Effects of enzymes and protein modifying reagents on the binding of H-prostaglandin E2 to porcine oxyntic mucosa

In the present study we have investigated the macromolecular nature of porcine oxyntic mucosal PGE₂ binding sites and the involvement of specific functional groups in the binding interaction. Incubation of oxyntic mucosal membranes with DNAse or RNAse did not influence binding. Phospholipase A₂ was strongly inhibitory while phospholipases C and D exerted variable effects. Trypsinzation of the membranes also reduced binding and this reduction was prevented by addition of soybean trypsin inhibitor. Neuraminidase and β-galactosidase treatments resulted in variable increases in binding activity. The increase in binding was due to an increase in binding affinity and/or binding site concentration. Protein modifying reagents acetic anhydride, N-ethylmaleimide and mercaptoethanol all reduced binding. These results suggest the importance of protein, lipid and carbohydrate components of the membrane in the binding interaction between PGE₂ and its binding site. The ability of mercaptoethanol and N-methylmaleimide to reduce binding suggest the involvement of both sulphydryl and disulphide groups in the PGE₂ binding reaction.     

Preclinical detection of metastatic melanoma

We present a patient with melanoma in whom the performance of combined immunoscintigraphy and immunolymphoscintigraphy indicated the presence of metastatic disease 16 months before clinical manifestation. This approach may be useful in the early detection of metastatic disease and the patient presented is a lesson that cutaneous foci of uptake should not be dismissed as false-positive in the absence of clinical correlation.     

Failure of anti-inflammatory agents to modulate infection-induced pelvic adhesive disease in rabbits

A suspension of Neisseria gonorrhoeae was injected into rabbit uterine horns to induce pelvic adhesive disease. There was no statistical difference in the formation of adhesions between the rabbits that were given anti-inflammatory agents and those that received no therapy. This study suggests that anti-inflammatory agents will offer no benefit as adjunctive therapy for the prevention of infection-induced pelvic adhesive disease.     

Studies on the mechanism of microbial N-demethylation of codeine by cell-free extracts of Cunninghamella bainieri

Cell-free extracts have been prepared from the fungus Cunninghamella bainieri which retain high levels of N-demethylase activity against codeine and other drug molecules. Extraction required both disruption and solubilization, indicating that the N-demethylase enzyme is probably membrane bound. The carbon monoxide-reduced u.v. difference spectrum of the extract suggests the presence of a cytochrome P-450. The effect of specific inhibitors and the generation of formaldehyde during the transformation suggest a mono-oxygenase mediated mechanism. Kinetic studies have shown that N-demethylation is unlikely to occur via the N-oxide intermediate. Instead it is proposed that N-demethylation proceeds via the transient N-hydroxymethyl intermediate.     

Specific binding of a stable prostacyclin analogue (Iloprost) to rat oxyntic mucosa

It has been reported that prostacyclin (PGI2) is the predominant species of prostanoid in rat oxyntic mucosa. However since PGI2 is inactivated under physiological conditions it has not been possible to demonstrate specific PGI2 binding to the rat stomach. Therefore a stable PGI2 analogue, Iloprost, was chosen as ligand in this study. Binding of labelled Iloprost to the 20,000 xg homogenate fraction of rat oxyntic mucosa was specific, dissociable, saturable and dependent upon the temperature and time of incubation. Neither tritiated PGE2 nor 6 keto PGF1 alpha displayed any significant specific binding to rat stomach. A Scatchard plot of the equilibrium binding data for Iloprost was curvilinear and could be resolved into at least two binding sites. The average parameters determined from Scatchard analysis were: dissociation constants of 1.8 X 10(-11) M and 7.1 X 10(-8) M and corresponding binding site concentrations of 12.0 pmole/mg and 4800 pmoles/mg protein respectively. PGI2 was less potent than unlabelled Iloprost in displacing 3H-Iloprost from its binding site. The addition of PGE2 to the incubation medium resulted in an increase in 3H-Iloprost binding. It is concluded that rat oxyntic mucosa has specific binding sites for PGI2-like agents but not for either PGE2 or 6 keto PGF1 alpha.     

Biological indicators for low temperature steam and formaldehyde sterilization: effect of variations in recovery conditions on the response of spores of Bacillus stearothermophilus NCIMB 8224 to low temperature steam and formaldehyde

Spores of a Bacillus stearothermophilus strain thought to be a good biological monitor for low temperature steam and formaldehyde (LTSF) sterilization have been subjected to different recovery environments following exposure to relevant LTSF treatments and prior to enumerating the numbers of surviving spores. The recovery treatments essentially consisted of holding samples at a temperature of 90°C for different time periods prior to plating out following exposure to 12 μg ml^-1 formaldehyde at temperatures between 63° and 83°C The data indicate that LTSF-exposed spores show a marked recovery when kept at 90°C prior to plating out; the extent of the recovery increases with increasing inactivation temperature between 73° and 83°C. The data bring into question the sporicidal activity of LTSF.     

16, 16 dimethyl prostaglandin E2 reduces histamine release from the stomach and protects the gastric mucosal barrier altered by ethanol in neutral solution.

Damage to the gastric mucosal barrier results in histamine release from intramucosal stores. Previous reports have shown that 16, 16 dimethyl prostaglandin E2 (dm PGE2) protects the stomach from injury by various damaging agents in either acidic or neutral solution. Furthermore histamine released in response to a damaging drug in an acidic medium was reduced by dm PGE2. Using the Heidenhain pouch dog preparation, the present study examined the action of dm PGE2 on ethanol-induced barrier breaking and histamine release in neutral solution. Topical ethanol treatment (15% w/v) damaged the gastric mucosal barrier as evidenced by increased net fluxes of Na+ and K+ and an increase in the histamine content of the fluid irrigating the histamine content of the fluid irrigating the Heidenhain pouch. Intravenous injection of dm PGE2 in the doses of 0.01, 0.10 and 1.00 microgram/kg one-half hour before ethanol administration significantly reduced the appearance of Na+, K+ and histamine. It is concluded that dm PGE2 effectively protects the canine gastric mucosa from damaging agents in neutral solution as evidenced by a reduction in the luminal appearance of Na+, K+ and histamine.     

The screening of some microorganisms for their ability to N-dealkylate drug molecules

Summary Selected microorganisms were screened for their ability to N-dealkylate drug molecules. The compounds studied enabled the investigation of N-alkyl groups in different chemical environments, including alkylaminoalkyl chains, saturated cyclic structures and in amide functions. Transformation products were extracted from the transformation mixtures, derivatised and analysed by gas liquid chromatography (GLC). For the purposes of screening, important transformation products were identified by comparison of their GLC retention data with similar derivatives of authentic standards. N-demethylation was effected by all test strains of the fungus Cunninghamella, except C. elegans, and also by three of the Streptomyces species tested. The Cunninghamella demonstrated the widest spectrum of transformation activity, N-demethylating the alkylaminoalkyl side chains of amitriptyline and chlorpromazine, the N-methylpiperidine function of codeine, and the cyclic amide function of diazepam. The N-demethylation of the latter substrate, which is only slightly water soluble, occurred in surprisingly high yield. There was no evidence that bulky groups such as N-dimethylallyl were celaved and selectivity for N-demethylation rather than O-demethylation was demonstrated when both N-and O-methyl functions were present in the same molecule. Comparisons are made with the mammalian metabolic pathways of the drug compounds studied.