Sphingosine synergistically stimulates tumor necrosis factor alpha-induced prostaglandin E2 production in human fibroblasts.

Sphingosine is a biologically active derivative of sphingomyelin. It affects diverse cellular functions and its mechanism(s) of action is poorly defined. Tumor necrosis factor alpha (TNF alpha) has recently been shown to rapidly induce sphingomyelin turnover, implicating this metabolic pathway in TNF alpha signal transduction. Because TNF alpha is known to induce prostaglandin E2 (PGE2) production in human fibroblasts, we tested the effect of sphingosine on TNF alpha-induced PGE2 production. We found that sphingosine enhanced TNF alpha-induced PGE2 production by as much as 18-fold over TNF alpha alone. Sphingosine appeared to stimulate TNF alpha-induced PGE2 production independent of TNF alpha-mediated interleukin 1 (IL-1) production, because anti-IL-1 antibodies and IL-1 receptor antagonist protein (IRAP) did not inhibit TNF alpha-induced PGE2 production or the stimulatory effect of sphingosine. TNF alpha stimulated PGE2 production to the same degree in normal and protein kinase C (PKC) downregulated cells in the presence and absence of sphingosine, indicating that neither TNF alpha nor sphingosine require active PKC to elicit their respective effects. The sphingosine analogues stearylamine and stearoyl-D-sphingosine had little or no effect on TNF alpha-mediated PGE2 production, supporting a specific role for sphingosine in the activation process. Short-term (1 min) exposure of cells to sphingosine dramatically increased TNF alpha-induced PGE2 production. A potential mechanism by which sphingosine could increase TNF alpha-induced PGE2 production involves enhancement of phospholipase A2 (PLA2) and/or cyclooxygenase (Cox) activity, the rate-limiting enzymes in PGE2 production. We found that both TNF alpha and sphingosine alone enhanced these enzymatic activities, and that sphingosine additively increased the effect of TNF alpha on phospholipase A2 activity. It appears that sphingosine affects TNF alpha-induced PGE2 production via a mechanism that is independent of PKC involvement, and that sphingosine may function as an endogenous second messenger capable of modulating the responsiveness of the cell to external stimuli.

Ceramide induces interleukin 6 gene expression in human fibroblasts.

We previously reported that ceramide, the immediate product of sphingomyelin hydrolysis, increases in response to interleukin (IL)-1 beta and plays a role in modulating IL-1 beta-mediated prostaglandin E2 production and cyclooxygenase gene expression in human fibroblasts (Ballou, L. R., C. P. Chao, M. A. Holness, S. C. Barker, and R. Raghow. 1992. J. Biol. Chem. 267:20044-20050). Here we describe the effects of ceramide in another IL-1 beta-mediated process in these cells, the induction of IL-6 production. We found that submicromolar concentrations of C2-ceramide induced IL-6 gene expression and protein production as effectively as IL-1 beta. Both D-erythro-C2-ceramide (a cell-permeable analogue of natural ceramide) and D-threo-C2-ceramide were potent inducers of IL-6 production, while neither L isomer of ceramide was effective. Compared with IL-1 beta-induced IL-6 production, cells treated with ceramide or exogenous sphingomyelinase induced 82 and 50% of maximal IL-1 beta-induced IL-6 levels by 6 h, respectively; by 24 h all three treatments induced similar levels of IL-6 production. Ceramide-induced IL-6 messenger RNA could be detected within 1 h of treatment and reached maximal levels by 24 h. These findings suggest that ceramide may play a role in the regulation of IL-6 gene expression.

Compensatory prostaglandin E2 biosynthesis in cyclooxygenase 1 or 2 null cells.

