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H J Leis

Publications and source records attributed to H J Leis.

At least 37 records · Page 2Linked to original sources
PubMed1997

Serum amyloid A (SAA) protein enhances formation of cyclooxygenase metabolites of activated human monocytes.

As serum amyloid A (SAA), an apolipoprotein associated with HDL during the acute-phase reaction may induce Ca2+ mobilization in human monocytes we raised the question whether SAA1 the predominant isoform of human acute-phase SAA is able to alter eicosanoid formation. In resting monocytes SAA1 was without effect on the secretion of cyclooxygenase metabolites while in calcium ionophore A23187- (0.5 and 2.5 microM) stimulated cells SAA1 led to a pronounced dose-dependent increase of TXA2, PGE2, and PGF2alpha. In addition a time-dependent increase of cyclooxygenase metabolites in between 1.5- and 3-fold in the presence of SAA1 was observed; apo A-I, the main HDL-apolipoprotein under non-acute-phase conditions, had no effect. Using sequence-specific anti-human SAA1 peptide (40-63) F(ab)2 fragments we could show that the proposed Ca2+-binding tetrapeptide Gly48-Pro49-Gly50-Gys51 of SAA1 is not responsible for enhanced biosynthesis of cyclooxygenase metabolites. Finally, we could demonstrate that human SAA1 is unable to bind Ca2+-ions, suggesting that SAA1 does not directly enhance eicosanoid biosynthesis via Ca2+ mobilization leading to enhanced phospholipase A2 activity.

Dinoprost↗
PubMed1997

Determination of homovanillic acid and vanillylmandelic acid in neuroblastoma screening by stable isotope dilution GC-MS.

A method for the quantitative determination of homovanillic acid (HVA) and vanillylmandelic acid (VMA), two metabolites of catecholamines, is presented. The assay is based on gas chromatography/electron impact mass spectrometry. The preparation of 13C-labeled VMA from [13C6]vanillin is described. Together with purchased deuterated HVA the 13C-labeled VMA is used as an internal standard in stable isotope dilution GC/MS. The method involves elution from soaked filter-papers, determination of creatinine content, extraction of HVA and VMA from eluted and urinary samples and derivatization to the di-and tri(trimethylsilyl) derivatives, respectively. The detection limits were found to be 4.0 pg for HVA and 0.8 pg for VMA. The method was applied to the routine determination of urinary HVA and VMA in a range from 5 to 100 ng HVA and VMA per microgram creatinine. The lower limits of pathological concentrations are set at 35 ng micrograms-1 creatinine for HVA and to 20 ng micrograms-1 creatinine for VMA, which are in close correlation with the values from other methods, but with the main advantage of reducing the amount of questionable or elevated results from 6.7% (high-performance liquid chromatography (HPLC) alone) to 0.9% (HPLC and GC/MS).

Calibration↗
PubMed1997

[3H]bradykinin receptor-binding, receptor-recycling, and receptor-internalization of the B2 bradykinin receptor in the murine osteoblast-like cell line MC3T3-E1.

