Being Born Large for Gestational Age is Associated with Increased Global Placental DNA Methylation.

Being born small (SGA) or large for gestational age (LGA) is associated with adverse birth outcomes and metabolic diseases in later life of the offspring. It is known that aberrations in growth during gestation are related to altered placental function. Placental function is regulated by epigenetic mechanisms such as DNA methylation. Several studies in recent years have demonstrated associations between altered patterns of DNA methylation and adverse birth outcomes. However, larger studies that reliably investigated global DNA methylation are lacking. The aim of this study was to characterize global placental DNA methylation in relationship to size for gestational age. Global DNA methylation was assessed in 1023 placental samples by LC-MS/MS. LGA offspring displayed significantly higher global placental DNA methylation compared to appropriate for gestational age (AGA; p < 0.001). ANCOVA analyses adjusted for known factors impacting on DNA methylation demonstrated an independent association between placental global DNA methylation and LGA births (p < 0.001). Tertile stratification according to global placental DNA methylation levels revealed a significantly higher frequency of LGA births in the third tertile. Furthermore, a multiple logistic regression analysis corrected for known factors influencing birth weight highlighted an independent positive association between global placental DNA methylation and the frequency of LGA births (p = 0.001).

Etoposide Upregulates Survival Favoring Sphingosine-1-Phosphate in Etoposide-Resistant Retinoblastoma Cells.

Improved knowledge of retinoblastoma chemotherapy resistance is needed to raise treatment efficiency. The objective of this study was to test whether etoposide alters glucosyl-ceramide, ceramide, sphingosine, and sphingosine-1-phosphate (sphingosine-1-P) levels in parental retinoblastoma cells (WERI Rb1) or their etoposide-resistant subclones (WERI EtoR). WERI Rb1 and WERI EtoR were incubated with 400 ng/ml etoposide for 24 h. Levels of glucosyl-ceramides, ceramides, sphingosine, sphingosine-1-P were detected by Q-TOF mass spectrometry. Statistical analysis was done by ANOVA followed by Tukey post-hoc test (p < 0.05). The mRNA expression of sphingolipid pathways enzymes in WERI Rb1, WERI EtoR and four human retinoblastoma tissue samples was analyzed by quantitative real-time PCR. Pathways enzymes mRNA expression confirmed similarities of human sphingolipid metabolism in both cell lines and tissue samples, but different relative expression. Significant up-regulation of sphingosine was seen in both cell lines (p < 0.001). Only sphingosine-1-P up-regulation was significantly increased in WERI EtoR (p < 0.01), but not in WERI Rb1 (p > 0.2). Both cell lines upregulate pro-apoptotic sphingosine after etoposide incubation, but only WERI EtoR produces additional survival favorable sphingosine-1-P. These data may suggest a role of sphingosine-1-P in retinoblastoma chemotherapy resistance, although this seems not to be the only resistance mechanism.

Arsenic-containing hydrocarbons: effects on gene expression, epigenetics, and biotransformation in HepG2 cells.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids found in fish and algae, elicit substantial toxic effects in various human cell lines and have a considerable impact on cellular energy levels. The underlying mode of action, however, is still unknown. The present study analyzes the effects of two AsHCs (AsHC 332 and AsHC 360) on the expression of 44 genes covering DNA repair, stress response, cell death, autophagy, and epigenetics via RT-qPCR in human liver (HepG2) cells. Both AsHCs affected the gene expression, but to different extents. After treatment with AsHC 360, flap structure-specific endonuclease 1 (FEN1) as well as xeroderma pigmentosum group A complementing protein (XPA) and (cytosine-5)-methyltransferase 3A (DNMT3A) showed time- and concentration-dependent alterations in gene expression, thereby indicating an impact on genomic stability. In the subsequent analysis of epigenetic markers, within 72 h, neither AsHC 332 nor AsHC 360 showed an impact on the global DNA methylation level, whereas incubation with AsHC 360 increased the global DNA hydroxymethylation level. Analysis of cell extracts and cell media by HPLC-mass spectrometry revealed that both AsHCs were considerably biotransformed. The identified metabolites include not only the respective thioxo-analogs of the two AsHCs, but also several arsenic-containing fatty acids and fatty alcohols, contributing to our knowledge of biotransformation mechanisms of arsenolipids.