Prostaglandin E2 (PGE2) production in immortalized, nontransformed cells derived from wild-type, cyclooxygenase 1-deficient (COX-1(-/-)) or cyclooxygenase 2-deficient (COX-2(-/-)) mice was examined after treatment with interleukin (IL)-1beta, tumor necrosis factor alpha, acidic fibroblast growth factor, and phorbol ester (phorbol myristate acetate). Compared with their wild-type counterparts, COX-1(-/-) or COX-2(-/-) cells exhibited substantially enhanced expression of the remaining functional COX gene. Furthermore, both basal and IL-1-induced expression of cytosolic phospholipase A2 (cPLA2), a key enzyme-regulating substrate mobilization for PGE2 biosynthesis, was also more pronounced in both COX-1(-/-) and COX-2(-/-) cells. Thus, COX-1(-/-) and COX-2(-/-) cells have the ability to coordinate the upregulation of the alternate COX isozyme as well as cPLA2 genes to overcome defects in prostaglandin biosynthetic machinery. The potential for cells to alter and thereby compensate for defects in the expression of specific genes such as COX has significant clinical implications given the central role of COX in a variety of disease processes and the widespread use of COX inhibitors as therapeutic agents.

Cyclic equibiaxial tensile strain induces both anabolic and catabolic responses in articular chondrocytes.

Mechanical disturbance is directly implicated in the development of osteoarthritis (OA) but the precise mode for degenerative changes is still largely unknown because of the complexity of the biomechanical and biochemical milieu in the articular joint. To investigate the effects of tensile strain on articular cartilage, cyclic equibiaxial tensile strain (CTS, 0.5 Hz, 10% strain) was applied to monolayer cultures of porcine articular chondrocytes by using a Flexercell strain unit. Overproduction of proinflammatory mediators and imbalanced expression of anabolic and catabolic genes were induced. The cellular secretion of nitric oxide (NO) and prostaglandin E(2) (PGE(2)), as well as the mRNA level of cyclooxygenase-2 (COX-2) were up-regulated in response to mechanical stimuli. Additionally, CTS resulted in an initial peak of anabolic response at 3 h of stretch with respect to the expression of type II collagen and aggrecan. After 12 h of CTS, the expression for these two cartilage-specific matrix proteins fell to control levels. A distinct catabolic response developed after 24 h of stretch with an increase in matrix metalloproteinase-1 (MMP-1). Interestingly, a parallel increase in transforming growth factor (TGF) beta3 was associated with the anabolic changes while an increase in expression of TGF beta1, the predominant isoform of the TGF family, appeared at 24 h. The expression at 24 h of MMP-1, an enzyme that degrades interstitial collagens as well as other cartilage matrix proteins and TGF beta1, may signify a shift towards matrix remodeling and potentially a change in matrix composition as a consequence of continuous CTS.

Interleukin 1 stimulates phosphatidylinositol kinase activity in human fibroblasts.

IL-1 mediates multiple cellular immune and inflammatory responses, but little is known of the intracellular biochemical mechanisms involved in IL-1 actions. We studied the effects of IL-1 on phosphatidylinositol (PtdIns) metabolism and confirmed reports indicating that IL-1 does not stimulate increased PtdIns turnover; however, we observed the accumulation of PtdIns-4-phosphate (PtdInsP) in response to IL-1. Using a fibroblast membrane preparation, we were able to detect stimulated PtdInsP accumulation within 10 s of IL-1 addition. Increased PtdInsP accumulation was due to stimulated PtdIns kinase activity, not the inhibition of PtdInsP hydrolysis by phospholipase(s). PtdIns kinase activity was magnesium dependent, increased as a function of IL-1 concentration, and specifically phosphorylated the D4 position of inositol. Stimulated PtdIns kinase activity could be detected at 10(-12) M IL-1 in fibroblast membranes, a concentration within the physiological range for IL-1 action; half-maximal activity was reached at approximately 10(-10) M IL-1. Heat denaturation of IL-1 or treatment of IL-1 with anti-IL-1 antibody abrogated the IL-1 effect. These findings demonstrate the direct, IL-1-mediated, stimulation of PtdIns kinase. IL-1-stimulated PtdIns kinase activity represents an important physiological regulatory effect by IL-1 as it could control the synthesis and/or maintenance of phosphorylated derivatives of PtdIns which comprise only a very small pool of substrates for the generation of the second messengers inositol 1,4,5-triphosphate and diacylglycerol.