Bradykinin (BK) has been demonstrated to induce inositol phosphate production, release of intracellular Ca2+, and prostaglandin E2 (PGE2) synthesis in the murine osteoblast-like cell line MC3T3-E1. Because cellular response to BK is a function of receptor affinity, receptor coupling, and receptor recycling, we investigated kinetic properties, specificity, and regulation at the BK-receptor level on intact, BK-sensitive MC3T3-E1 cells. Our results clearly demonstrate the existence of a single category of binding sites for [3H]BK (kD =366+/-98 pM; Bmax =45.3+/-6.6 fmol/mg of protein). Displacement studies with various BK analogs gave a rank order compatible with a B2 BK-receptor type (BK > Lys-BK > [Hyp3]-BK > Met-Lys-BK > HOE140 > Tyr-BK > Tyr8-BK > D-Arg, [Hyp3, Thi5,8, D-Phe7]-BK > [D-Phe7]-BK > des-Arg9-BK > des-Arg9, [Leu8]-BK = angiotensin II). No atypic high-affinity binding sites for the B1 receptor agonist des-Arg9-BK could be observed. Prestimulation of MC3T3-E1 cells with BK resulted in the disappearance of accessible B2 receptors at the cell surface by internalization. Postexposure of BK-pretreated cells to ligand-free medium resulted in almost complete receptor restoration within 30 minutes, exhibiting an intermediate state of two categories of binding sites (kD1 =444+/-37 pM, Bmax1 =9.2+/-0.3 fmol/mg of protein and kD2 =2.7+/-0.28 pM, Bmax2 =24.2+/-0.2 fmol/mg of protein), probably representing coupled and uncoupled B2 receptors. Prolonged stimulation with BK (2.5-5 h) also revealed the temporal occurrence of two categories of binding sites after 2.5 h (kD1 =228+/-3.5 pM; Bmax1 =15.6+/-0.6 fmol/mg of protein; kD2 =2.7+/-0.25 nM; Bmax2 =40.7+/-1.5 fmol/mg of protein), whereas low-affinity binding sites disappeared after 5 h.

3T3 Cells↗
PubMed1997

Phenotypic heterogeneity of osteoblast-like MC3T3-E1 cells: changes of bradykinin-induced prostaglandin E2 production during osteoblast maturation.

We have examined clonal murine calvarial MC3T3-E1 cells obtained from different sources to compare their osteoblastic features (alkaline phosphatase [ALP], cyclic adenosine monophosphate [cAMP] response to parathyroid hormone, prostaglandin E2 (PGE2) and PGE1, bradykinin-induced production of PGE2). It was found that the sublines investigated showed large variation of the above-mentioned parameters, which may be attributed to distinct differentiated stages of osteoblast development. Increase of ALP activity was paralleled by an increase in cAMP accumulation in response to the above-mentioned agents. The most striking difference was observed with bradykinin-induced production of PGE2. Early stage cells (low ALP) produced high levels of PGE2, whereas cells with high ALP activity showed no bradykinin stimulation at all. This was consistent with the results of specific binding of 3H-bradykinin to its receptor and also correlated well with the bradykinin-induced signal transduction sequence (inositol triphosphate liberation and elevation of intracellular calcium levels). This was confirmed by Northern blot analysis of bradykinin receptor mRNA expression. These results indicate that the widely used osteoblast-like cell line MC3T3-E1 is synonymous for multiple sublines, representing different stages of osteoblast development. These sublines were most likely emerging from the early stage cell line due to the applied culture conditions. Moreover, distinct biochemical features are displayed in correlation to the differentiation stage, thus providing a useful model to study the molecular mechanism of osteoblast maturation.

3T3 Cells↗
PubMed1996

Stable isotope labelling and determination of ketroprofen in human plasma and urine by gas chromatography/negative ion chemical ionization mass spectrometry.

A method for the quantitative measurement of the non-steroidal anti-inflammatory drug ketoprofen in human plasma and urine is presented. The assay is based on gas chromatography/negative ion chemical ionization mass spectrometry. The preparation of stable isotope-labelled ketoprofen for use as an internal standard is described. After solvent extraction from the acidified matrix, urine samples were analysed as pentafluorobenzyl esters, whereas plasma samples required further derivatization to the hydroxylamine-trimethylsilyl derivatives. The detection limit was found to be 5 pg in both cases. The method was applied to the pharmacokinetics of ketoprofen in man after epidermal application.

Administration, Topical↗
PubMed1996

Method for the determination of vitamin K1(20) in human plasma by stable isotope dilution/gas chromatography/mass spectrometry.

