In human in-vitro fertilization (IVF)-embryo transfer, the in-vitro culture environment differs from in-vivo conditions in that the oxygen concentration is higher, and in such conditions the mouse embryos show a higher concentration of reactive oxygen species (ROS) in simple culture media. ROS are believed to cause damage to cell membranes and DNA fragmentation in somatic cells. This study was conducted to ascertain the level of H2O2 concentration within embryos and the morphological features of cell damage induced by H2O2. A total of 62 human oocytes and embryos (31 fragmented, 15 non-fragmented embryos, 16 unfertilized oocytes) was obtained from the IVF-embryo transfer programme. The relative intensity of H2O2 concentrations within embryos was measured using 2',7'-dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescence imaging and DNA fragmentation was observed with transmission electron microscopy and an in-situ apoptosis detection kit. The H2O2 concentrations were significantly higher in fragmented embryos (72.21 +/- 9.62, mean +/- SEM) compared to non-fragmented embryos (31.30 +/- 3.50, P < 0.05) and unfertilized oocytes (30.75 +/- 2.67, P < 0.05). Apoptosis was observed only in fragmented embryos, and was absent in non-fragmented embryos. Electron microscopic findings confirmed apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres. We conclude that there is a direct relationship between increased H2O2 concentration and apoptosis, and that further studies should be undertaken to confirm these findings.

Mass screening for pancreatic cancer using CA 19-9 levels in asymptomatic subjects is ineffective because of a very low positive predictive value, despite its high sensitivity and specificity.

Annexin II tetramer (AIIt) is an important endothelial cell surface protein receptor for plasminogen and t-PA. AIIt, a heterotetramer, is composed of two p36 subunits (called annexin II) and two p11 subunits. In this report, we have compared the ability of the isolated p36 and p11 subunits to stimulate t-PA-dependent [Glu]plasminogen activation. The fluid-phase recombinant p11 subunit stimulated the rate of t-PA-dependent activation of [Glu]plasminogen about 46-fold compared to an approximate stimulation of 2-fold by the recombinant p36 subunit and 77-fold by recombinant AIIt. The stimulation of t-PA-dependent activation of [Glu]plasminogen by the p11 subunit was Ca2+-independent and inhibited by epsilon-aminocaproic acid. [Glu]Plasminogen bound to a p11 subunit affinity column and could be eluted with epsilon-aminocaproic acid. Both AIIt and the p11 subunit protected t-PA and plasmin from inactivation by PAI-1 and alpha2-antiplasmin, respectively. A peptide to the C terminus of the p11 subunit (85-Y-F-V-V-H-M-K-Q-K-G-K-K-96) inhibited the p11-dependent stimulation of t-PA-dependent plasminogen activation. In addition, a deletion mutant of the p11 subunit, missing the last two C-terminal lysine residues, retained only about 15% of the activity of the wild-type p11 subunit. Similarly, a mutant AIIt composed of the wild-type p36 subunit and the p11 subunit deletion mutant possessed about 12% of the wild-type activity. These results, therefore, suggest that the C-terminal lysine residues of the p11 subunit bind plasminogen and participate in the stimulation of t-PA-dependent activation of plasminogen by AIIt.

One of the major physiological functions of the proteolytic enzyme plasmin is the degradation and solubilization of fibrin, the major constituent of blood clots. Plasmin has a broad trypsin-like specificity and the production of plasmin from its precursor plasminogen is precisely regulated (reviewed in Refs. 1-5). One way in which plasmin activity is localized to the fibrin clot involves a fibrin-specific mechanism for the conversion of plasminogen to plasmin by tissue-type plasminogen activator (t-PA).1 For example, recent studies have shown that by virtue of its ability to bind both t-PA and plasminogen, fibrin acts as a template that promotes the formation of a t-PA⅐fibrin⅐plasminogen ternary complex. The catalytic efficiency of t-PA-dependent conversion of plasminogen to plasmin is determined by the stability of the ternary complex (6). Thus, fibrin is both a substrate of plasmin and a template for plasmin production. Fibrin also plays a role in the plasmin-dependent stimulation of plasmin formation. For example, the partial proteolysis of fibrin results in the transient generation of new carboxyl-terminal lysine residues that act as high affinity binding sites for the lysine-binding sites of plasminogen (7, Recently, the endothelial cell-surface Ca 2ϩ -binding protein, annexin II, has also been shown to stimulate the t-PA-dependent formation of plasmin from plasminogen (13,14). Annexin II was originally identified as an intracellular Ca 2ϩ -and phospholipid-binding protein and subsequent studies suggested that this protein was potentially involved in the regulation of membrane trafficking events such as exocytosis or endocytosis (reviewed in Ref. 15). Annexin II can exist in cells as both a monomer or as a heterotetramer. The heterotetramer, called annexin II tetramer (AIIt) consists of two annexin II molecules and two molecules of an 11-kDa regulatory subunit referred to as the p11 light chain. The binding of the p11 light chain regulates many of the in vitro activities of annexin II and the biochemical properties of AIIt are distinct from the annexin II monomer (16,17). In many cells such as Madin-Darby canine kidney cells, bovine intestinal epithelial cells, and calf pulmonary arterial endothelial cells, 90 -95% of the total cellular annexin II is present in the heterotetrameric form (18,19). Annexin II and AIIt have been shown to exist on the extracellular surface of many cells although the relative extracellular distribution of the two forms of the protein has not been quantified (13, 20 -23).In the present report, we have compared the kinetics of annexin II and AIIt-dependent activation of t-PA-mediated plasminogen activation. These experiments establish the presence of AIIt on the HUVEC surface and that AIIt is a potent in vitro activator of t-PA-mediated plasminogen activation. EXPERIMENTAL PROCEDURESMaterials-Fibrinogen was obtained from Sigma and further purified by gel permeation chromatography on Superose 12 to remove