Temperature-sensitive glucosamine auxotroph of Saccharomyces cerevisiae.

Temperature-sensitive revertants were isolated from Saccharomyces cerevisiae D-glucosamine auxotrophs previously obtained in this laboratory (W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). The auxotrophs lack the enzyme 2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (EC 5.3.1.19), and the revertants appear to be temperature sensitive in the formation of enzyme activity. The enzyme they produce under permissive conditions decays in activity at a rate comparable to that of the wild-type enzyme, and it has similar kinetic properties. The homozygous diploid mutant fails to sporulate at the nonpermissive temperature. Temperature shift experiments were carried out in an effort to determine what effect glucosamine deficiency had on mannoprotein secretion as reflected in the formation of external asparaginase. Although the results were complicated by the slow decay of the residual ketol-isomerase activity, they did show that mannoprotein synthesis or secretion was altered when the internal pool of D-glucosamine was depleted.

The isozyme-specific effects of cyclooxygenase-deficiency on bone in mice.

Prostaglandin E(2) (PGE(2)) plays a critical role in skeletal physiology and bone loss. PGE(2) production is regulated in vivo by at least two cyclooxygenase (COX) isozymes, COX-1 and COX-2. The purpose of this study was to investigate the in vivo effects of the selective deletion of COX-1 or COX-2 on bone mineral density (BMD), bone microarchitecture and bone strength in wild type (WT), COX-1(-/-) and COX-2(-/-) mice. Using a LUNAR PIXImus, BMD was measured in 18 (WT), 18 COX-1(-/-) and 16 COX-2(-/-) mice. COX-1(-/-) mice exhibited significantly higher BMD (0.0506 g/cm(2) +/- 0.0014 g/cm(2)) than either WT (0.0493 g/cm(2) +/- 0.0019, P < or = 0.05) or COX-2(-/-) (0.0473 g/cm(2) +/- 0.0034, P < or = 0.01) mice. COX-2(-/-) mice had significantly lower BMD than WT (P < or = 0.01) or COX-1(-/-) (P < or = 0.01). Flexure stress of the femurs, determined by breaking the bones with three-point bending, correlated with bone density. Although plasma levels of both Ca(2+) and PTH were comparable in wild type and COX-1(-/-) mice, both were elevated in COX-2(-/-) mice consistent with primary hyperparathyroidism. These studies suggest that COX enzymes are important regulators of BMD and bone strength in mice. The beneficial effect of absence of the COX-1 enzyme on skeletal parameters may be secondary to decreases in PGE(2). On the other hand, primary hyperparathyroidism and lower bone magnesium content may account for the lower BMD and impairments in bone strength of COX-2(-/-) mice. Further elucidation of the effects of the COX pathway on bone remodeling may provide important information on potential therapeutic targets for preventing and/or treating osteoporosis.

Induction of the mitogen-activated p70 S6 kinase by adenovirus E1A.

Adenovirus E1A proteins can transform primary cells in culture in conjunction with other oncogenes, such as E1B or activated ras. The modulation of various cell cycle regulators by E1A is thought to be involved in this transformation process. In this paper we show that E1A enhances the expression of the mitogen-inducible p70 S6 kinase (p70s6k), a kinase which is essential for G1 progression. p70s6k mRNA and protein levels are enhanced 3-4-fold in various E1A-expressing cell lines. Similarly, the activity of p70s6k is enhanced in E1A-expressing cells in a manner partially independent of enhanced expression of p70s6k. The induction of p70s6k correlates with the presence of conserved region 1 (CR1) of E1A and with morphological transformation by E1A. These results suggest that induction of p70s6k by E1A might be involved in transformation by E1A.

Ceramide signalling and the immune response.