Breaking the Barrier - Potent Anti-Inflammatory Activity following Efficient Topical Delivery of Etanercept using Thermoresponsive Nanogels.

Topical administration permits targeted, sustained delivery of therapeutics to human skin. Delivery to the skin, however, is typically limited to lipophilic molecules with molecular weight of < 500 Da, capable of crossing the stratum corneum. Nevertheless, there are indications protein delivery may be possible in barrier deficient skin, a condition found in several inflammatory skin diseases such as psoriasis, using novel nanocarrier systems. METHODS: Water in water thermo-nanoprecipitation; dynamic light scattering; zeta potential measurement; nanoparticle tracking analysis; atomic force microscopy; cryogenic transmission electron microscopy; UV absorption; centrifugal separation membranes; bicinchoninic acid assay; circular dichroism; TNFα binding ELISA; inflammatory skin equivalent construction; human skin biopsies; immunohistochemistry; fluorescence microscopy; western blot; monocyte derived Langerhans cells; ELISA Results: Here, we report the novel synthesis of thermoresponsive nanogels (tNG) and the stable encapsulation of the anti-TNFα fusion protein etanercept (ETR) (~150 kDa) without alteration to its structure, as well as temperature triggered release from the tNGs. Novel tNG synthesis without the use of organic solvents was conducted, permitting in situ encapsulation of protein during assembly, something that holds great promise for easy manufacture and storage. Topical application of ETR loaded tNGs to inflammatory skin equivalents or tape striped human skin resulted in efficient ETR delivery throughout the SC and into the viable epidermis that correlated with clear anti-inflammatory effects. Notably, effective ETR delivery depended on temperature triggered release following topical application. CONCLUSION: Together these results indicate tNGs hold promise as a biocompatible and easy to manufacture vehicle for stable protein encapsulation and topical delivery into barrier-deficient skin.

Enhanced topical delivery of dexamethasone by ß-cyclodextrin decorated thermoresponsive nanogels.

Highly hydrophilic, responsive nanogels are attractive as potential systems for the topical delivery of bioactives encapsulated in their three-dimensional polymeric scaffold. Yet, these drug carrier systems suffer from drawbacks for efficient delivery of hydrophobic drugs. Addressing this, ß-cyclodextrin (ßCD) could be successfully introduced into the drug carrier systems by exploiting its unique affinity toward dexamethasone (DXM) as well as its role as topical penetration enhancer. The properties of ßCD could be combined with those of thermoresponsive nanogels (tNGs) based on dendritic polyglycerol (dPG) as a crosslinker and linear thermoresponsive polyglycerol (tPG) inducing responsiveness to temperature changes. Electron paramagnetic resonance (EPR) studies localized the drug within the hydrophobic cavity of ßCD by differences in its mobility and environmental polarity. In fact, the fabricated carriers combining a particulate delivery system with a conventional penetration enhancer, resulted in an efficient delivery of DXM to the epidermis and the dermis of human skin ex vivo (enhancement compared to commercial DXM cream: ∼2.5 fold in epidermis, ∼30 fold in dermis). Furthermore, DXM encapsulated in ßCD tNGs applied to skin equivalents downregulated the expression of proinflammatory thymic stromal lymphopoietin (TSLP) and outperformed a commercially available DXM cream.

Increased global placental DNA methylation levels are associated with gestational diabetes.

