Expression of prothrombin and protease activated receptors in human myometrium during pregnancy and labor

As thrombin is proposed to be involved in stimulating myometrial contractility during labor and preterm labor, we aimed to investigate the expression of prothrombin (F7), the precursor of thrombin, its receptors, the protease-activated receptor (PAR) family (F2R, F2RL1, F2RL2, and F2RL3), and prothrombinase FGL2 in human myometrium during pregnancy and labor. Messenger RNA and protein were isolated from human pregnant laboring and nonlaboring myometrial tissue and from human primary myometrial smooth muscle cells. Semiquantitative RT-PCR, real-time fluorescence RT-PCR, Western blotting, and fluorescence microscopy were performed to determine the expression levels of F7, FGL2, F2R, F2RL1, F2RL2, and F2RL3 in the myometrial tissues and cells. The expression of mRNA and protein for these molecules is reported for the first time in human myometrium at term pregnancy, at labor, and in the nonpregnant state. Importantly, an increase in F2R and a significant increase in F2RL3 mRNA expression at labor were demonstrated. Statistically significant increases in F2R and F2RL3 protein expression was also detected in human myometrium at labor. Furthermore, FGL2 mRNA expression at labor, and FGL2 protein expression at term pregnancy and at labor was observed in this tissue for the first time. The expression of F7, FGL2, F2R, F2RL1, F2RL2, and F2RL3 in human myometrium reveals that all the machinery necessary for thrombin activation and cellular activity is present in the myometrium during pregnancy and labor. These data, in conjunction with the demonstrated increase in F2R and F2RL3 expression at labor, suggest a principal role for these molecules in the regulation of myometrial function at labor, including preterm labor.     

The Fusarium Graminearum virulence factor FGL targets an FKBP12 immunophilin of wheat

Wheat scab, caused by the fungal pathogen Fusarium graminearum is a devastating disease worldwide. Despite an extensive and coordinated effort to investigate this pathosystem, little progress has been made to understand the molecular basis of host–pathogen interactions, for example how the pathogen causes disease in plant. Recently, a secreted lipase (FGL1) has been identified from the fungus and shown to be an important virulence factor; however, the intrinsic function of FGL1 in plant is unknown. Here, we report the identification of the molecular components that may possibly be involved in the FGL virulence pathway using yeast two hybrid system. FGL gene was amplified from a local virulent strain (F15) and shown to be 99.5% identical to the original published FGL at the amino acid level. We showed that transient expression of this FGL gene by Agroinfiltration in tobacco leaves causes cell death further implicating the role of FGL in virulence. To identify FGL initial physical target in plant, we screened two wheat cDNA libraries using the FGL protein as the bait. From both libraries, a small FKBP-type immunophilin protein, designated wFKBP12, was found to physically interact with FGL. The direct interaction of FGL with wFKBP12 was confirmed in living onion epidermal cells by biomolecular fluorescence complementation (BiFC) assay. To investigate further, we then used wFKBP12 protein as bait and identified an elicitor-responsive protein that contains a potential Ca^2 + binding domain. Semi-quantitative PCR showed that this elicitor-responsive gene is down-regulated during the F. graminearum infection suggesting that this protein may be an important component in FGL virulence pathway. This work serves as an initial step to reveal how fungal lipases act as a general virulence factor.     

Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase

The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K(d) approximately 1 nm) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K(d) = 260-500 nm) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.     

Characterisation of the NUCKS gene on human chromosome 1q32.1 and the presence of a homologous gene in different species

The NUCKS gene is located on human chromosome 1q32.1 and consists of seven exons and six introns. The gene lacks a TATA box but contains two Inr elements, two GC boxes, and one consensus-binding site for E2F-1. NUCKS is expressed in all human adult and foetal tissues investigated, and has all the features of being a housekeeping gene. Both data searches and Western immunoblotting experiments show that a homologous protein is present in fish, amphibians, and birds but not in insects and yeast, suggesting that NUCKS is a vertebrate specific gene. In all the species investigated, the protein contains several consensus phosphorylation sites for cyclin-dependent kinases and CK-2, and we have shown that the fish protein (like mammalian NUCKS) indeed is a substrate for CDK1 and CK-2 in vitro. The NUCKS protein is also conserved with respect to a DNA-binding domain previously characterised in mammals, and two putative bipartite nuclear localisation signals.     

Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12-p13.

