Lipoxin A-induced inhibition of human natural killer cell cytotoxicity: studies on stereospecificity of inhibition and mode of action

Human leukocyte-derived lipoxin A (LXA; 5S,-6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid) inhibits the cytotoxic activity of human natural killer (NK) cells. LXA and three of its isomers were prepared by total organic synthesis and assayed for activity with human NK cells. Dose-response studies showed that biologically derived LXA and synthetic LXA were equally effective in inhibiting NK cell cytotoxicity. 6S-LXA, with its 6S-OH group in an (S) configuration, proved to be approximately half as potent as LXA. In contrast, 6S-11-trans-LXA and 11-trans-LXA displayed virtually no inhibitory activities. The methyl esters of both LXA and 6S-LXA proved to be more potent than their corresponding free acids. Thus, LXA inhibition of NK cells displays clear-cut stereochemistry. In the absence of putative inhibitors, NK cells bind to their targets to form conjugates. This event is followed by polarization of the NK Golgi apparatus, which moves towards the plasma membrane that is in contact with the target cell. However, in the presence of either the methyl ester or free acid of LXA, the Golgi apparati of NK cells bound to their targets were randomly oriented. In contrast, neither 6S-11-trans-LXA nor the potent NK inhibitor prostaglandin E2 affected the polarization. Furthermore, although prostaglandin E2 resulted in a decrease in NK-target cell binding efficiency, LXA and its isomers failed to affect conjugate formation. Together these results indicate that LXA-induced inhibition of NK cytotoxicity does not act on NK cell binding but may block cytotoxicity by disrupting "signals" involved in the specific orientation of the Golgi. Thus, this latter event may appear to be important in cytotoxicity.     

The action of lipoxin-A on glomerular microcirculatory dynamics in the rat

Intrarenal administration of 750 ng/kg/min of LX-A in euvolemic rats resulted in significant increases in single nephron GFR (38.4 +/- 1.7 to 45.5 +/- 3.0 nl/min) and plasma flow rate (95 +/- 6 to 127 +/- 9 nl/min). The latter was due to a dramatic fall in afferent arteriolar resistance. Mean transcapillary hydraulic pressure difference increased from 33 +/- 1 to 43 +/- 3 mmHg (p less than 0.05) and the glomerular capillary ultrafiltration coefficient fell from 0.060 +/- 0.013 to 0.033 +/- 0.005 nl/(s X mmHg) (p less than 0.05). These responses to LXA in the renal microcirculation are in sharp contrast to those previously observed for the leukotrienes, and thus may represent the first example of counterregulatory (constrictor/dilator) vascular interactions within the lipoxygenase pathways.     

Site-specific gap-misrepair mutagenesis by O4-ethylthymine

The mutagenicity of the DNA base O-alkylation adduct, O4-ethylthymine, specifically incorporated into the plasmid vector pUC8 at the unique SalI and HincII recognition sites, was studied in vivo. Escherichia coli, Micrococcus luteus and AMV DNA polymerases catalyze the incorporation of O4-ethylTMP against template adenine and guanine residues, resulting in DNA sequence alteration during subsequent replication in the host E. coli K-12 strain JM83. The greatest mutation frequency was observed with error-prone AMV DNA polymerase. High levels of cognate restriction endonuclease-resistant mutant plasmid isolates were obtained by gap replication repair in the presence of O4-ethylTTP. The yields of mutant isolates were dependent upon the relative concentration of the competing pyrimidine deoxynucleoside triphosphates, TTP and dCTP, in the misreplication reaction. Repair of incorporated O4-ethylTMP of plasmid DNA by in vitro treatment with specific alkyltransferase, prior to transformation in the host, effectively increases the mutagenic efficiency of the adduct. The results obtained are consistent with the high miscoding potential O4-ethylthymine observed in in vitro studies and its ability to base-pair with noncomplementary guanine residues in DNA.     

