Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Nucleolar fibrillar centers in mouse spermatid nucleoli

The nucleoli of young spermatids of mice are described. They exhibit a very special shape resembling a "padlock" in which three different areas can be distinguished: (a) a compact zone corresponding to the fibrillar component, (b) the granular component and (c) a fibrillar center of low density. Fibrillar and granular components usually appear segregated. This nucleolus has been reconstructed based on serial sectioning. When the silver impregnation technique is employed, both fibrillar and granular components show a positive reaction, although the fibrillar center is free of granules. The morphology of the fibrillar center seems to be similar to that reported in other cells. The possibility that these fibrillar centers correspond to the nucleolar organizer is discussed.     

Formation of knots in partially replicated DNA molecules

Bacterial plasmids with two origins of replication in convergent orientation are frequently knotted in vivo. The knots formed are localised within the newly replicated DNA regions. Here, we analyse DNA knots tied within replication bubbles of such plasmids, and observe that the knots formed show predominantly positive signs of crossings. We propose that helical winding of replication bubbles in vivo leads to topoisomerase-mediated formation of knots on partially replicated DNA molecules.     

Bi-directional replication and random termination

Two-dimensional (2D) agarose gel electrophoresis was used to study termination of DNA replication in a shuttle vector, YRp7′, when it replicated in Escherichia coli, Saccharomyces cerevisiae and Xenopus egg extracts. In E.coli, the 2D gel patterns obtained were consistent with uni-directional replication initiated at a specific site, the ColE1 origin. In consequence, termination also occurred precisely at the ColE1 origin. In Xenopus egg extracts, the particular shape of the bubble arc as well as the triangular smear detected to the left of the simple-Y pattern indicated random initiation and termination. In S.cerevisiae, initiation occurred at the ARS1 origin and replication proceeded in a bi-directional manner. However, termination did not always occur at a specific site 180° across from the origin, but almost all along the south hemisphere of the plasmid. Inversion, deletion or replacement of DNA sequences located throughout this hemisphere did not eliminate random termination. Analysis of the replication intermediates of another yeast plasmid bearing a different origin, ARS305, also exhibited random termination. We propose that the random termination events observed in S.cerevisiae could be due to an asynchronous departure of both forks from the bi-directional origin in addition to differences in the rate of fork progression. These observations could be extended to all bi-directional origins.     

Replication fork reversal occurs spontaneously after digestion but is constrained in supercoiled domains

Replication fork reversal was investigated in undigested and linearized replication intermediates of bacterial DNA plasmids containing a stalled fork. Two-dimensional agarose gel electrophoresis, a branch migration and extrusion assay, electron microscopy, and DNA-psoralen cross-linking were used to show that extensive replication fork reversal and extrusion of the nascent-nascent duplex occurs spontaneously after DNA nicking and restriction enzyme digestion but that fork retreat is severely limited in covalently closed supercoiled domains.