Ceramide, produced through either the induction of SM hydrolysis or synthesized de novo transduces signals mediating differentiation, growth, growth arrest, apoptosis, cytokine biosynthesis and secretion, and a variety of other cellular functions. A generalized ceramide signal transduction scheme is shown in Fig. 2 in which ceramide is generated through the activation of distinct SMases residing in separate subcellular compartments in response to specific stimuli. Clearly, specificity of cellular responses to ceramide depends upon many factors which include the nature of the stimulus, co-stimulatory signals and the cell type involved. Ceramide derived from neutral SMase activation is thought to be involved in modulating CAPK and MAP kinases, PLA2 (arachidonic acid mobilization), and CAPP while ceramide generated through acid SMase activation appears to be primarily involved in NF-kappa B activation. While there is no apparent cross-talk between these two ceramide-mediated signalling pathways, there is likely to be significant cross-talk between ceramide signalling and other signal transduction pathways (e.g., the PKC and MAP kinase pathways). Other downstream targets for ceramide action include Cox, IL-6 and IL-2 gene expression, PKC zeta, Vav, Rb, c-Myc, c-Fos, c-Jun and other transcriptional regulators. Many, if not all, of these ceramide-mediated signalling events have been identified in the various cells comprising the immune system and are integral to the optimal functioning of the immune system. Although the role of the SM pathway and the generation of ceramide in T and B lymphocytes have only recently been recognized, it is clear from these studies that signal transduction through SM and ceramide can strongly affect the immune response, either directly through cell signalling events, or indirectly through cytokines produced by other cells as the result of signalling through the SM pathway. An overview of the signalling mechanisms coupling ceramide to the modulation of the immune response is depicted in Fig. 3 and shows how ceramide may play pivotal roles in regulating a number of complex processes. The SM pathway represents a potentially valuable focal point for therapeutic control of immune responses, perhaps for either enhancement of the activity of T cells in the elimination of tumors, or the down-regulation of lymphocyte function in instances of autoimmune disease. The recent explosion of knowledge regarding ceramide signalling notwithstanding, a number of critical questions need to be answered before a comprehensive, mechanistic understanding can be formulated relative to the incredibly varied effects of ceramide on cell function. For example, (i) how is a structurally simple molecule like ceramide able to mediate so many different, and sometimes paradoxical, physiological responses ranging from cell proliferation and differentiation to inhibition of cell growth and apoptosis, (ii) what are the molecular identities and modes of activation of the various SMase isoforms, (iii) what determines the distribution of the unique isoforms of SMase in cells of different lineages or at different stages of differentiation, (iv) what is the relative contribution of ceramide generated through SM hydrolysis versus de novo synthesis, and (v) by what means does ceramide interact with specific intracellular targets? Although a number of ceramide-activatable kinases, phosphatases, and their protein substrates have been identified, a more extensive search for additional cellular targets will be indispensable in determining the phosphorylation cascades linking the activation of the SM pathway to the regulation of nuclear events. Clearly, cross-talk between ceramide-induced signal transduction cascades and other signalling pathways adds to the inherent difficulty in distinguishing the specific effects of complex, intertwining signalling pathways.

Cholecalciferol induces prostaglandin E2 biosynthesis and transglutaminase activity in human keratinocytes.

