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R Nüsing

Publications and source records attributed to R Nüsing.

9 recordsLinked to original sources
PubMed1992

Induction of thromboxane synthase and prostaglandin endoperoxide synthase mRNAs in human erythroleukemia cells by phorbol ester.

The effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the mRNA levels of two enzymes, thromboxane synthase (TXS) and prostaglandin endoperoxide synthase (PES), responsible for the synthesis of thromboxane A2 from arachidonic acid, were studied in human erythroleukemia (HEL) cells by RNA blot analysis. TPA induced both TXS and PES mRNAs in HEL cells in a dose-dependent manner at 36 h. The half-maximal and maximal effects for the induction of both mRNAs were at approximately 3 x 10(-9) M and at 10(-8) M, respectively. TXS and PES mRNA levels increased in a time-dependent fashion by TPA, and reached to 7- and 3.5-fold of the control, respectively after 48 h of TPA treatment. These results suggest that expression of TXS and PES genes in HEL cells were simultaneously stimulated by TPA.

Cyclooxygenase Inhibitors↗
PubMed1992

Regulation of cyclooxygenase and thromboxane synthase in human monocytes.

Stimulation of human monocytes by lipopolysaccharide or phorbol ester resulted in an increase in thromboxane-B2 and prostaglandin-E2 production, whereas interleukin 1, tumour necrosis factor alpha and leukotriene C4 exerted no effects. Inhibitors of protein kinase C suppressed these increases. The activity of cyclooxygenase was induced 3.2-fold by an 8-h stimulation, whereas thromboxane-synthase and prostaglandin-E-isomerase activities remained unchanged. A glucocorticoid, dexamethasone, blocked both basal and induced prostanoid release, as well as cyclooxygenase activity. By immunoprecipitation, we were able to demonstrate an enhanced de novo synthesis of cyclooxygenase protein induced by lipopolysaccharide and phorbol ester. Dexamethasone suppressed cyclooxygenase synthesis, whereas thromboxane synthase was induced. For cyclooxygenase, we calculated a half-life of 3.2 h in human monocytes, and for thromboxane synthase, a half-life of 28 h. These results suggest that the regulation of differential prostanoid production mainly occurs by up and down regulation of cyclooxygenase.

Cells, Cultured↗
PubMed1992

Localization of thromboxane synthase in human tissues by monoclonal antibody Tü 300.

Using the monoclonal antibody Tü 300 we localized thromboxane synthase, a secondary enzyme of the arachidonic acid cascade, employing the alkaline phosphatase anti-alkaline phosphatase method and indirect double labelling immunofluorescence in frozen sections of human tissues. Aside from platelets, the source of the antigen, all cells of the mononuclear phagocytic system were positive, including epithelioid cells and associated giant cells, starry sky macrophages, dendritic cells of T-cell areas, Langerhans cells and Kupffer cells. In addition, some epithelial cells such as epithelia of tonsillar crypts, reticular epithelia of the thymic cortex and ductular epithelia in liver, pancreas, female breast and salivary glands showed occasional focal reactivity for thromboxane synthase. We suggest that the mAb Tü 300 is a key marker for the macrophage system and the thromboxane generating system in normal and pathological conditions. It may detect functional activities of as yet unknown significance in some specialized epithelial cells.

Blood Platelets↗
PubMed1990

Immunoaffinity purification of human thromboxane synthase.

A recently produced monoclonal antibody against human thromboxane synthase was used to purify the enzyme from platelets in a one-step procedure with good yields. The isolated protein exhibited a single band of about 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained one heme/mol. Although the visible spectrum of the oxidized enzyme displayed a peak at 418 nm like the previously isolated enzyme after dithionite reduction and CO addition, it shifted to 419 nm but not to 450 nm where only a small shoulder could be detected. Its catalytic activity was only 1-5% of the previous preparations, but with the same Km of about 10 microM and a ratio of thromboxane B2: 12-hydroxyheptadecatrienoic acid of 1:1. Studies with EPR spectrometry and inhibitors confirmed that only a minor part of the enzyme was in its native heme-thiolate conformation, whereas the major part had been converted to the inactive P420 form by the elution procedure. The amino acid analysis revealed 46% hydrophobic residues. According to the sequence of 26 amino acids from the N-terminus and two tryptic peptides no homology to one of the cytochrome P450 monooxygenases, or to cyclooxygenase, or to prostacyclin synthase was detected.

Amino Acid Sequence↗
PubMed1990

Production and characterization of polyclonal and monoclonal antibodies against human thromboxane synthase.

Polyclonal and monoclonal antibodies (MoAbs) were raised against human platelet thromboxane (Tx) synthase. Neither the antiserum nor the MoAbs inhibited the enzyme activity significantly. Three MoAbs, Tü 300, Kon 6, and Kon 7, were purified and further characterized. They are monospecific as shown by activity precipitation or Western blot analysis, and recognized different epitopes on Tx-synthase. Tü 300 could precipitate the enzyme and recognized conformational epitopes, whereas Kon 6 and Kon 7 only reacted in Western blots. Antibody Tü 300 can be used in immunohistology but shows no crossreactivity with Tx-synthase from other species. In human lung tissue staining with peroxidase, coupled Tü 300 was only found in alveolar macrophages.

Antibodies↗
PubMed1990

Immunohistochemical localization of thromboxane synthase in human tissues.

Thromboxane A2 is formed mainly in platelets where we have isolated thromboxane synthase as a cytochrome P450-like haemoprotein. A rabbit antiserum prepared against this enzyme was monospecific according to Western blot analysis and was used for immunostaining of human tissues. In liver the Kupffer cells were clearly stained and in the connective tissue, histiocytes and monocytic cells were positive. In lung, alveolar macrophages contained most of the enzyme, whereas Type I and II cells were negative. Kidney mesangial cells were stained and in lymphatic tissue dendritic reticular cells as well as invading macrophages were positive. These were also seen in the crypts of tonsils and surprisingly also the epithelium. Placenta again showed staining of the Hofbauer cells and of the endothelial lining of the vessels. Umbilical cord and uterus only contained weak antigenicity in dendritic cells.

Female↗
PubMed1990

Immunoquantitation of thromboxane synthase in human tissues.

Although the formation of TxB2 has been reported for different cells such as monocytes, lymphocytes, granulocytes or fibroblasts and tissues like lung and liver, the corresponding enzyme has not yet been measured. We have established an enzyme-linked immunosorbent assay (ELISA) for measurement of human Tx-synthase with a monospecific polyclonal and a purified monoclonal antibody. ELISA data were verified by either western blots or immunohistochemistry. Next to platelets, blood monocytes were found to have the highest content of Tx-synthase, whereas lung fibroblasts and promyelocytic cells possess only very low levels. In granulocytes and lymphocytes neither Tx-synthase nor its activity could be demonstrated. Among different tissues, lung and liver showed the highest content of Tx-synthase. These data correlate TxB2-synthase activity in a given human tissue with its enzyme content of Tx-synthase for the first time. In view of the frequent involvement of TxA2 in pathological conditions, the technique developed may prove valuable for the characterization of disease states, particularly inflammatory events.

Blotting, Western↗