Protein tyrosine phosphorylation has been implicated in the growth and functional responses of hematopoietic cells. Recently, approaches have been developed to characterize the protein tyrosine phosphatases that may contribute to regulation of protein tyrosine phosphorylation. One novel protein tyrosine phosphatase was expressed predominantly in hematopoietic cells. Hematopoietic cell phosphatase encodes a 68-kDa protein that contains a single phosphatase conserved domain. Unlike other known protein tyrosine phosphatases, hematopoietic cell phosphatase contains two src homology 2 domains. We also cloned the human homolog, which has 95% amino acid sequence identity. Both the murine and human gene products have tyrosine-specific phosphatase activity, and both are expressed predominantly in hematopoietic cells. Importantly, the human gene maps to chromosome 12 region p12-p13. This region is associated with rearrangements in approximately 10% of cases of acute lymphocytic leukemia in children.     

Fibrinogen-like protein 1, a hepatocyte derived protein is an acute phase reactant

Fibrinogen-like protein 1 (FGL1) is a hepatocyte derived protein that is upregulated in regenerating rodent livers following partial hepatectomy. It has been implicated as a mitogen for liver cell proliferation. In this study, we show that recombinant human IL-6 induces FGL1 expression in Hep G2 cells in a pattern similar to those of acute phase reactants. Following induction of acute inflammation in rats by subcutaneous injection of turpentine oil, serum FGL1 levels are also enhanced. Although, a recent report suggests that FGL1 associates almost exclusively with the fibrin matrix, we report here that approximately 20% of the total plasma FGL1 remains free. The enhancement of FGL1 levels in vitro by IL-6 and its induction after turpentine oil injection suggest that it is an acute phase reactant. Its presence in bound and free forms in the blood also implies biological roles that extend beyond the proposed autocrine effect it has on hepatocytes during regeneration.     

Cloning of a complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homologous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19

The human B1 (CD20) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In order to determine whether this structure is also expressed by murine B cells, cDNA clones that encode the mouse equivalent of the B1 molecule were isolated. The longest murine cDNA clone isolated, pmB1-1, contained a 1.4-kb insert with an 873 base pair open reading frame that encodes a protein of 32 kDa. The predicted mouse B1 protein contains three hydrophobic domains that may span the membrane four times and shares a 73% amino acid sequence homology with the human B1 protein. The pmB1-1 cDNA probe was used to examine mB1 mRNA expression. Northern blot analysis indicated that pmB1-1 hybridized with two mRNA species of 2.3 and 3.0 kb that were expressed only in murine spleen lymphocytes, in B lineage cell lines representing mature B cells, and were weakly expressed in one of two plasmacytoma cell lines. pmB1-1 failed to hybridize with RNA isolated from murine T cell lines, thymus, and nonlymphoid tissues. Southern blot analysis indicated that mB1 was encoded by a single copy gene. In situ hybridization localized the mB1 gene to chromosome 19 band B, a region that also contains the genes that encode the Ly-1, Ly-10, and Ly-12 Ag. These results suggest that only B cells express this heretofore undescribed murine cell-surface protein that is structurally homologous with the membrane-embedded human B1 Ag.     

Isolation of a human gene with protein sequence similarity to human and murine int-1 and the Drosophila segment polarity mutant wingless.

An expressed gene sequence which was identified by the isolation of a methylation free CpG island from human chromosome 7 has been cloned from a human lung cDNA library. The deduced protein sequence contains 360 amino acids and has several features of a secreted protein; it is cysteine rich with a signal peptide sequence and two potential asn-linked glycosylation sites. The protein sequence shows marked similarity with human and murine int-1 and their Drosophila homolog wingless (Dint-1). This human int-1 related protein, int-1 and Dint-1 have diverse patterns of expression, but the inferred structural similarities suggest that some of the functional characteristics of these proteins may be shared.     

Plasma cell membrane glycoprotein PC-1. cDNA cloning of the human molecule, amino acid sequence, and chromosomal location

The murine cell membrane glycoprotein PC-1 is a homodimer with restricted tissue distribution, being first characterized in plasma cells. We now describe the isolation of cDNA clones encoding the human homolog of the murine PC-1 protein, its complete amino acid sequence, and its chromosomal location. Overall, the amino acid sequence of the human protein is about 80% identical to the murine protein, although the extent of homology varies in different domains. It had not been possible to assign a definitive amino terminus to the murine protein. Comparison of the murine and human sequence necessitates reassignment of the amino terminus, resulting in a cytoplasmic tail of 24 amino acids rather than 58 amino acids as previously published for the mouse. The sequence of several independently obtained cDNA clones indicates that the 3' end of the mRNA is subject to alternative splicing. Southern blots suggest a single copy gene. In situ chromosomal hybridization localizes the gene for human PC-1 to chromosome 6q22-q23, a common site for deletions in human lymphoid neoplasia.