In this study, we examined the effects of cholecalciferol, a primary keratinocyte metabolite and precursor of the hydroxylated form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], on prostaglandin E2 (PGE2) production in human keratinocytes by examining its respective effects on cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A2 (cPLA2) expression, the rate-limiting enzymes regulating PGE2 biosynthesis and differentiation of keratinocytes. Cholecalciferol induced PGE2 production, whereas 1alpha,25(OH)2D3 had no effect on PGE2 production both in normal human epidermal keratinocytes and in the immortalized human keratinocyte cell line, HaCaT. In HaCaT cells, neither COX-1 mRNA nor protein was detectable without stimulation and COX-1 expression did not increase in response to cholecalciferol treatment. Although cPLA2 mRNA and protein were constitutively expressed in untreated HaCaT cells, expression levels did not increase in response to cholecalciferol treatment; however, unlike COX-1 and cPLA2 expression, COX-2 mRNA and COX-2 protein expression increased in response to cholecalciferol treatment. Calphostin C, a potent protein kinase C inhibitor, significantly reduced cholecalciferol-induced PGE2 production by inhibiting cholecalciferol-enhanced COX-2 mRNA and protein expression. These results indicate that (i) 1alpha,25(OH)2D3 does not induce PGE2 biosynthesis in keratinocytes, (ii) cholecalciferol-induced PGE2 production is primarily COX-2 dependent, and (iii) cholecalciferol enhances both COX-2 mRNA and protein expression, via a protein kinase C-dependent mechanism in human keratinocytes. Furthermore, cholecalciferol increased total cellular transglutaminase activity dose dependently, suggesting a potential role for cholecalciferol in regulating the differentiation of human keratinocytes.

Transforming growth factor-beta inhibits interferon-gamma-induced HLA-DR expression by cultured human fibroblasts.

This study shows the induction of HLA-DR (DR) in fibroblasts by IFN-gamma and investigates the molecular mechanisms involved in the further DR down-regulation by TGF-beta 1. Kinetics of DR induction on human dermal fibroblasts by IFN-gamma showed that 1 hr of exposure was required to induce detectable levels of DR, and maximal DR expression was achieved only after 2 days of exposure to IFN-gamma. TGF-beta 1 inhibited DR induction by IFN-gamma, although complete inhibition never could be achieved, even with high concentrations of TGF-beta 1 and low concentrations of IFN-gamma. Inhibition was not accounted for by reduction in cell numbers, as TGF-beta 1 stimulated growth of the fibroblasts. Inhibition of DR induction was seen only if TGF-beta 1 was added during the first 24 hr of IFN-gamma treatment. TGF-beta 1 inhibited equally well if the cells were pretreated for as little as 1 hr and then washed before addition of IFN-gamma. TGF-beta 1 did not cause an overall suppression of protein synthesis. Northern blot analysis revealed that TGF-beta 1 greatly reduced the steady-state level of DR beta mRNA induced by IFN-gamma at 24 hr, and then DRP transcripts became undetectable at later stages. It is concluded that early intracellular signals must build up to stimulate maximum DR synthesis, which, later on, are inactivated or degraded by the action of TGF-beta 1. We suggest that these mechanisms regulating DR gene transcription involve the action of genes coding for specific IFN-gamma-inducible transcriptional factors that are turned on and off in an expeditious manner.

Cyclooxygenase-1 and cyclooxygenase-2 in the mouse ductus arteriosus: individual activity and functional coupling with nitric oxide synthase.

1. Prenatal patency of the ductus arteriosus is maintained by prostaglandin (PG) E(2), conceivably in concert with nitric oxide (NO). Local PGE(2) formation is sustained by cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2), a possible exception being the mouse in which COX1, or both COXs, are reportedly absent. Here, we have examined the occurrence of functional COX isoforms in the near-term mouse ductus and the possibility of COX deletion causing NO upregulation. 2. COX1 and COX2 were detected in smooth muscle cells by immunogold electronmicroscopy, both being located primarily in the perinuclear region. Cytosolic and microsomal PGE synthases (cPGES and mPGES) were also found, but they occurred diffusely across the cytosol. COX1 and, far more frequently, COX2 were colocalised with mPGES, while neither COX appeared to be colocalized with cPGES. 3. The isolated ductus from wild-type and COX1-/- mice contracted promptly to indomethacin (2.8 micro M). Conversely, the contraction of COX2-/- ductus to the same inhibitor started only after a delay and was slower. 4. N(G)-nitro-L-arginine methyl ester (L-NAME, 100 micro M) weakly contracted the isolated wild-type ductus. Its effect, however, increased three- to four-fold after deleting either COX, hence equalling that of indomethacin. 5. In vivo, the ductus was patent in all mice foetuses, whether wild-type or COX-deleted. Likewise, no genotype-related difference was noted in its postnatal closure. 6. We conclude that the mouse ductus has a complete system for PGE(2) synthesis comprising both COX1 and COX2. The two enzymes respond differently to indomethacin but, nevertheless, deletion of either one results in NO upregulation. PGE(2) and NO can function synergistically in keeping the ductus patent.

