Quantitation of lyso-platelet activating factor molecular species from human neutrophils by mass spectrometry

Two physicochemical methods have been developed for the quantitative analysis of lyso-platelet activating factor (lyso-PAF) based on gas-liquid chromatography-mass spectrometry (GLC/MS) and fast atom bombardment-mass spectrometry (FAB/MS) using stable isotope dilution. After addition of deuterated internal standards, lyso-PAF produced from neutrophils was purified by silicic acid chromatography and thin-layer chromatography (TLC). The GLC/MS assay employed phospholipase C or hydrofluoric acid for hydrolysis of the phosphocholine moiety to yield ether monoglycerides. Condensation of monoglycerides with acetone yielded the 1-O-alkyl-2,3-isopropylidene glycerol which could be analyzed by GLC/MS. The ions corresponding to M-15 fragments for both the labeled and unlabeled derivatives were monitored in a selected ion recording mode. Standard curves were found to be linear over the range tested (10-2000 ng) with a limit of detection found to be below 200 pg injected on column. For the FAB/MS assay, the unmodified lyso-PAF was well suited for direct analysis; however, the limit of detection (S/N greater than 3) using a glycerol matrix was found to be 5 ng placed on the probe tip. It was found that human neutrophils contain approximately 300 pg/10(6) cells which increased 2-3-fold during the 5-min period following challenge with 1.9 microM calcium ionophore, A23187. Two molecular species of lyso-PAF were identified as hexadecyl and octadecyl ethers at sn-1 with the octadecyl molecular species of lyso-PAF predominating in abundance after stimulation.     

A COMPARISON OF DIFFERENT METHODS OF AGE DETERMINATION IN THE SHARPTOOTH CATFISH CLARIAS GARIEPINUS

SUMMARY

Growth of the sharptooth catfish Clarias gariepinus from four southern African impoundments was estimated by back-calculating age from vertebrae and spines. Length frequency analysis supported the growth estimates derived from vertebrae but not those from spines.- Spines under-estimated the age of fish from all four dams; this discrepancy increased with age. Ring formation occurred in the winter months (April to July) and not at the time of spawning (November to February) or low water levels (August to December).

A critical review of ageing studies shows a lack of consistency in the choice of formula used for back-calculation. The correct choice of formula for age and growth estimates derived from back-calculation must be based upon detailed analysis of fish size versus body part size. Unquestioning acceptance of the Lee equation with its “correction factor” should he avoided.     

The arthropod,but not the vertebrate host or its environment,dictates bacterial community composition of fleas and ticks

Bacterial community composition in blood-sucking arthropods can shift dramatically across time and space. We used 16S rRNA gene amplification and pyrosequencing to investigate the relative impact of vertebrate host-related, arthropod-related and environmental factors on bacterial community composition in fleas and ticks collected from rodents in southern Indiana (USA). Bacterial community composition was largely affected by arthropod identity, but not by the rodent host or environmental conditions. Specifically, the arthropod group (fleas vs ticks) determined the community composition of bacteria, where bacterial communities of ticks were less diverse and more dependent on arthropod traits—especially tick species and life stage—than bacterial communities of fleas. Our data suggest that both arthropod life histories and the presence of arthropod-specific endosymbionts may mask the effects of the vertebrate host and its environment.     

IK inactivation in squid axons is shifted along the voltage axis by changes in the intracellular pH.

The inactivation curve of the delayed rectifier in internally perfused squid giant axons is shifted along the voltage axis by changes in the pH of the internal perfusate. The amplitude of the shift is 9.5 mV per pH unit (6 less than or equal to pHi less than or equal to 10). No saturation of the effect was observed at either end of the pH range. This result suggests that the inactivation gating mechanism has several titratable groups accessible to protons from the intracellular side of the membrane.     

Binding of platelet activating factor to albumin

Binding of platelet activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) to albumin is an important facet of the biological activity of this phospholipid. Measurement of that binding has been hampered by the physical nature of the lipid, which made estimation of the free and bound concentrations difficult. With the use of ultracentrifugation to generate an albumin gradient and to produce a region free of protein, the successful measurement of free PAF and PAF bound to albumin was accomplished. This study has demonstrated that PAF binds to albumin at four binding sites and that the average equilibrium dissociation constant for this binding is 1.10(-7) M. Consideration of these data has led to the hypothesis that the receptor active form of PAF is the albumin-PAF complex, rather than free PAF.