Identification of a new endogenous platelet-activating factor-like molecule in gingival crevicular fluid.

Periodontal disease is an inflammatory disease and the major cause of tooth loss in adults. Bacteria and their products are the causative agents of this disease. Endogenous molecules mediate the inflammatory process and play a major role in its amplification and perpetuation as well as in the ensuing tissue destruction. The relationship between platelet-activating factor (PAF) and periodontal disease has not so far been examined thoroughly. We have isolated a phospholipid molecule with PAF-like activity from gingival crevicular fluid. This molecule, purified on HPLC, causes washed platelet aggregation with EC50 value 0.1 microM, based on phosphorus determination. It acts through PAF-receptors and is inactivated by PAF-acetylhydrolase. In addition, this phospholipid presents biological activity towards human platelets. The combination of the results obtained from the chemical and enzymic treatments, the biological assays as well as results from the electrospray analysis, leads to the conclusion that this phospholipid is a hydroxyl-PAF analogue with relative molecular mass 703. This PAF-like molecule may be implicated in periodontal disease.

Enhanced in-vitro activity of liposome-trapped penicillin-G against Pseudomonas aeruginosa.

The growth inhibition of four Pseudomonas aeruginosa strains by liposome-trapped penicillin-G was investigated. There were indications of an association of the efficacy of liposomal penicillin-G with the nature of the 0-antigenic polymeric side chain. Namely, P28-800 and PCF-95 strains, characterized by a rough polysaccharide chain, were the most susceptible, whereas strain P28-0, possessing an intact lipopolysaccharide, resisted the activity of the entrapped drug. Among the rough strains, P642, a beta-lactamase producer, was not affected by the encapsulated drug. The composition of liposomes seems to have a significant impact in arresting the growth of the P. aeruginosa strain.

Structural properties of the cAMP-dependent protein kinase associated with rat synaptic plasma membranes--I. Configuration in situ and membrane association-derived influences on [3H]cAMP binding.

1. A geometry of molecular ordering in situ characterizes the synaptosomal membrane-bound cAMP kinase, which is revealed from the expression of two equal-size [3H]cAMP-binding domains of differing sensitivity to physical (freeze-thaw) and chemical (reconstitution following salt-effected peripheral protein depletion) treatment of the membrane. 2. Cross-linking of rat synaptosomal membrane proteins with glutaraldehyde revealed after electrophoretic (agarose-polyacrylamide-SDS) resolution and "Western blot" transfer a series of bands with in situ (i.e. on the Western blots) [3H]cAMP-binding capacity. The molecular sizes of these protein bands corresponded closely with those of the cAMP kinase subunit assemblies (R2C2)2, (R2C)2, R2C2, R2C and RC. 3. The subunit assembly (R2C2)2 was also revealed after cross-linking with glutaraldehyde of the purified (commercial preparation) cytosol-derived cAMP kinase II reconstituted in lecithin liposomes. 4. The results support consideration of an operative in vivo configurational shifting between assembly forms and are discussed in the light of the outlined possibility that such shiftings might be involved in the regulation of the membrane-bound cAMP kinase.

Outer membrane alterations in multiresistant mutants of Pseudomonas aeruginosa selected by ciprofloxacin.

Spontaneous mutants of Pseudomonas aeruginosa selected by ciprofloxacin were studied for outer membrane alterations. Acquisition of ciprofloxacin resistance was at least partially related to defects in lipopolysaccharide synthesis. When ciprofloxacin resistance was combined with resistance to beta-lactams and aminoglycosides, several alterations in outer membrane proteins were noted.

Effect of calcium on the binding in vitro of [3H]cAMP to synaptosomal membranes from rat brain.

1. The binding of [3H]cAMP in vitro to synaptosomal membranes from rat brain was resolved in two components; one saturable at 20 nM cAMP with dissociation constant (KD) of 4.7 nM, and another nonsaturable within the 5-133 nM cAMP concentration range with an estimated KD value of 0.26 microM. 2. MgATP at concentration of 0.4 mM effected complete inhibition of the binding of [3H]cAMP to synaptosomal membranes throughout the used concentration range. This and the above finding indicate that the studied binding was focused on to the cAMP kinase on the membrane. 3. Calcium at concentrations of 0.1 and 10 mM stimulated a transient 20-30% increase of [3H]cAMP binding to the membranes which was influenced, as regards its time of appearance, by the concentration of cAMP. 4. The stimulation by calcium of the binding of [3H]cAMP to the membranes was inversely related to the phosphorylation of an Mr = 80,000 membrane protein, indicating stimulation of a negative effector function of cAMP--through cAMP-mediated phosphorylation--in the phosphorylation by calcium of this substrate. Moreover, the temporal displacement by cAMP of the peak of [3H]cAMP binding, produced similar temporal displacement of the inhibitory effect of cAMP on the Mr = 80,000 substrate phosphorylation. 5. These results suggest interaction in vitro of calcium and cAMP in modulation of the activity of cAMP kinase on the synaptosomal membranes.

Targeting of liposomes containing methotrexate towards Tetrahymena pyriformis cells.

The uptake of liposomes containing methotrexate by Tetrahymena pyriformis cells was investigated with the aim of producing liposome-cell association enabling methotrexate to be introduced into the cytoplasm of intact cells. Incubation of liposomes containing methotrexate with tetrahymena pyriformis cells resulted in a time and concentration-dependent uptake of entrapped methotrexate by the cells. The uptake by Tetrahymena pyriformis cells (at 1 hr) of liposomes prepared by phospholipids and gangliosides extracted from Tetrahymena pyriformis cells was approximately three fold higher than that of liposomes prepared by commercial phospholipids. Approximately 90% of liposome uptake could be inhibited by cytochalasin B and also by NaN3 and 2-deoxyglucose. This was consistent with the uptake being the result of endocytosis. The remaining uptake was probably the result of adhesion of liposomes to the cell membrane. The rate of efflux vs time of methotrexate entrapped in liposomes was much slower than that of free methotrexate which reinforces the concept that endocytosis is the main mode of liposomes uptake by the cells. Liposomes containing methotrexate at concentrations as low as 4.5 microM effectively inhibited the activity of dihydrofolate reductase which was used as a function parameter in this study. Similar inhibition of the enzyme activity by free methotrexate was achieved only at concentrations as high as 880 microM. The influence of liposomes lipid composition on the targeting of liposomes to Tetrahymena pyriformis cells was discussed.