Dynamics of cell cycle regulators: artifact-free analysis by recultivation of cells synchronized by centrifugal elutriation.

Studies on the molecular properties of cell cycle regulators in animal cells require cell preparations highly enriched in particular cell cycle phases. Centrifugal elutriation is frequently used to synchronize cells because this technique was thought to cause only minimal distortions in protein expression or metabolic functions. However, in primary chicken erythroblasts, we consistently observed artefacts in mitotic cyclin mRNA expression and p70 S6 kinase activity, which were clearly caused by the elutriation procedure. Therefore, we modified the standard protocol by reseeding various elutriated fractions into preconditioned medium, a process termed recultivation, and harvesting after an appropriate amount of time. This avoided the pleiotropic effects caused by stress and lack of growth factor supply during elutriation. Using this recultivation procedure, highly synchronous progression starting from any given cell cycle phase could be achieved for a variety of cell types, including primary, factor-dependent cells of hematopoietic origin. Mitotic cyclin expression and S6 kinase activity was found to be normal again in recultivated cultures, as opposed to elutriated ones. Finally, monitoring of mitosis-specific cyclin A degradation in recultivated G2 phase cells showed that recultivation provided an excellent tool to follow cells through M phase into G1 without the requirement for a chemical cell cycle block.

Cyclooxygenase-2 mediates the febrile response of mice to interleukin-1beta.

Various lines of evidence have implicated cyclooxygenase (COX)-2 as a modulator of the fever induced by the exogenous pyrogen lipopolysaccharide (LPS). Thus, treatment with specific inhibitors of COX-2 suppresses the febrile response without affecting basal body (core) temperature (T(c)). Furthermore, COX-2 gene-ablated mice are unable to develop a febrile response to intraperitoneal (i.p.) LPS, whereas their COX-1-deficient counterparts produce fevers not different from their wild-type (WT) controls. To extend the apparently critical role of COX-2 for LPS-induced fevers to fevers produced by endogenous pyrogens, we studied the thermal responses of COX-1- and COX-2 congenitally deficient mice to i.p. and intracerebroventricular (i.c.v.) injections of recombinant murine (rm) interleukin (IL)-1beta. We also assessed the effects of one selective COX-1 inhibitor, SC-560, and two selective COX-2 inhibitors, nimesulide (NIM) and dimethylfuranone (DFU), on the febrile responses of WT and COX-1(-/-) mice to LPS and rmIL-1beta, i.p. Finally, we verified the integrity of the animals' responses to PGE2, i.c.v. I.p. and i.c.v. rmIL-1beta induced similar fevers in WT and COX-1 knockout mice, but provoked no rise in the T(c)s of COX-2 null mutants. The fever produced in WT mice by i.p. LPS was not affected by SC-560, but it was attenuated and abolished by NIM and DFU, respectively, while that caused by i.p. rmIL-1beta was converted into a T(c) fall by DFU. There were no differences in the responses to i.c.v. PGE2 among the WT and COX knockout mice. These results, therefore, further support the notion that the production of PGE2 in response to pyrogens is critically dependent on COX-2 expression.

The febrile response to lipopolysaccharide is blocked in cyclooxygenase-2(-/-), but not in cyclooxygenase-1(-/-) mice.