BACKGROUND: Gestational diabetes mellitus (GDM) is associated with adverse pregnancy outcomes. It is known that GDM is associated with an altered placental function and changes in placental gene regulation. More recent studies demonstrated an involvement of epigenetic mechanisms. So far, the focus regarding placental epigenetic changes in GDM was set on gene-specific DNA methylation analyses. Studies that robustly investigated placental global DNA methylation are lacking. However, several studies showed that tissue-specific alterations in global DNA methylation are independently associated with type 2 diabetes. Thus, the aim of this study was to characterize global placental DNA methylation by robustly measuring placental DNA 5-methylcytosine (5mC) content and to examine whether differences in placental global DNA methylation are associated with GDM. METHODS: Global DNA methylation was quantified by the current gold standard method, LC-MS/MS. In total, 1030 placental samples were analyzed in this single-center birth cohort study. RESULTS: Mothers with GDM displayed a significantly increased global placental DNA methylation (3.22 ± 0.63 vs. 3.00 ± 0.46 %; p = 0.013; ±SD). Bivariate logistic regression showed a highly significant positive correlation between global placental DNA methylation and the presence of GDM (p = 0.0009). Quintile stratification according to placental DNA 5mC levels revealed that the frequency of GDM was evenly distributed in quintiles 1-4 (2.9-5.3 %), whereas the frequency in the fifth quintile was significantly higher (10.7 %; p = 0.003). Bivariate logistic models adjusted for maternal age, BMI, ethnicity, recurrent miscarriages, and familiar diabetes predisposition clearly demonstrated an independent association between global placental DNA hypermethylation and GDM. Furthermore, an ANCOVA model considering known predictors of DNA methylation substantiated an independent association between GDM and placental DNA methylation. CONCLUSIONS: This is the first study that employed a robust quantitative assessment of placental global DNA methylation in over a thousand placental samples. The study provides large scale evidence that placental global DNA hypermethylation is associated with GDM, independent of established risk factors.

Incorporation and visualization of azido-functionalized N-oleoyl serinol in Jurkat cells, mouse brain astrocytes, 3T3 fibroblasts and human brain microvascular endothelial cells.

The synthesis and biological evaluation of azido-N-oleoyl serinol is reported. It mimicks biofunctional lipid ceramides and has shown to be capable of click reactions for cell membrane imaging in Jurkat and human brain microvascular endothelial cells.

Sphingosine-1-phosphate and FTY720 as anti-atherosclerotic lipid compounds.

All stages of atherosclerosis have been identified as a chronic vascular inflammatory disease. In the last few years there is increasing evidence that endogenous lysophospholipids such as sphingosine-1-phosphate (S1P) have potent anti-inflammatory properties. The S1P analogue FTY720 that has been developed as a potent, orally active, immunosuppressant in the field of transplantation and autoimmune disease has interesting effects on inflammatory processes in the arterial vessel wall. S1P targets five specific S1P receptors (S1P(1-5)), which are ubiquitously expressed. S1P(1-3) receptor expression is identified in arterial vessels. S1P and FTY720 show potent silencing effects on some vascular proinflammatory mechanisms in endothelial and vascular smooth muscle cells. In addition, the interaction of monocytes with the vessel wall is inhibited. As shown recently, FTY720 can effectively reduce the progression of atherosclerosis in apolipoprotein E-deficient mice having a high-cholesterol diet. It is not entirely clear which S1P receptor subtype is mainly involved in this process. However, it is currently speculated that the S1P(3) and probably the S1P(1) is involved in the anti-atherosclerotic effects of FTY720. This review summarizes the current knowledge about S1P- and FTY720-effects on mechanisms of vascular inflammatory disease. In addition S1P receptor subtypes are identified which might be interesting for molecular drug targeting.

Characterization of the postjunctional alpha 2C-adrenoceptor mediating vasoconstriction to UK14304 in porcine pulmonary veins.

BACKGROUND AND PURPOSE: In terms of postjunctional alpha(2)-adrenoceptors in the pulmonary circulation, no evidence is available with regard to the receptor subtypes mediating vasoconstriction. Therefore, we characterized the alpha(2)-adrenoceptor subtypes mediating contraction in isolated porcine pulmonary veins. EXPERIMENTAL APPROACH: alpha-adrenoceptor-mediated vasoconstriction was studied using a tissue bath protocol. mRNA profile and relative quantification of alpha(2)-adrenoceptor subtypes were determined in porcine pulmonary veins using reverse-transcriptase polymerase chain reaction (RT-PCR) and real-time PCR. KEY RESULTS: In porcine pulmonary veins, noradrenaline, phenylephrine (alpha(1)-adrenoceptor agonist), UK14304 and clonidine (alpha(2)-adrenoceptor agonists) caused concentration-dependent contractions. The rank order of agonist potency was: NA approximately UK14304 approximately clonidine > phenylephrine. UK14304 responses were antagonised by MK912 (noncompetitive antagonist parameter pD'(2): 10.1), rauwolscine (pK(B): 9.5), yohimbine (pK(B): 9.1), WB4101 (pK(B): 8.7), ARC239 (pK(B): 7.5), prazosin (pK(B): 7.1) and BRL44408 (pK(B): 7.0). Antagonist potencies fitted best with radioligand binding data (pK(i)) at the human recombinant alpha(2C)-adrenoceptor (r(2)=0.96, P=0.0001). Correlation with alpha(2B)-adrenoceptors was lower (r(2)=0.74, P>0.01) and no correlation was obtained with alpha(2A)-adrenoceptors. Moreover, RT-PCR studies in porcine pulmonary veins showed mRNA signals for alpha(2A)- and alpha(2C)-adrenoceptors, but not for alpha(2B)-adrenoceptors, whilst real-time PCR studies indicated a prominent expression of alpha(2C)-adrenoceptor mRNA. CONCLUSIONS AND IMPLICATIONS: Postjunctional alpha(2C)-adrenoceptors mediated contraction in porcine pulmonary veins. alpha(1)-Adrenoceptors also seem to be present in this tissue. Since alpha(2)-adrenoceptor responsiveness is increased when pulmonary vascular tone is elevated, alpha(2C)-adrenoceptor antagonists may be beneficial in diseases such as pulmonary hypertension or congestive heart failure.