A method for the quantitative determination of vitamin K1(20) (VK), an essential cofactor in the carboxylation of clotting factors, is presented. The assay is based on gas chromatography/electron impact mass/spectrometry. The preparation of deuterium-labelled vitamin K1(20) for use as an internal standard is described. The method involves extraction of VK from human plasma and its derivatization to the heptafluorobutyryl ester after reduction of one carbonyl group with zinc. The detection limit was found to be 1.0 pg and the limit of quantitation 2.0 pg ml-1 plasma. This permits the measurement of vitamin K1(20) even in small quantities of plasma, which is highly desirable in investigations dealing with clotting abnormalities in neonates and infants.

Calibration↗
PubMed1996

Inhibition of prostanoid formation in intact cells by 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a blocker of Ca(2+)-ATPases.

1 The blocker of endoplasmic reticulum Ca(2+)-ATPase, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ) was shown to inhibit formation of prostaglandin E2 and prostacyclin in the osteoblast-like cell lines, MC3T3-E1 and ROS 17/2.8, respectively, in a dose-dependent manner with an IC50 of 0.5-1 microM. Inhibition was observed with various stimuli (arachidonic acid, bradykinin, melittin and calcium ionophore, A23187). 2 This effect was also observed in human platelets, where BHQ dose-dependently blocked thromboxane biosynthesis and formation of 12-hydroxy-heptadecatrienoic acid after stimulation with arachidonic acid, but not formation of 12-hydroxy-eicosatetraenoic acid. 3 Inhibition of prostaglandin E2 formation in MC3T3-E1 cells was not observed with thapsigargin after stimulation with arachidonic acid, A23187 or melittin, whereas bradykinin-induced prostaglandin E2 biosynthesis was blocked. 4 Taken together, the results suggest a direct inhibitory action of BHQ on the cyclo-oxygenase in these three cell systems.

Arachidonic Acid↗
PubMed1995

Quantification of 7-dehydrocholesterol in plasma and amniotic fluid by liquid chromatography/particle beam-mass spectrometry as a biochemical diagnostic marker for the Smith-Lemli-Opitz syndrome.

The qualitative and quantitative determination of cholesterol and 7-dehydrocholesterol (7-DHC) in plasma as a biochemical diagnostic marker for the Smith-Lemli-Opitz syndrome by liquid chromatography/particle beam interface-mass spectrometry (LC/PB-MS) is presented. Baseline separation of cholesterol and 7-DHC is achieved on a silica column with hexane+ethanol (99: 1 v/v) as mobile phase within a 10 min analysis. Recoveries of cholesterol and 7-DHC in a simple two-phase extraction system are nearly 100%. The absolute limit of detection using LC/PB-MS is approximately 10 ng. The method presented allows extraction, analysis and quantification of cholesterol and 7-DHC within 15 min without the necessity of sample derivatization.

Amniotic Fluid↗
PubMed1995

The regulatory role of thyroid hormones in bone cell growth and differentiation.

The clinical effects of thyroid hormones on bone in hypo- and hyperthyroidism are well known but their fundamental role in the regulation of bone remodeling is still poorly understood. In this review the current literature is summarized and experimental data from our laboratory are presented. The direct stimulation of bone resorption by thyroid hormones in organ culture, which in part is mediated by prostaglandins and TGF-beta, and the effect of different agents thereon are reviewed. More recent data concerning thyroid hormone action in the osteoblastic cell line MC3T3E1, are summarized. From their effect on proliferation and alkaline phosphatase activity, we conclude that thyroid hormones accelerate osteoblastic differentiation. The regulation of the transcriptional expression of certain genes by nuclear T3 receptors and their effect on osteoblastic target genes like IGF-I are reviewed. In addition a novel role of triiodothyronine as inhibitor of growth factor induced transcriptional expression of regulatory genes (c-fos, c-jun) is suggested.

Alkaline Phosphatase↗
PubMed1995

On the inhibition of prostanoid formation by SK&F 96365, a blocker of receptor-operated calcium entry.

