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Sialic acids comprise a family of terminal sugars essential for a variety of biological recognition systems. N‐Propanoylmannosamine, an unphysiological sialic acid precursor, is taken up and metabolized by mammalian cells resulting in oligosaccharide‐bound N‐propanoylneuraminic acid. N‐Propanoylmannosamine, applied to endogenously hyposialylated subclones of the myeloid leukemia HL60 and of the B‐cell lymphoma BJA‐B, both deficient in UDP‐N‐acetylglucosamine 2‐epimerase, is efficiently metabolized to CMP‐N‐propanoylneuraminic acid resulting in up to 85% of glycoconjugate‐associated sialic acids being unphysiological N‐propanoylneuraminic acid. Thus, UDP‐N‐acetylglucosamine 2‐epimerase‐deficient cell lines provide an important experimental progress in engineering cells to display an almost homogeneous population of defined, structurally altered sialic acids.

Sialic acids comprise a family of terminal sugars essential for a variety of biological recognition systems. N-Propanoylmannosamine, an unphysiological sialic acid precursor, is taken up and metabolized by mammalian cells resulting in oligosaccharide-bound N-propanoylneuraminic acid. N-Propanoylmannosamine, applied to endogenously hyposialylated subclones of the myeloid leukemia HL60 and of the B-cell lymphoma BJA-B, both deficient in UDP-N-acetylglucosamine 2-epimerase, is efficiently metabolized to CMP-N-propanoylneuraminic acid resulting in up to 85% of glycoconjugate-associated sialic acids being unphysiological N-propanoylneuraminic acid. Thus, UDP-N-acetylglucosamine 2-epimerase-deficient cell lines provide an important experimental progress in engineering cells to display an almost homogeneous population of defined, structurally altered sialic acids.

We have used DNase I footprinting to examine the effect of a novel naphthylquinoline dimer, designed as a triplex‐specific bis‐intercalator, on the stability of intermolecular DNA triplexes. We find that this compound efficiently promotes triplex formation between the 9‐mer oligonucleotide 5′‐TTTTTTCTT and its oligopurine duplex target at concentrations as low as 0.1 μM, enhancing the triplex stability by at least 1000‐fold. This compound, which is the first reported example of a triplex bis‐intercalator, is about 30 times more potent than the simple monofunctional ligand.

The biosynthesis of leukotrienes (LT) C 4and B 4is followed by an export of these mediators into the extracellular space. This transport was characterized using plasma membrane vesicles prepared from mastocytoma cells and identified as an ATP-dependent primary active process. The apparent K m-values were 110 nM for LTC 4and 48 μM for ATP. The transport rate was highest for LTC 4, whereas LTD 4, LTE 4, and N-acetyl-LTE 4were transported with relative rates of 31, 12 and 8%, respectively, at a concentation of 10nM. LTB 4transport was also dependent on ATP, LTC 4transport with inhibited by LTD 4receptor antagonists (IC 10= 1.0 μM for MK-571 and 1.3 μM for LY245769) and by the inhibitor of leukotriene biosynthesis MK-886 (IC 10= 1.8 μM). The ATP-dependent export carrier for leukotrienes in leukotriene-synthesizing cells represents a novel member of the family of ATP-dependent exit pumps.

Arachidonate metabolites are potent mediators generated in endotoxin shock. Following endotoxin administration (15 mgkg) into unanesthetized rats, we found a rapid biliary secretion of peptide leukotrienes. Analysis of bile for peptide leukotrienes included organic solvent extractions, reversed phase-HPLC, radioimmunoassay (RIA), and spectrophotometry. The major immunoreactive endogenous leukotriene (LT) from bile was eluted between LTC 4and LTD 4in three chromatographic systems. It corresponded thereby to a biliary metabolite of injected LTC 4and LTD 4which in turn showed the ultraviolet spectrum of a peptide leukotriene. This demonstration of endotoxin-induced generation of peptide LTs in vivo was possible by sequential HPLC and RIA analyses in bile into which peptide LTs are eliminated from blood.

2-Deoxy-2-fluoro-D-galactose (dGalF), added to the medium of primary cultured rat hepatocytes, inhibited N-glycosylation on membrane (gp 120) and secretory glycoproteins ( α1-macroglobulin) in a concentration dependent manner. Complete inhibition of N-glycosylation was achieved at concentrations of 1 mM and above. At identical concentrations, 2-deoxy-2-fluoro-D-glucose (dGlcF) caused only incomplete inhibition of N-glycosylation. dGalF reduced incorporation of D-[2,6- 3H]mannose into lipid-linked oligosaccharides indicating interference with their assembly in the dolichol cycle.

