The urokinase plasminogen activator receptor (uPAR) is a multifunctional, GPI-linked receptor that modulates cell adhesion/migration and fibrinolysis. We mapped the interaction sites between soluble uPAR (su-PAR) and high molecular mass kininogen (HK). Binding of biotin-HK to suPAR was inhibited by HK, 56HKa, and 46HKa with an IC 50 of 60, 110, and 8 nM, respectively. We identified two suPAR-binding sites, a higher affinity site in the light chain of HK and 46HKa (His 477 -Gly 496 ) and a lower affinity site within the heavy chain (Cys 333 -Lys 345 ). HK predominantly bound to suPAR fragments containing domains 2 and 3 (S-D2D3). Binding of HK to domain 1 (S-D1) was also detected, and the addition of S-D1 to S-D2D3 completely inhibited biotin-HK or -46HKa binding to suPAR. Recent investigations indicate that high molecular mass kininogen (HK)1 binds to endothelial cell membranes through an interaction with a multiprotein receptor complex comprising at least cytokeratin 1, gC1qR, and the urokinase plasminogen activator receptor (uPAR) (1-5). The three proteins co-localize on the endothelial cell membrane (6). The same three proteins form a receptor complex for factor XII (7), but binding of factor XII in vivo is likely limited both by the low plasma concentration of free Zn 2ϩ , which is below the requirement for FXII binding and by the much higher plasma concentration of HK (7). Binding of HK to this multiprotein receptor complex predominates, localizing prekallikrein (PK) to the cell surface. The plasma concentration of PK and the ambient free Zn 2ϩ concentration in plasma also prevent FXI from binding to HK under conditions where platelets or other cells are not activated (8). PK bound to HK on endothelial cells is proteolyzed by membrane-expressed prolylcarboxypeptidase to form plasma kallikrein (9, 10). This multiprotein receptor complex thereby regulates the assembly and activation of the plasma kallikrein/ kinin system.The requirements for HK binding to each component of this receptor complex and the effects of other biologically relevant ligands, e.g. urokinase, on this binding have not been well delineated. It is known that both the heavy and light chains of HK interact with a region of cytokeratin 1 coded by exon 1 (2). Antibody to this region completely inhibits HK binding to cytokeratin 1 as well as to the receptor complex (6). Likewise, some antibodies to gC1qR and uPAR completely block HK binding to the proteins individually as well as when they are part of the complex expressed on endothelial cells (6, 8). However, it is unclear whether each component of the cellular complex recognizes discrete or overlapping portions of HK and whether each molecule of HK binds to more than one component of the complex at the same time. To begin to address these issues, we sought to identify the regions in HK and uPAR required for binding. The results indicate the existence of multiple potential sites of interaction between this ligand and receptor and provide insight into the assembly of HK on its multireceptor co...

Few directed searches have been undertaken to identify the genes involved in vertebrate lens formation. In the frog Xenopus, the larval cornea can undergo a process of transdifferentiation to form a new lens once the original lens is removed. Based on preliminary evidence, we have shown that this process shares many elements of a common molecular/genetic pathway to that involved in embryonic lens development. A subtracted cDNA library, enriched for genes expressed during cornea-lens transdifferentiation, was prepared. The similarities/identities of specific clones isolated from the subtracted cDNA library define an expression profile of cells undergoing cornea-lens transdifferentiation ("lens regeneration") and corneal wound healing (the latter representing a consequence of the surgery required to trigger transdifferentiation). Screens were undertaken to search for genes expressed during both transdifferentiation and embryonic lens development. Significantly, new genes were recovered that are also expressed during embryonic lens development. The expression of these genes, as well as others known to be expressed during embryonic development in Xenopus, can be correlated with different periods of embryonic lens induction and development, in an attempt to define these events in a molecular context. This information is considered in light of our current working model of embryonic lens induction, in which specific tissue properties and phases of induction have been previously defined in an experimental context. Expression data reveal the existence of further levels of complexity in this process and suggests that individual phases of lens induction and specific tissue properties are not strictly characterized or defined by expression of individual genes.