1. The proposed blocker of receptor-operated calcium channels, SK&F 96365 was shown to inhibit formation of prostaglandin E2 in two osteoblast-like cell lines, MC3T3-E1 and UMR-106 in a dose-dependent manner at an IC50 of 3-4 microM. Inhibition was observed with various stimuli (arachidonic acid, bradykinin and calcium ionophore A23187). 2. This effect was also observed in human platelets, where SK&F 96365 dose-dependently blocked thromboxane biosynthesis and formation of 12-hydroxy-heptadecatrienoic acid after stimulation with arachidonic acid (IC50 = 4 microM). 3. The compound had no effect on 12-hydroxy-eicosatetraenoic acid production by human platelets. Additionally, linoleic acid oxidation by soybean 15-lipoxidase was not impaired by SK&F 96365. The results thus provide evidence for cyclo-oxygenase inhibition by SK&F 96365 at concentrations used to block receptor-operated calcium influx.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1995

Lysine modification of LDL or lipoprotein(a) by 4-hydroxynonenal or malondialdehyde decreases platelet serotonin secretion without affecting platelet aggregability and eicosanoid formation.

The effects of lysine-modified atherogenic plasma lipoproteins, known to be constituents of human atherosclerotic plaques, were studied on platelet function in vitro. LDL and lipoprotein(a) [Lp(a)] modified with secondary breakdown products of lipid peroxidation (4-hydroxy-2,3-trans-nonenal [HNE] 0.1 to 10 mmol/L or malondialdehyde [MDA] 1 to 50 mmol/L) induced neither spontaneous platelet aggregation nor secretion of 5-hydroxytryptamine (5-HT) from platelet aminestorage granules. Incubation of platelets with HNE- or MDA-modified LDL or Lp(a) (up to 1200 micrograms protein/mL) prior to thrombin (0.2 U/mL)- or collagen (2 micrograms/mL)-induced aggregation did not enhance platelet aggregability or formation of eicosanoids, ie, thromboxane A2 or prostaglandins E2 and F2 alpha. In contrast to native lipoproteins, HNE- or MDA-modified LDL and Lp(a) (approximately 20% to 30% of total apolipoprotein lysine residues modified) exerted a pronounced dose-dependent inhibition of 5-HT release from activated platelets in the following order: HNE LDL (50%) > HNE Lp(a) (40%) > MDA LDL (20%) > MDA Lp(a) (5%). Preincubation of human blood platelets with acetylated LDL or Lp(a) (approximately 60% to 70% of total lysine residues modified) prior to aggregation impaired serotonin secretion by 50% compared with native LDL or Lp(a). These findings suggest that the interaction of platelets with aldehyde-modified atherogenic plasma lipoproteins should not necessarily be considered as proatherogenic with respect to the effects observed in our in vitro studies.

Acetylation↗
PubMed1995

Effects of heparin and hirudin on thrombin generation and platelet aggregation after intrinsic activation of platelet rich plasma.

The effects of unfractionated heparin (UH) and recombinant hirudin (rH) on prothrombin activation, free thrombin generation, and platelet aggregation induced by endogenously generated thrombin after intrinsic activation of platelet rich plasma were compared. Free thrombin generation and platelet aggregation were assessed simultaneously by delaying fibrinogen polymerisation with GPRP. UH more effectively inhibited prothrombin activation and free thrombin generation than rH. Increasing concentrations of rH had hardly any effect on the peak amount of free thrombin, while in the presence of 400 nM UH only traces of free thrombin were detected. Comparison of TAT and THC (thrombin-hirudin complex) generated until the onset of platelet aggregation on a molar basis showed that much more thrombin was inactivated in the presence of rH than in plasma containing UH. The explosive generation of free thrombin in hirudinized plasmas was accompanied by a markedly steeper aggregation curve as compared to heparinized plasmas. The generation of thromboxane B2 was markedly delayed in the presence of UH but not influenced in the presence of rH. Our results suggest that UH is more effective than rH in inhibiting prothrombin activation after intrinsic activation of platelet rich plasma, while rH prevents clotting more by direct inactivation of already generated thrombin. The inability of even high concentrations of rH to prevent the explosive generation of free thrombin might contribute to the observed inefficiency of rH to inhibit platelet aggregation.

