Characterization of a ceramide kinase activity from human leukemia (HL-60) cells. Separation from diacylglycerol kinase activity

This laboratory first provided evidence for a potential signal transduction pathway involving sphingomyelin and its derivatives (Kolesnick, R.N., and Clegg, S. (1988) J. Biol. Chem. 263, 6534-6537). Recently, this laboratory demonstrated the existence of the novel sphingolipid ceramide 1-phosphate in human leukemia (HL-60) cells. Ceramide 1-phosphate was synthesized from ceramide derived from sphingomyelin but not glycosphingolipids. This suggested that a specific pathway extended from sphingomyelin to ceramide 1-phosphate. The present studies provide additional support for this notion by demonstrating the existence of a ceramide kinase activity distinct from diacylglycerol (DG) kinase in HL-60 cells. Microsomal membranes contained a kinase activity that phosphorylated ceramide but not 1,2-DG in the presence of physiologic and higher Ca2+ concentrations (60 nM-3 mM). Kinetic analyses demonstrated an apparent Vmax for ceramide and ATP of 70 pmol.min-1.mg protein-1; apparent Km values were 45 and 25 microM, respectively. The pH optimum was within the physiologic range (pH 6-8). Magnesium but not other divalent cations (Mn2+, Ba2+, Cd2+, Zn2+) also stimulated ceramide phosphorylation. Magnesium also induced 1,2-DG phosphorylation. Since DG kinase is a Mg2(+)-stimulable enzyme that may utilize ceramide as substrate, additional studies separated calcium-dependent ceramide kinase from DG kinase activity. 1,2-DGs competitively inhibited magnesium- but not calcium-dependent ceramide phosphorylation. Hence, calcium-dependent ceramide kinase activity neither utilized DG as substrate nor was inhibited by DG. These activities were physically separable. Both activities were solubilized by n-octyl-beta-D-glucopyranoside and stabilized by glycerol. Ceramide kinase activity bound weakly to a DEAE-cellulose anion exchange column and eluted with 4-fold purification as a single peak of activity in the flow-through and 0.05 M NaCl elutions. In contrast, the majority of DG kinase activity bound more tightly and was recovered as a broad peak in the 0.2-0.35 M NaCl elutions. These studies demonstrate the existence of a ceramide kinase activity in HL-60 cells which is functionally and physically separable from DG kinase. These studies provide further support for the notion of a specific pathway from sphingomyelin to ceramide 1-phosphate.     

Light spectra blocking films reduce numbers of western flower thrips,Frankliniella occidentalis (Thysanoptera: Thripidae) in strawberry,Fragaria x ananassa

1. Frankliniella occidentalis is a pest of horticultural crops, including commercial strawberry (Fragaria x ananassa). Control is challenging because certain populations are resistant to insecticides and, in strawberry, now relies on the application of biocontrols. However, this approach is not always successful if F. occidentalis populations overwhelm biocontrols. We investigated whether targeted spectral modifications to cladding materials could reduce numbers of F. occidentalis, in strawberry flowers.
2. Five UV-attenuating plastic-film materials were tested in three, 6-week, semi-field tunnel experiments containing strawberry plants. F. occidentalis were introduced into tunnels from a laboratory culture and subsequent numbers that developed in strawberry flowers were recorded.
3. Limiting UV-A radiation to the crop significantly reduced the numbers of adult and larval F. occidentalis in strawberry flowers. The numbers of adult (and larvae) in flowers were reduced by 42 (47)%, 54 (41)%, 70 (73)%, and 82 (73)% in UV350, UV370, UV400, and UV430-attenuating films, respectively, compared with the UVopen (control) film. However, no damage to strawberry fruits was observed regardless of the film treatment.
4. Incorporating UV-attenuating films as tunnel cladding can suppress F. occidentalis numbers in strawberry. Reducing populations of F. occidentalis in crops is likely to enable the more successful use of other non-chemical control strategies.