A binding site for the kringle II domain of prothrombin in the apple 1 domain of factor XI.

Previously we defined binding sites for high molecular weight kininogen (HK) and thrombin in the Apple 1 (A1) domain of factor XI (FXI). Since prothrombin (and Ca(2+)) can bind FXI and can substitute for HK (and Zn(2+)) as a cofactor for FXI binding to platelets, we have attempted to identify a prothrombin-binding site in FXI. The recombinant A1 domain (rA1, Glu(1)-Ser(90)) inhibited the saturable, specific and reversible binding of prothrombin to FXI, whereas neither the rA2 domain (Ser(90)-Ala(181)), rA3 domain (Ala(181)-Val(271)), nor rA4 domain (Phe(272)-Glu(361)) inhibited prothrombin binding to FXI. Kinetic binding studies using surface plasmon resonance showed binding of FXI (K(d) approximately 71 nm) and the rA1 domain (K(d) approximately 239 nm) but not rA2, rA3, or rA4 to immobilized prothrombin. Reciprocal binding studies revealed that synthetic peptides (encompassing residues Ala(45)-Ser(86)) containing both HK- and thrombin-binding sites, inhibit (125)I-rA1 (Glu(1)-Ser(90)) binding to prothrombin, (125)I-prothrombin binding to FXI, and (125)I-prothrombin fragment 2 (Ser(156)-Arg(271)) binding to FXI. However, homologous prekallikrein-derived peptides (encompassing Pro(45)-Gly(86)) did not inhibit FXI rA1 binding to prothrombin. The peptides Ala(45)-Arg(54), Phe(56)-Val(71), and Asp(72)-Ser(86), derived from sequences of the A1 domain of FXI, acted synergistically to inhibit (125)I-rA1 binding to prothrombin. Mutant rA1 peptides (V64A and I77A), which did not inhibit FXI binding to HK, retained full capacity to inhibit rA1 domain binding to prothrombin, and mutant rA1 peptides Ala(45)-Ala(54) (D51A) and Val(59)-Arg(70) (E66A), which did not inhibit FXI binding to thrombin, retained full capacity to inhibit rA1 domain binding to prothrombin. Thus, these experiments demonstrate that a prothrombin binding site exists in the A1 domain of FXI spanning residues Ala(45)-Ser(86) that is contiguous with but separate and distinct from the HK- and thrombin-binding sites and that this interaction occurs through the kringle II domain of prothrombin.

Distribution of leukotriene B4 receptors in human hematopoietic cells.

Leukotriene B4 (LTB4), a product of arachidonic acid metabolism, plays an important role in inflammatory responses. We have cloned from human erythroleukemia cells, a G protein-coupled receptor, designated P2Y(7), which was later identified as the receptor for LTB4 (B-LTR). We have investigated the distribution of LTB4 receptors in various hematopoietic cells. Northern blotting and reverse transcription-coupled polymerase chain reaction (RT-PCR) analyses using radiolabeled LTB4 receptor cDNA as a probe indicated the presence of LTB4 receptor mRNA in peripheral blood leukocytes but not in platelets. Flow cytometry analysis of peripheral blood cells using specific LTB4 receptor antibodies revealed that monocytes, granulocytes, and lymphocytes, but not platelets, express LTB4 receptors. RT-PCR-Southern hybridization analysis revealed that peripheral blood leukocytes and human umbilical vein endothelial cells express the LTB4 receptor. Of the hematopoietic cell lines tested, promonocytic U937 cells, promyelocytic HL-60 cells, K562 cells, and human erythroleukemia cells express the LTB4 receptor. These results suggest a physiological role for the LTB4 receptor in the stimulation of monocytes, neutrophils, and endothelial cells.

Computer-based oral health records on the World Wide Web.