Various lines of evidence have implicated inducible cyclooxygenase-2 (COX-2) in fever production. Thus, its expression is selectively enhanced in brain after peripheral exogenous (e.g., lipopolysaccharide [LPS]) or endogenous (e.g., interleukin-1) pyrogen administration, while selective COX-2 inhibitors suppress the fever induced by these pyrogens. In this study, we assessed the febrile response to LPS of congenitally constitutive COX-1 (COX-1-/-) and COX-2 (COX-2-/-)-deficient C57BL/6J-derived mice. COX-1+/- and COX-2+/- mice were also evaluated; controls were wild-type C57BL/6J mice (Jackson Labs.). All the animals were pretrained daily for two weeks to the experimental procedures. LPS was injected intraperitoneally at 1 microgram/mouse; pyrogen-free saline (PFS) was the vehicle and control solution. Core temperatures (Tcs) were recorded using thermocouples inserted 2 cm into the colon. The presence of the COX isoforms was determined in cerebral blood vessels immunocytochemically after the experiments, without knowledge of the functional results. The data showed that the wild-type, COX-1+/-, and COX-1-/- mice all responded to LPS with a 1 degrees C rise in Tc within 1 h; the fever gradually abated over the next 4 h. By contrast, COX-2+/- and COX-2-/- mice displayed no Tc rise after LPS. PFS did not affect the Tc of any animal. It would appear therefore that COX-2 is necessary for LPS-induced fever production.

Obesity is induced in mice heterozygous for cyclooxygenase-2.

In mice heterozygous for the cyclooxygenase-2 gene (COX-2+/-) the body weight was enhanced by 33% as compared to homozygous COX-2-/- mice. The weights of the gonadal fat pads in COX-2+/- mice were enhanced by 3.5 to 4.7 fold as compared to COX-2-/- mice and by 1.5 to 3.5 fold as compared to wild-type controls+/+ Serum leptin levels and leptin release by cultured adipose tissue of COX-2+/- mice were both elevated as compared to either control or COX-2-/- animals. The basal release of PGE2 or 6 keto PGF1alpha per fat pad over a 24 h incubation of adipose tissue was reduced by 80% and 95% respectively in tissue from COX-2-/- mice. NS-398, a specific COX-2 inhibitor, inhibited leptin release by 27% in adipose tissue from control mice, 31% in tissue from COX-1-/- mice and by 23% in tissue from COX-2+/- mice while having no effect on leptin release by adipose tissue from COX-2-/- mice. These data indicate that heterozygous COX-2 mice develop obesity which is not secondary to a defect in leptin release by adipose tissue.

Separation of yeast asparagine-linked oligosaccharides by high-performance anion-exchange chromatography.

Oligosaccharides obtained from Saccharomyces cerevisiae mannoproteins by digestion with endo-N-acetyl-beta-D-glucosaminidase H were fractionated by anion-exchange chromatography, by elution with 50-100mM NaOH without or with a sodium-acetate gradient, and detected with a pulsed amperometric detector (PAD). The elution times of homologous oligosaccharides fell on a straight line having a slope characteristic of the structural type. The response of the PAD detector per mole of oligosaccharide increased about 2-fold going from Man3GlcNAc to Man13GlcNAc, and appeared to depend primarily on the oxidation of the reducing-end N-acetylglucosamine unit common to all the oligosaccharides. The digestion of a Man10GlcNAc with jack-bean alpha-mannosidase was monitored by injecting portions of the crude reaction mixture, and the intermediates were characterized by their elution positions and n.m.r. spectra in the anomeric proton region. One commercial jack-bean alpha-mannosidase preparation contained a novel endolytic activity that released N-acetylglucosamine from the reducing ends of the oligosaccharides and was shown to convert P----6 alpha Man----6 alpha Man----6 beta Man----4 alpha beta GlcNAc to P----6 alpha Man----6 alpha Man----6 alpha beta Man plus free N-acetylglucosamine. Another commercial jack-bean alpha-mannosidase converted the Man10GlcNAc to a Man3GlcNAc having the structure alpha Man----6 beta Man----4 alpha beta GlcNAc, [formula: see text] whereas the Oerskovia sp. alpha-mannosidase converted the same oligosaccharide to a Man4GlcNAc having the structure alpha Man----6 alpha Man----6 beta Man----4 alpha beta GlcNAc. [formula: see text]