidine stem from its ability to stimulate the epithelial proliferative activities through the enhancement of EGF and PDGF receptors expression.

Differential expression of salivary mucin bacterial aggregating activity with caries status.

1. The low and high mol. wt mucin forms were isolated from saliva of caries-resistant (CR) and caries-susceptible (CS) individuals, and assessed for their bacterial aggregating potential towards S. mutans and S. sanguis, the common cariogenic microorganisms encountered in the oral cavity. 2. The high mol. wt mucin from both groups of subjects exhibited similar protein and carbohydrate content, but the level of covalently bound fatty acids was significantly lower in the CR group. The mucin from CR group showed only a weak inhibitory potential, and no inhibitory activity was observed with the mucin of CS group. 3. The low mol. wt mucins from both groups, while displaying compositional similarities, showed a marked variation in the bacterial aggregating activity. With both bacteria, the activity of the mucin from CR group was at least 128-fold greater than that of CS group. 4. The conversion of the high mol. wt mucin to a low mol. wt form through the action of salivary protease produced in both groups enhancement in mucin's bacterial aggregating capacity. This enhancement was, however, considerably less pronounced in the case of mucin from CS group. 5. The results for the first time demonstrate that the bacterial aggregating epitope of salivary mucins is expressed to a greater extent in CR individuals, and that this epitope is apparently more accessible to bacteria in the low mol. wt mucin form.