The impact of skin viability on drug metabolism and permeation -- BSA toxicity on primary keratinocytes.

For testing cutaneous absorption of drugs, ingredients of cosmetics and also for risk assessment of industrial compounds predictable in vitro test protocols are under investigation using excised skin or reconstructed human epidermis. Since the metabolizing enzymes expressed by viable skin can influence the absorption behaviour of substances by changing their structure and thereby their physicochemical characteristics, the metabolic capacity should be considered in the design of the test protocols of compounds susceptible to metabolism. Then data, generated using viable reconstructed epidermis may reflect the in vivo situation. Interestingly, bovine serum albumin (BSA) commonly used in receptor media in permeation studies to facilitate solubility of highly lipophilic substances strongly inhibited the metabolism of topically applied prednicarbate in reconstructed epidermis. Here, we show that 5% BSA is toxic to reconstructed epidermis and keratinocytes which was consistent with the earlier findings. While media toxicity (deficiency media) was at least partly the cause of both apoptotic and necrotic processes in keratinocytes, BSA only slightly increased the rate of necrotic cells. Moreover, caspase inhibitors did not reduce BSA toxicity. Yet, the results show that BSA toxicity on keratinocytes has to be carefully considered if this protein is used in permeation studies with reconstructed epidermis.

Sphingosine 1-phosphate is involved in cytoprotective actions of calcitriol in human fibroblasts and enhances the intracellular Bcl-2/Bax rheostat.

Calcitriol is originally known to decrease proliferation rates of several carcinoma cells, partly via induction of apoptosis. On the other hand, the secosteroid is revealed to protect some cell types like thyrocytes, HL-60 cells and melanocytes against programmed cell death. Here we report that calcitriol despite its strong antiproliferative effect on human dermal fibroblasts did not induce apoptosis in these cells. In contrast, calcitriol possessed an antiapoptotic action in dermal fibroblasts. Thus, the ability of the apoptotic stimuli TNFalpha/actinomycin and C2-ceramides (C2-Cer) to induce programmed cell death was drastically diminished in the presence of calcitriol. Moreover, we identified sphingosine 1-phosphate (S1P) as a downstream mediator of calcitriol for its cytoprotective property. Thus, the secosteroid could not protect fibroblasts from apoptosis in the presence of N,N-dimethylsphingosine (DMS), which inhibits sphingosine kinase, the crucial enzyme to form S1P. Like calcitriol, S1P in different concentrations did not induce fibroblast apoptosis and moreover drastically decreased the rates of apoptotic cells after treatment with TNFalpha1/actinomycin. As S1P has been identified to modify the Bcl-2/ Bax ratio in epithelial cells and keratinocytes, we also measured the expression of these proteins in dermal fibroblasts revealing an increased Bcl-2 level after stimulation with S1P while the Bax protein expression was not modified. In conclusion, calcitriol H was revealed to protect human fibroblasts from apoptosis by formation of S1P resulting in a changed Bcl-2/Bax ratio.

Glucocorticoids for human skin: new aspects of the mechanism of action.