The biosynthesis of leukotrienes (LT) C4and B4is followed by an export of these mediators into the extracellular space. This transport was characterized using plasma membrane vesicles prepared from mastocytoma cells and identified as an ATP‐dependent primary active process. The apparent Km‐values were 110 nM for LTC4and 48 μM for ATP. The transport rate was highest for LTC4, whereas LTD4, LTE4, and N‐acetyl‐LTE4were transported with relative rates of 31, 12 and 8%, respectively, at a concentation of 10nM. LTB4transport was also dependent on ATP, LTC4transport with inhibited by LTD4receptor antagonists (IC10= 1.0 μM for MK‐571 and 1.3 μM for LY245769) and by the inhibitor of leukotriene biosynthesis MK‐886 (IC10= 1.8 μM). The ATP‐dependent export carrier for leukotrienes in leukotriene‐synthesizing cells represents a novel member of the family of ATP‐dependent exit pumps.

The ATP‐dependent transport of the three 3H‐labeled, amphiphilic cations quinidine, N‐(n‐pentyl)‐quinidinium, and N‐(4',4'‐azo‐n‐pentyl) ‐21‐deoxyajmalinium was studied in rat canalicular plasma membrane vesicles. N‐Alkylation of quinidine with an n‐pentyl residue resulted in a permanently charged cationic substrate for ATP‐dependent transport which exhibited a 10‐fold higher transport rate relative to quinidine. The Kmvalue was 0.4 μM for N‐(n‐pentyl)‐quinidinium and 5 μM for quinidine. The permanently cationic and photolabile derivative of ajmaline, N‐(4',4'‐azo‐n‐pentyl)‐21‐deoxyajmalinium, was also an efficient substrate and served to label canalicular membrane proteins with molecular masses of 143 kDa and 108 kDa. ATP‐dependent transport of the permanently charged amphiphilic cations was inhibited by the P‐glycoprotein inhibitors and substrates quinidine, verapamil, and daunorubicin. The data demonstrate that N‐alkylation of quinidine and ajmaline results in most efficient substrates for mdr1P‐glycoprotein‐mediated ATP‐dependent transport.

The distinct ATP‐dependent transporters for taurocholate, leukotriene C4, and daunorubicin, studied in rat liver canalicular membrane vesicles, are sensitive to inhibition by cyclosporin A and its non‐immunosuppressive analog PSC 833. Kivalues for cyclosporin A were 0.2, 3.4 and 1.5 μM for the transport of taurocholate, leukotriene C4, and daunorubicin, respectively. The corresponding Kivalues for PSC 833 were 0.6, 29, and 0.3 μM. Both inhibitors were competitive with respect to the three substrates. The cyclosporins serve as new and potent tools to interfere with different potency with the distinct ATP‐dependent export carriers in the hepatocyte canalicular membrane.

Arachidonate metabolites are potent mediators generated in endotoxin shock. Following endotoxin administration (15 ) into unanesthetized rats, we found a rapid biliary secretion of peptide leukotrienes. Analysis of bile for peptide leukotrienes included organic solvent extractions, reversed phase‐HPLC, radioimmunoassay (RIA), and spectrophotometry. The major immunoreactive endogenous leukotriene (LT) from bile was eluted between LTC4and LTD4in three chromatographic systems. It corresponded thereby to a biliary metabolite of injected LTC4and LTD4which in turn showed the ultraviolet spectrum of a peptide leukotriene. This demonstration of endotoxin‐induced generation of peptide LTs in vivo was possible by sequential HPLC and RIA analyses in bile into which peptide LTs are eliminated from blood.

2‐Deoxy‐2‐fluoro‐D‐galactose (dGalF), added to the medium of primary cultured rat hepatocytes, inhibited N‐glycosylation on membrane (gp 120) and secretory glycoproteins (α1‐macroglobulin) in a concentration dependent manner. Complete inhibition of N‐glycosylation was achieved at concentrations of 1 mM and above. At identical concentrations, 2‐deoxy‐2‐fluoro‐D‐glucose (dGlcF) caused only incomplete inhibition of N‐glycosylation. dGalF reduced incorporation of D‐[2,6‐3H]mannose into lipid‐linked oligosaccharides indicating interference with their assembly in the dolichol cycle.