A genomic clone encoding the Purkinje cell-specific L7 protein has been isolated and utilized to drive the expression of beta-galactosidase in mice. Three independent transgenic lines, germ line transformed with an L7-beta-galactosidase fusion gene, exhibit beta-galactosidase expression in both cerebellar Purkinje cells and retinal bipolar neurons. This distribution is the same as that previously determined for the L7 protein by immunohistochemistry. The transgenic murine lines can be used to obtain populations of marked Purkinje and bipolar neurons. Similar L7 promoter constructs can be used to express other foreign genes specifically in these two classes of neurons.

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

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Aldolase C is regarded as the brain-specific form of fructose-1,6-bisphosphate aldolase whereas aldolase A is regarded as muscle-specific. In situ hybridization of mouse central nervous system using isozyme-specific probes revealed that aldolase A and C are expressed in complementary cell types. With the exception of cerebellar Purkinje cells, aldolase A mRNA is found in neurons; aldolase C message is detected in astrocytes, some cells of the pia mater, and Purkinje cells. We isolated aldolase C genomic clones that span the entire protein coding region from 1.5 kb 5 to the transcription start site to 0.5 kb 3 to the end of the last exon. The bacterial gene, lacZ, was inserted in two different locations and the constructs tested in transgenic mice. When the protein coding sequences were replaced with lacZ, three of five transgenic lines expressed ␤-galactosidase only in cells of the pia mater; one line also expressed in astrocyte-like cells. When lacZ was inserted into the final exon (and all structural gene sequences were retained) transgene expression was observed in astrocytes in all regions of the central nervous system as well as in pial cells. Thus, with the exception of Purkinje cell expression, the behavior of the full-length transgene mimics the endogenous aldolase C gene. The results with the shorter transgene suggest that additional enhancer elements exist within the intragenic sequences. The absence of Purkinje cell staining suggests that the cis elements required for this expression must be located outside of the sequences used in this study.