Topical glucocorticoids have always been considered first-line drugs for inflammatory diseases of the skin and bronchial system. Applied systemically, glucocorticoids are used for severe inflammatory and immunological diseases and the inhibition of transplant rejection. Owing to the progress in molecular pharmacology, the knowledge of the mechanism of action has increased during the last years. Besides distinct genomic targets, which are due to the activation of specific cytoplasmatic receptors resulting in the (trans-) activation or (trans-) repression of target genes, there are non-genomic effects on the basis of the interference with membrane-associated receptors as well as with membrane lipids. In fact, various glucocorticoids appear to differ with respect to the relative influence on these targets. Thus, the extended knowledge of glucocorticoid-induced cellular signalling should allow the design and development of even more specifically acting drugs - as it has been obtained with other steroids, e.g. estrogens for osteoporosis prevention.

New chimera proteins for fluorescence correlation spectroscopy.

A new class of chimera proteins has been developed. They are ideally suited for detection by fluorescence correlation spectroscopy (FCS), a new technology to analyze molecular interactions. The molecular structure of these chimera proteins consists of four domains: a N-terminal (His)6-tag for affinity chromatography followed by an eight amino acid epitope for immunodetection, a polypeptide affinity domain (ADF) for target specific interaction and a C-terminal Green Fluorescent Protein (GFPuv) for reporting of interaction with the target by FCS. We designed, prepared and characterized a prototype of ADF-GFP proteins capable of specific interaction with DNA fragments bearing nuclear factor (NF)-kappaB sites. ADF NF-kappaB p50 and a non-DNA-binding deletion mutant (p35) combined with GFPuv were inserted in a procaryotic vector and expressed in E. coli. Following affinity purification the fluoroproteins p50-GFPuv and p35-GFPuv were employed in specific protein-protein and protein-DNA interaction studies. FCS analysis as well as EMSA showed that p50-GFPuv revealed a fully functional ADF. We present a model for the preparation of GFP fusion proteins capable of specific interaction with proteins, lipids or nucleic acids. The rational design allows any polypeptide fragment to be incorporated into the chimeric protein. So a new series of bio-molecules with different binding specificities and assays can be developed.

Glucocorticoids mediate differential anti-apoptotic effects in human fibroblasts and keratinocytes via sphingosine-1-phosphate formation.

Glucocorticoids are potent anti-inflammatory and immunomodulatory drugs which also induce growth inhibition in a variety of cell types. For this reason long-term treatment of inflammatory skin diseases may result in irreversible skin atrophy. To elucidate whether the antiproliferative action of glucocorticoids in fibroblasts is accompanied by induction of apoptosis we investigated the influence of dexamethasone (DEX) on both parameters. Interestingly, we revealed that growth inhibitory concentrations of this glucocorticoid did not induce fibroblast apoptosis. Moreover, DEX protected these cells from apoptosis induced by tumor necrosis factor alpha (TNFalpha)/actinomycin, UV-irradiation, and cell permeable ceramides. These findings are in contrast to the lack of anti-apoptotic effects detected in keratinocytes. Although DEX inhibited TNFalpha mediated nuclear factor-kappa (NF-kappaB) activity in fibroblasts, this mechanism was not involved in its cytoprotection as it was verified by specific NF-kappaB inhibitors. Therefore, we looked for alternative intracellular mediators. Coincubation of fibroblasts with the sphingosine kinase inhibitor N,N-dimethylsphingosine, which blocks formation of the sphingolipid degradation product sphingosine-1-phosphate (S1P), abrogated the protective glucocorticoid effect almost completely. As preincubation with S1P reduced the number of apoptotic cells after stimulation with TNFalpha/actinomycin and moreover DEX increased the intracellular S1P content a role of this sphingolipid in the cytoprotection by DEX is suggested.

Transcriptional activity of potent glucocorticoids: relevance of glucocorticoid receptor isoforms and drug metabolites.

As compared to standard glucocorticoids (GC), prednicarbate (PC) is favorable in the treatment of eczema due to its high benefit/risk ratio. The remarkable anti-inflammatory effects of PC are in strong contrast to its reported low glucocorticoid receptor (GR) binding affinity. In transfected COS-7 cells we related the transcriptional potencies of PC, its metabolites and conventional GC to their receptor binding properties. Moreover, the expression pattern of the human GR isoform hGRalpha and its mutual dominant negative inhibitor hGRbeta in skin cells have been investigated as well as the influence of hGRbeta on receptor binding and transactivation. hGRalpha mRNA and protein was largely overexpressed in skin cells. hGRbeta showed no influence on hGRalpha binding and transactivation. Concentration response curves indicated the greater transactivation potency of betamethasone 17-valerate followed by dexamethasone and prednisolone 17-ethylcarbonate. Native PC appeared almost as potent as dexamethasone. With both a strong correlation was observed between transactivation and GR binding.