No abstract

Urokinase plasminogen activator (uPA) and PA inhibitor type 1 (PAI-1) are elevated in acute lung injury, which is characterized by a loss of endothelial barrier function and the development of pulmonary edema. Two-chain uPA and uPA-PAI-1 complexes (1-20 nM) increased the permeability of monolayers of human pulmonary microvascular endothelial cells (PMVECs) in vitro and lung permeability in vivo. The effects of uPA-PAI-1 were abrogated by the nitric-oxide synthase (NOS) inhibitor L-NAME (N D -nitro-L-arginine methyl ester). Two-chain uPA (1-20 nM) and uPA-PAI-1 induced phosphorylation of endothelial NOS-Ser 1177 in PMVECs, which was followed by generation of NO and the nitrosylation and dissociation of ␤-catenin from VE-cadherin. uPA-induced phosphorylation of eNOS was decreased by anti-low density lipoprotein receptor-related protein-1 (LRP) antibody and an LRP antagonist, receptor-associated protein (RAP), and when binding to the uPA receptor was blocked by the isolated growth factor-like domain of uPA. uPAinduced phosphorylation of eNOS was also inhibited by the protein kinase A (PKA) inhibitor, myristoylated PKI, but was not dependent on PI3K-Akt signaling. LRP blockade and inhibition of PKA prevented uPA-and uPA-PAI-1-induced permeability of PMVEC monolayers in vitro and uPA-induced lung permeability in vivo. These studies identify a novel pathway involved in regulating PMVEC permeability and suggest the utility of uPAbased approaches that attenuate untoward permeability following acute lung injury while preserving its salutary effects on fibrinolysis and airway remodeling.

Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine β-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674–680, 1994). This paper reports the cloning and nucleotide sequencing of the S. typhimurium apeE gene as well as some properties of the esterase that it encodes. The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens. The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C6 to C16 fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing Salmonella spp. from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates thatEscherichia coli K-12 does not contain a close analog ofapeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E. coli.

No abstract

There is a clinical need for pragmatic approaches to measure integrated hemostatic reactions in whole blood rapidly, using small volumes of blood. The authors have applied T2 magnetic resonance (T2MR) to assess coagulation reactions based on partitioning of red blood cells and proteins that occurs during fibrin formation and platelet-mediated clot contraction. T2MR is amenable to measuring clotting times, individual coagulation factors, and platelet function. T2MR also revealed a novel "hypercoagulable" signature characterized by fibrin clots almost insusceptible to fibrinolysis that surround tessellated arrays of polyhedral erythrocytes ("third peak"). This signature, which develops under conditions associated with intense clot formation in vitro, may help identify patients at risk of developing thrombosis and for monitoring antithrombotic therapies in the future.

Previous studies have identified intervening sequences that encode homing endonucleases within the genes encoding several archaeal DNA polymerases. We report the sequence of the gene encoding the DNA polymerase of Methanococcus voltae and describe evidence that it lacks analogous intervening sequences.Recent studies have identified the presence of in-frame insertions in the structural genes encoding the DNA polymerases of the archaeon Thermococcus littoralis (7,10) and Pyrococcus species strain GB-D (17). In both cases, the primary translation product is processed to yield an internal protein(s) (termed the intein[s] [11]) and the active mature protein formed by the joining of the external sequences (termed the extein [11]). Similar protein-splicing events have been described for the production of the mature 69-kDa subunit of the vacuolar ATPase of Saccharomyces cerevisiae (3,4,6,8) and Candida tropicalis (5) and the RecA protein from Mycobactenium tuberculosis and Mycobacterium leprae (2). In each of these systems, the inteins have sequence similarity to homing endonucleases, and in a few cases endonuclease activity has been directly established (13).To explain the finding that alleles without inteins are found in related species, it has been suggested that inteins may spread through lateral transfer (13). Thus, the pattern of specific intein types among species can provide information on the history of genetic exchange between species and, in addition, provide a perspective on the evolution of genome content and organization.We report the DNA sequence of a DNA polymerase gene from a methanogen, a representative of the Euryarchaeota branch of the domain Archaea (16). We identified the Methanococcus voltae DNA polymerase gene through an analysis of an open reading frame immediately upstream of the methanogen S-layer structural gene (9). A search of protein sequence databases by the National Center of Biotechnology Information Blastp program (1) yielded highest sequence similarities to the DNA polymerases of Pyrococcus furiosus, Pyrococcus species strain GB-D, and T littoralis (identity to each, 45%). The complete sequence of the M. voltae DNA polymerase gene can be obtained from GenBank (accession no. L33366).The a-type DNA polymerases have characteristic interspersed regions that are thought to comprise functional domains (15). These motifs are easily identified in the M. voltae DNA polymerase and are present in the same order as that observed in a-type enzymes (Fig. 1). For comparison, similar regions for the human a-type DNA polymerases are shown.While the length of the presumptive primary translation product of the M. voltae gene (823 amino acids) differs substantially from those of the primary translation products of the genes encoding the Pyrococcus species strain GB-D and T. * Corresponding author.littoralis DNA polymerases (1,311 and 1,702 amino acids, respectively), the lengths of the mature forms of these polymerases are more similar (775 and 774 amino acids, respectively) to that of the M. voltae tran...