Investigations determined that the cell matrix-associated prekallikrein (PK) activator is prolylcarboxypeptidase. PK activation on human umbilical vein endothelial cell (HUVEC) matrix is inhibited by antipain (IC 50 = 50 W WM) but not antifactor XIIa antibody, 3 mM benzamidine, 5 mM iodoacetic acid or iodoacetamide, or 3 mM N-ethylmaleimide. Corn trypsin inhibitor (IC 50 = 100 nM) or Fmoc-aminoacylpyrrolidine-2-nitrile (IC 50 = 100 W WM) blocks matrix-associated PK activation. Angiotensin II (IC 50 = 100 W WM) or bradykinin (IC 50 = 3 mM), but not angiotensin 1^7 or bradykinin 1^5, inhibits matrix-associated PK activation. ECV304 cell matrix PK activator also is blocked by 100 W WM angiotensin II, 1 W WM corn trypsin inhibitor, and 50 W WM antipain, but not angiotensin 1^7. 1 mM angiotensin II or 300 W WM Fmoc-aminoacylpyrrolidine-2-nitrile indirectly blocks plasminogen activation by inhibiting kallikrein formation for single chain urokinase activation. On immunoblot, prolylcarboxypeptidase antigen is associated with HUVEC matrix. These studies indicate that prolylcarboxypeptidase is the matrix PK activator. ß
Fetal calf serum (FCS) and PMA (phorbol 12-myristate-13-acetate) specifically stimulate the synthesis of heparan sulfate proteoglycan in endothelial cells. Staurosporine and n-butanol, kinase inhibitors, abolish the PMA effect. Forskolin and 8-bromo adenosine 3':5'-cyclic monophosphate, activators of, respectively, adenylate cyclase and protein kinase A cannot reproduce the PMA effect. The kinetics of cell entry into S phase of the endothelial cells was determined by DNA synthesis ([3H]-thymidine and Br-dU incorporation), and flow cytometry. The mitogenic effect of fetal calf serum is abolished by PMA. Also, PMA pre-treatment inhibits the enhanced synthesis of heparan sulfate proteoglycan after a second PMA exposure. Remarkably, the stimulation of heparan sulfate proteoglycan synthesis by fetal calf serum and PMA seems to be mainly restricted to G1 phase. Therefore fetal calf serum and PMA cause an enhanced synthesis of heparan sulfate proteoglycan, and PMA causes a cell cycle block at G1 phase.
Dissemination of a malignant tumour is the result of a cascade of events beginning with detachment of cells from primary tumour followed by extravasation and growth at secondary sites. The differences in metastatic ability could be attributed to properties intrinsic to the various tumour types. Thus the clonal selection of tumour cells from successive metastases apparently results in cells better equipped for survival and formation of colonies in secondary sites, indicating that survival is not a random phenomenon. Many studies of malignant cells have correlated the overexpression of adhesion receptors such as integrins and the production of cysteine proteases and glycosidases with the progression of malignancy. The interaction of cysteine proteases with basement membrane components has been implicated in tumour invasion, activation of hormones and growth factors. On the other hand, the expression of the heparanase gene and its protein has been associated with the metastatic potential of several human and mouse tumour cell lines. This study aimed to investigate the correlations between the metastatic properties of clones with high and low metastatic potential and their ability to adhere to the extracellular matrix and to degrade proteins and sulphated glycosaminoglycans present there. Clonal selection of the B16F10 cell line was performed, and the clones were examined for the expression of an integrin-type laminin receptor. A significantly higher level was detected in a high metastatic clone. Enzymatic assays showed higher activity for alpha-d-N-acetylglucosaminidase, beta-d-N-acetylgalactosaminidase and beta-d-glucuronidase in conditioned medium from low metastatic clones compared with that from high metastatic clones. However, higher endopeptidase activity was observed in conditioned medium from high metastatic clones. In summary, these results showed a positive correlation between high metastatic potential and endopeptidase secretion. Similarly, a positive correlation was observed between low metastatic cells and the secretion of glycosaminoglycan-degrading glycosidases.
Presented by A.C.M. PaivaDentate granule cells are generally considered to be relatively resistant to excitotoxicity and have been associated to robust synaptogenesis after neuronal damage. Synaptic reorganization of dentate granule cell axons, the mossy fibers, has been suggested to be relevant for hyperexcitability in human temporal lobe epilepsy and animal models. A recent hypothesis has suggested that mossy fiber sprouting is dependent on newly formed dentate granule cells. However, we have recently demonstrated that cycloheximide (CHX) can block the mossy fiber sprouting that would be otherwise induced by different epileptogenic agents and do not interfere with epileptogenesis in those models. Here, we investigated cell damage and neurogenesis in the dentate gyrus of pilocarpine-or kainate-treated animals with or without the co-administration of CHX. Dentate granule cells were highly vulnerable to pilocarpine induced-status epilepticus (SE), but hardly damaged by kainate induced-SE. CHX-pretreatment markedly reduced the number of injured neurons after pilocarpine-induced SE. Induction of SE dramatically increased the mitotic rate of KA and KA + CHX treated animals. Induction of SE in animals injected with pilocarpine alone led to increases of between two to sevenfold in the mitotic rate of dentate granule cells as compared to increases of between five and thirtyfold for pilocarpine+CHX animals. These observations indicate that in presence of cycloheximide the increase of the mitotic rate after pilocarpine-induced SE may be due to protection of a vulnerable precursor cell population that would otherwise degenerate. We further suggest that the mossy fiber sprouting and neurogenesis of granule cells are not necessarily related events. - ( September 14, 1999 ) . The α1-adrenoceptor antagonist indoramin was used in the rat vas deferens and aorta, against contractions induced by noradrenaline. Indoramin behaved as a competitive antagonist yielding pA 2 values of 7.38 ± 0.05 in rat vas deferens and 6.78 ± 0.14 in aorta. In the presence of cocaine (6µM), the potency (pA 2 ) of indoramin in antagonizing the contractions of the vas deferens to noradrenaline was increased to 8.72 ± 0.07 while its potency remained pratically unchanged in the aorta (6.69 ± 0.12).
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