Depolarisation induces rapid and transient formation of intracellular sphingosine-1-phosphate.

Formation of sphingosine-1-phosphate (SPP) by sphingosine kinase serves as a signalling pathway for various membrane receptors. Here, we show that membrane depolarisation is another mechanism by which this pathway can be activated. Formation of [(3)H]SPP as well as levels of endogenous SPP were rapidly and transiently increased in PC12 pheochromocytoma cells depolarised with high KCl. Time course and maximum were similar to those induced by bradykinin. Depolarisation-induced SPP production was also observed in RINm5F insulinoma cells, dependent on extracellular Ca(2+) and fully suppressed by verapamil, thus apparently caused by Ca(2+) influx via voltage-gated Ca(2+) channels. Studies with sphingosine kinase inhibitors and overexpression of sphingosine kinase revealed a partial contribution of this pathway to depolarisation-induced noradrenaline release and Ca(2+) increase.

1Alpha,25-dihydroxyvitamin D3 protects human keratinocytes from apoptosis by the formation of sphingosine-1-phosphate.

Owing to its ability to induce growth arrest and differentiation of keratinocytes, 1alpha,25-dihydroxyvitamin D3 and its analogs are useful for the treatment of hyperproliferative skin diseases, such as psoriasis vulgaris. It has been implicated that the 1alpha,25-dihydroxyvitamin D3-induced differentiation of keratinocytes is mediated, at least in part, by the formation of ceramides; however, ceramides have also been identified to induce apoptosis in many cells, including keratinocytes. Therefore, it was of interest to investigate the influence of 1alpha,25-dihydroxyvitamin D3 on apoptosis in keratinocytes. Most interestingly, physiological concentrations of 1alpha,25-dihydroxyvitamin D3 did not induce apoptosis in keratinocytes, despite the formation of ceramides. Moreover, 1alpha,25-dihydroxyvitamin D3 appeared cytoprotective and made keratinocytes resistant to apoptosis induced by ceramides, ultraviolet irradiation, or tumor necrosis factor-alpha. The cytoprotective effect was accompanied by the formation of the sphingolipid breakdown product sphingosine-1-phosphate, which prevented apoptosis in analogy to 1alpha,25-dihydroxyvitamin D3. The effect of 1alpha,25-dihydroxyvitamin D3 was specific as the almost inactive precursor cholecalciferol neither induced sphingosine-1-phosphate formation nor prevented cells from apoptosis. Besides this, the cytoprotective aptitude of 1alpha,25-dihydroxyvitamin D3 was completely abolished by the sphingosine kinase inhibitor N,N-dimethylsphingosine, which blocked sphingosine-1-phosphate formation. Moreover, sphingosine-1-phosphate was able to restore the cytoprotective effect of 1alpha,25-dihydroxyvitamin D3 in the presence of N,N-dimethylsphingosine. Taken together, here we report for the first time that 1alpha,25-dihydroxyvitamin D3 protects keratinocytes from apoptosis and additionally this cytoprotection is mediated via the formation of sphingosine-1-phosphate.

Stimulation of nuclear sphingosine kinase activity by platelet-derived growth factor.

Subcellular fractionation revealed that a significant fraction of total sphingosine kinase, the enzyme that phosphorylates sphingosine to form the bioactive lipid metabolite sphingosine-1-phosphate, resides in the nuclei of Swiss 3T3 cells, localized to both the nuclear envelope and the nucleoplasm. Platelet-derived growth factor, in addition to rapidly stimulating cytosolic sphingosine kinase, also induced a large increase in nucleoplasm-associated activity after 12-24 h that correlated with progression of cells to the S-phase of the cell cycle and translocation of sphingosine kinase-green fluorescent protein fusion protein to the nuclear envelope. Our results add sphingosine kinase to the growing list of lipid-metabolizing enzymes associated with the nucleus, and suggest that sphingosine-1-phosphate may also play a role in signal transduction in the nucleus.