Identification of a novel 7-cis-11-trans-lipoxin A4 generated by human neutrophils: total synthesis, spasmogenic activities and comparison with other geometric isomers of lipoxins A4 and B4

Addition of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) and the ionophore A23187 (2.5 microM) to human neutrophils led to the formation of both lipoxin A4 and lipoxin B4 as well as a novel 5,6,15-trihydroxyeicosatetraenoic acid. The new compound was identified using an improved isolation and detection system and its basic structure was determined by physical methods. On the basis of biosynthetic considerations, geometric isomers of lipoxin A4 and lipoxin B4 were prepared by total synthesis. Comparison of these synthetic materials with the neutrophil-derived product showed that the new compound is (5S,6R,15S)-trihydroxy-9,11,13-trans-7-cis-eicosatetraenoic acid or the 7-cis-11-trans-isomer of LXA4 (7-cis-11-trans-LXA4). LXA4, 11-trans-LXA4, 7-cis-LXA4 and 7-cis-11-trans-LXA4 all evoked dose-dependent (0.1-10 microM) contractions of the guinea pig lung strip, whereas 6-cis-LXB4 and 6-cis-8-trans-LXB4 relaxed this preparation. LXA4 and 7-cis-LXA4 were approx. 10-times more potent than the compounds with 11-trans geometry. However, all four double-bond isomers of LXA4 caused contractions which, based upon pharmacological evidence, appeared to involve specific activation of the same site as cysteinyl-containing leukotrienes. In conclusion, 7-cis-11-trans-LXA4 was isolated and identified as a novel biologically active eicosanoid formed by human neutrophils.     

Trihydroxytetraenes: A novel series of compounds formed from arachidonic acid in human leukocytes

Addition of 15L-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) to human leukocytes led to the formation of a novel series of compounds containing four conjugated double bonds. The yield of tetraenes was increased approx. 100-fold when ionophore A23187 (5 μM) was added simultaneously with 15-HPETE. The structure of the major tetraene was established by physical methods as well as by chemical degradation and found to be 5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid.     

Comparison of European and US biological indicators for ethylene oxide sterilization

Summary Biological indicators (BIs) are used to monitor ethylene oxide (EO) gas sterilization processes for medical devices. Several European and United States BIs for EO sterilization were evaluated for resistance according to both United States Pharmacopeia (USP) XXI and United Kingdom's (UK) tests for D-values. US BIs areB. subtilis var. niger spores on paper strips or disc carriers while European BIs use aluminum strips, quartz sand, or cotton yarn. Numerous BIs per run and runs per lot, as well as 2–3 different lots of BIs from each manufacturer, were examined. Both British and US BIs met their respective label claims for rates of inactivation when tested against British and USP EO test parameters, respectively. However, Danish BIs, on cotton yarn or quartz sand, were not inactivated following USP specifications during the exposure dwell times tested (600 mg L^–1 EO, 54°C, 60% RH, 0–110 min). The Danish BIs will require further testing in order for us to determine if theirB. subtilis spores are unusually resistant to EO or if the spore carrier substrates protect the spores from the sterilizing gas. In conclusion, the British and American BIs for EO sterilization are equivalent in resistance despite differences in carrier substrate, recovery conditions, calculation methods for D-values, and the labeled sterilization conditions for use.     

Determination of the configurations of lactic and glyceric acids from human serum and urine by capillary gas—liquid chromatography

The separation of the enantiomers of lactic and glyceric acids can be achieved by capillary gas chromatography on SP-1000 using the corresponding O-acetylated menthyl esters. The structures of the derivatives were proved by proton magnetic resonance spectroscopy and mass spectrometry. The method has been used for the determination of the absolute configurations of lactic and glyceric acids isolated from serum and urine from different patients.     

Microbiological quality of frozen cauliflower, corn, and peas obtained at retail markets.

The microbiological quality of blanched frozen cauliflower, cut corn, and peas at the retail level was determined. At 35 degrees C, mean aerobic plate count (APC) values for cauliflower, corn, and peas, respectively, were 30,000, 6,100, and 4,700 per g; at 30 degrees C, the mean APC values were 45,000, 8,500, and 6,800 per g, respectively. Geometric means for coliform, Escherichia coli, and Staphylococcus aureus counts for all three vegetables were less than 10 per g.     

Microbiological quality of some spices and herbs in retail markets.

The microbiological quality of 10 spices or herbs was determined by a national survey at the retail level. Aerobic plate count values for the 10 products ranged from less than 100 to 3.1 X 10(8) per g; mean values of the individual spices or herbs ranged from 1,400 to 820,000 per g. Coliform counts ranged from less than 3 to 1.1 X 10(6) per g; however, mean values were less than 20 per g for all products. Escherichia coli counts ranged from less than 3 to 2,300 per g. Except for celery seed, which had a mean value of 7 per g, all mean values were less than 3 per g. Yeast and mold counts were made for 5 of the 10 products. Mean values were generally low; the highest mean (290 per g) was obtained for cinnamon.