Recently, the World Wide Web has emerged as a platform for computer-based oral health records. Web-based patient records can make teledentistry an instant reality. Because an increasing number of dental care providers can access Web pages, traditional barriers to exchanging information are dropping. Web-based records also make cumulative, longitudinal patient records possible. Sophisticated security mechanisms can ensure the integrity and confidentiality of patient information. Because Web-based systems are simpler to install and configure, the cost of operating them may be reduced. However, their development is complex, difficult, and expensive because the Web was not developed as a programming environment. Furthermore, the technologies underlying the Web are constantly evolving, forcing developers to continuously reengineer their systems. In addition, several policy questions, such as storage of and access to computer-based patient records, have to be answered. This article describes CMSWeb, a Web-based clinical information system implemented at Temple University School of Dentistry.

Distribution of P2Y receptor subtypes on haematopoietic cells.

1. RT-PCR-southern hybridization analyses with radiolabelled P2Y receptor cDNAs as probes indicated that the peripheral blood leukocytes and the human umbilical vein endothelial cells express P2Y1, P2Y2, P2Y4 and P2Y6 receptors. 2. Of the haematopoietic cell lines tested, promonocytic U937 cells express P2Y2 and P2Y6, but not P2Y1 or P2Y4; promyelocytic HL-60 cells express the P2Y1, P2Y2 and P2Y6 receptors but not the P2Y4 receptor; K562 cells express P2Y1 but not P2Y2, P2Y4 or P2Y6; and Dami cells express P2Y1, P2Y2, P2Y4 and P2Y6 receptors. 3. Of the peripheral blood leukocytes tested, polymorphonuclear cells express P2Y4 and P2Y6 but not P2Y1 or P2Y2 receptors; monocytes express P2Y1, P2Y2, P2Y4 and P2Y6 receptors and lymphocytes express P2Y1, P2Y2, P2Y4 and P2Y6 receptors. 4. These results suggest a physiological role for different P2Y receptor subtypes in the extracellular nucleotide-mediated stimulation of monocytes, neutrophils, lymphocytes and endothelial cells.

Molecular cloning of a novel P2 purinoceptor from human erythroleukemia cells.

Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y7, has 352 amino acids and shares 23-30% amino acid identity with the P2Y1-P2Y6 purinoceptors. The P2Y7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 +/- 6 nM), much less for UTP and ADP (approximately 1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.

Characterization of P2 purinergic receptors on human erythro leukemia cells.

We have investigated the nature of the nucleotide receptors on human erythro leukemia (HEL) cells, a cell line with some megakaryocytic properties, using a combination of pharmacological, photoaffinity labeling, and molecular biological techniques. Fura-2 loaded HEL cells responded to 2-methylthio ATP, ATP, 2-methylthio ADP, ADP and UTP with an increase in intracellular calcium. 2 Methylthio ADP was the most potent agonist. When external calcium was chelated with EDTA, calcium responses were observed indicating the mobilization of intracellular stores. These responses showed evidence of both homologous and heterologous receptor desensitization. In photoaffinity labeling experiments, beta-[32P]-AzPET-ADP was incorporated into three protein species with mobilities corresponding to M(r) approximately 55 kDa (doublet) and approximately 43 kDa. Labeling of approximately 55 kDa proteins was specifically inhibited by ADP, while that of the approximately 43 kDa was inhibited specifically by UTP. Nucleotide sequence analysis of the positive clones obtained by screening the HEL cell cDNA library with mouse P2U cDNA revealed that the P2U receptor from HEL cells is identical to the previously cloned human P2U receptor. These experiments suggest that the HEL cells contain a P2Y purinoceptor responding to ADP, in addition to a P2U receptor and possibly also a third P2 purinoceptor with a unique agonist profile.

Mapping of the P2U purinergic receptor gene to human chromosome 11q 13.5-14.1.

We mapped a human P2U purinergic receptor gene to chromosome 11q13.5-14.1. Oligonucleotide primers complementary to a part of the human P2U purinergic receptor cDNA were used to amplify a region from genomic DNAs from a panel of mouse/human somatic cell hybrid cell lines, each containing a single human chromosome. A PCR product of the expected size (378 bp) resulted from a single hybrid cell line containing human chromosome 11. The gene was further localized to a region of chromosome 11 using a sub-chromosomal hybrid panel containing different segments of chromosome 11. Based on the specific PCR product obtained and its Southern hybridization to the P2U receptor cDNA, the human P2U receptor gene was localized to chromosome 11q13.5-14.1.