sn-1,2-diacylglycerols and phorbol diesters: uptake, metabolism, and subsequent assimilation of the diacylglycerol metabolites into complex lipids of cultured cells

The cell-permeable diacylglycerol mediators have been shown to mimic partially the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured cells. In order to evaluate the metabolic stability of the lipid mediators, several radiolabeled diacylglycerols were synthesized and their uptake and intracellular fate in cultured HL-60 (human promyelocytic leukemia) cells was compared with TPA. In addition to whole cell assessment, the stability of diacyl lipids and TPA was evaluated in a buffer/water system and in the presence of serum and subcellular fractions. The compounds studied include 1,2-dioleoyl-sn-glycerol (DiOG), 1-oleoyl-2-acetyl-sn-glycerol (OaG), 1-palmitoyl-2-acetyl-sn-glycerol (PaG), the ether-linked analog 1-palmityl-2-acetyl-sn-glycerol (ePaG), and TPA. TPA was comparatively stable to lipase hydrolysis in all systems examined. First, the data show that within 5 min at pH 7.9, nearly 50% of the PaG (originally greater than 92% 1,2-isomer) had isomerized, and rapid formation of the 1,3-isomer also occurred with OaG and ePaG. The metabolism of OaG and PaG by serum hydrolases, using a reaction medium containing 10% serum, was chiefly by acetate hydrolysis; however, fatty acid was also liberated. After a 60-min incubation 68% of the [14C]OaG was converted, by serum enzymes, to monooleoylglycerol plus oleic acid. Heat-inactivation of serum reduced the enzymatic formation of fatty acid by 60-70%. ePaG was also metabolized by serum enzymes, but the ether-linked alkylglycerol product was stable. The results of cell-free studies (postmitochondrial supernatant) showed that cellular enzymes were present that could, like serum, convert the diacylglycerols to monoacylglycerols and free fatty acids. Studies using cultured cells showed that radiolabeled OaG, PaG, and ePaG were rapidly taken up by the cells and metabolized. Labeled metabolic products from the diacylglycerols appeared, in a time-dependent manner, in cellular phospholipids and triacylglycerols. The results from experiments employing 1-acyl-2-acetyl-sn-[3H]glycerol and [3H]acyl-2-acetyl-sn-glycerol indicate that the intracellular mode of mediator metabolism is via complete hydrolysis with subsequent incorporation of 3H-acyl groups into complex lipids. Data are also presented which show that a substantial amount of cellular lipid acyl group modification occurs and large amounts of glycerol are produced when cells are cultured with OaG. Collectively, these results demonstrate that the diacylglycerol mediators, when compared with TPA, are not stable and are metabolized by both serum and cellular enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid evolution of a young L1 (LINE-1) clade in recently speciated rattus taxa

L1 elements are retrotransposons that have been replicating and evolving in mammalian genomes since before the mammalian radiation. Rattus norvegicus shares the young L1_mlvi2 clade only with its sister taxon, Rattus cf moluccarius. Here we compared the L1_mlvi2 clade in these recently diverged species and found that it evolved rapidly into closely related but distinct clades: the L1_mlvi2-rm clade (or subfamily), characterized here from R. cf moluccarius, and the L1_mlvi2-rn clade, originally described in R. norvegicus. In addition to other differences, these clades are distinguished by a cluster of amino acid replacement substitutions in ORF I. Both rat species contain the L1_mlvi2-rm clade, but the L1_mlvi2-rn clade is restricted to R. norvegicus. Therefore, the L1_mlvi2-rm clade arose prior to the divergence of R. norvegicus and R. cf moluccarius, and the L1_mlvi2-rn clade amplified after their divergence. The total number of L1_mlvi2-rm elements in R. cf moluccarius is about the same as the sum of the L1_mlvi2-rm and L1_mlvi2-rn elements in R. norvegicus. The possibility that L1 amplification is in some way limited so that the two clades compete for replicative supremacy as well as the implications of the other distinguishing characteristic of the L1_mlvi2-rn and L1_mlvi2-rm clades are discussed. Received: 6 November 1996 / Accepted: 12 April 1997     

Macrophytic primary production and nutrient concentrations in a deltaic floodplain marsh of the Lower Paraná River

Biomass changes across an annual cycle were followed at two sampling sites in the floodplain marsh of the Lower Paraná River: close to the river-shore and 800 m inside the floodplain marsh, both dominated by Scirpus californicus and Cyperus giganteus. Tidal influence determines a daily exchange of water between the river and the floodplain marsh.Estimated net primary production was higher in the river (2820 against 1770 g m^–2). Contents of nitrogen and phosphorus in plant tissue decreased from the river to the floodplain (0.62 to 0.45% N and 0.18 to 0.14% P). In spite of the important water exchange between the river and the floodplain, a decrease in nitrate, oxygen and suspended matter, and an increase in soluble reactive phosphorus in the water were observed from the river towards the floodplain marsh.A primary production gradient exists from the river to the inner floodplain marsh, where production is nitrogen-limited, sustained mainly on nutrients supplied by the river. Floodplain marshes are nitrate sinks, probably through denitrification losses and macrophyte uptake.     

Ceramide glycosylation potentiates cellular multidrug resistance.

Ceramide glycosylation, through glucosylceramide synthase (GCS), allows cellular escape from ceramide-induced programmed cell death. This glycosylation event confers cancer cell resistance to cytotoxic anticancer agents [Liu, Y. Y., Han, T. Y., Giuliano, A. E., and M. C. Cabot. (1999) J. Biol. Chem. 274, 1140-1146]. We previously found that glucosylceramide, the glycosylated form of ceramide, accumulates in adriamycin-resistant breast carcinoma cells, in vinblastine-resistant epithelioid carcinoma cells, and in tumor specimens from patients showing poor response to chemotherapy. Here we show that multidrug resistance can be increased over baseline and then totally reversed in human breast cancer cells by GCS gene targeting. In adriamycin-resistant MCF-7-AdrR cells, transfection of GCS upgraded multidrug resistance, whereas transfection of GCS antisense markedly restored cellular sensitivity to anthracyclines, Vinca alkaloids, taxanes, and other anticancer drugs. Sensitivity to the various drugs by GCS antisense transfection increased 7- to 240-fold and was consistent with the resumption of ceramide-caspase-apoptotic signaling. GCS targeting had little influence on cellular sensitivity to either 5-FU or cisplatin, nor did it modify P-glycoprotein expression or rhodamine-123 efflux. GCS antisense transfection did enhance rhodamine-123 uptake compared with parent MCF-7-AdrR cells. This study reveals that GCS is a novel mechanism of multidrug resistance and positions GCS antisense as an innovative force to overcome multidrug resistance in cancer chemotherapy.