Blood Platelets↗
PubMed1994

Quantitative measurement of amphetamine in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry using (2H5)amphetamine as internal standard.

A selected ion monitoring stable isotope dilution gas chromatographic/negative ion chemical ionization mass spectrometric assay for amphetamine in human plasma is described. The preparation of (2H5)amphetamine from the intact target molecule and its use as an internal standard for quantitative gas chromatography/mass spectrometry are demonstrated. Derivatization to the highly electrophoric pentafluorobenzoate with pentafluorobenzoyl chloride in hexane resulted in high yield of prominent negative ions with methane as a moderating gas. The pentafluorobenzoate was also found to be superior to fluoroacyl derivatives with respect to sample handling due to its lower volatility. The method presented permits detection of amphetamine in the low picogram per millilitre range and should thus be highly useful for its trace level determination in forensic, toxicological and pharmaceutical applications.

Amphetamine↗
PubMed1994

Extracellular Ca2+ sensing by the osteoblast-like cell line, MC3T3-E1.

The present study was undertaken to clarify the role of extracellular calcium on osteoblast activation. It was found that bradykinin and thrombin induced synthesis of prostaglandin E2 was strongly dependent on the concentration of extracellular calcium in the osteoblast-like cell line, MC3T3-E1. Moreover, this effect was not related to Ca2+ influx, since it was even potentiated by Ni2+ and Co2+, which was not due to intracellular activity of Ni2+, as judged by studies with 63Ni2+. Ba2+, Mg2+ and Sr2+ had no effect. Cd2+ caused dose-dependent synthesis of prostaglandin E2, which was shown to correlate with its cytotoxic properties. The results thus strongly suggest the presence of a divalent cation sensor in osteoblast-like MC3T3-E1 cells.

Animals↗
PubMed1993

Cyclooxygenase pathway in dermal fibroblasts from patients with metabolic disorders of peroxisomal origin.

Cyclooxygenase metabolism was studied in fibroblasts from patients with metabolic disorders of peroxisomal origin (adrenomyeloneuropathy, X-linked adrenoleukodystrophy, cerebrohepatorenal syndrome of Zellweger and rhizomelic chondrodysplasia punctata). In response to arachidonic acid (6.25-100 microM) or calcium ionophore A23187 (2.5-20 microM) prostaglandin E2 and 6-keto-prostaglandin F1 alpha are the main cyclooxygenase metabolites formed. No formation of thromboxane B2 or 2,3-dinor-thromboxane B2 was found. Apparently due to the heterogeneous nature of peroxisomal disorders no uniform pattern of cyclooxygenase metabolism and eicosanoid concentrations in cell lines from patients with peroxisomal defects was found.

6-Ketoprostaglandin F1 alpha↗
PubMed1992

Quantitative measurement of N-acetyl-L-aspartic acid in urine by gas chromatography with negative-ion chemical ionization mass spectrometry.

A highly specific and sensitive assay for N-acetyl-L-aspartic acid has been developed. The trideuterated compound was synthesized and used as an internal standard for gas chromatography with negative-ion chemical ionization mass spectrometry. Urine samples were acidified and extracted with ethyl acetate, and the compounds converted into their pentafluorobenzyl ester derivatives. Under these conditions, sub-picogram amounts of the pure derivatives could be detected. Thus, only microliter volumes of urine samples have to be processed to achieve reliable quantification of "basal" levels of N-acetyl-L-aspartic acid.

Aspartic Acid↗
PubMed1991

Deuterium-labelling and quantitative measurement of ketotifen in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry.

A gas chromatographic/mass spectrometric assay for the anti-anaphylactic agent Ketotifen is described. The method is based on negative ion chemical ionization, utilizing the electrophoric nature of the underivatized drug. A simple procedure for deuteration of the intact target molecule is presented, thus enhancing specificity by internal standardization via stable isotope dilution. The assay was applied to pharmacokinetics of the drug in man.

Deuterium↗