An improved high-performance liquid chromatographic method for the determination of sphingosine-1-phosphate in complex biological materials.

Sphingosine-1-phosphate (SPP) has been proposed to act both as an intracellular second messenger and as an extracellular mediator via specific cell surface receptors. Based on the increasing diverse cellular roles methods to quantify endogenous and exogenous SPP are highly required. Here, we report a rapid HPLC method that allows quantification of SPP in the picomolar range even in complex biological systems. A two-step lipid extraction serves to separate SPP from most interfering phospholipids and sphingolipids. Importantly, dihydrosphingosine-1-phosphate (dihydro-SPP), not detectable in all cultured cells and biological samples in considerable amounts, possesses equal extraction properties and therefore is an ideal internal standard. Following extraction SPP and dihydro-SPP are converted to fluorescent isoindol derivatives by ortho-phthaldialdehyde (OPA) and separated by HPLC using a gradient program with methanol and 0.07 M K2HPO4 as eluents. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.5 pM to 0.2 nM. The identity of SPP and dihydro-SPP was confirmed by the use of the ion pair reagent tetraammoniumsulfate, which induced a shift of both peaks but did not alter peak areas. Moreover, enzymatic conversions to sphingosine and sphinganine by bovine intestinal mucosa alkaline phosphatase (AP) excluded the existence of overlapping compounds. Levels of SPP were determined in a variety of biological samples like serum, thrombocytes, primary keratinocytes and several cell lines. Furthermore, we were able to detect increases of intracellular SPP levels in human keratinocytes after exposure to 1alpha,25-dihydroxyvitamin D3(1,25-(OH)2D3) for which a stimulation of sphingosine kinase activity has been recognized.

Cutaneous inflammation and proliferation in vitro: differential effects and mode of action of topical glucocorticoids.

The nonhalogenated double ester of prednisolone, prednicarbate (PC), is the first topical glucocorticoid with an improved benefit/risk ratio verified clinically and in vitro. To evaluate if this is due to unique characteristics of this steroid, a new compound created according to an identical concept, prednisolone 17-ethylcarbonate, 21-phenylacetate (PEP), and the new halogenated monoester desoximetasone 21-cinnamate (DCE) were tested and compared to PC, desoximetasone (DM) and betamethasone 17-valerate (BMV). Isolated foreskin keratinocytes served for in vitro investigations of anti-inflammatory processes in the epidermis, fibroblasts of the same origin were used to investigate the atrophogenic potential. Inflammation was induced by TNFalpha, resulting in an increased interleukin 1alpha (Il-1alpha) synthesis. As quantified by ELISA, all drugs significantly reduced Il-1alpha production. But PC and BMV appeared particularly potent, followed by DM and the two new congeners, which revealed minor anti-inflammatory activity. Glucocorticoid esters including PEP are rapidly degraded in keratinocytes (85% within 12 h). Hence, a ribonuclease protection assay of Il-1alpha mRNA was performed allowing short incubation times and thus minimizing biodegradation. This assay confirmed the anti-inflammatory potency of native PC and BMV. In contrary DCE and PEP did not reduce Il-1alpha mRNA to a significant extent. Therefore PEP acts as a prodrug only. In fibroblasts, Il-1alpha and Il-6 syntheses indicate proliferation and inflammation, respectively. Whereas PC and PEP inhibited Il-1alpha and Il-6 production in fibroblasts only to a minor extent, cytokine synthesis was strongly affected by the conventional glucocorticoids BMV and DM, but also by DCE. The minor unwanted effect of PC and PEP on fibroblasts was also reflected by their low influence on cell proliferation as derived from (3)H-thymidine incorporation. Again, more pronounced antiproliferative features were seen with the halogenated glucocorticoids. In the following, the correlation between antiphlogistic effects in keratinocytes (suppression of Il-1alpha) and antiproliferative effects in fibroblasts (suppression of Il-1alpha and Il-6; (3)H-thymidine incorporation) was analyzed. Here, PC is revealed as the only glucocorticoid with an improved benefit/risk ratio. Native PEP is shown to be almost ineffective and DCE presents exactly the opposite features of PC. It is tempting to speculate if this is due to different glucocorticoid receptor subtypes or different signaling pathways in keratinocytes and fibroblasts.