Molecular defect in factor IXBm Lake Elsinore. Substitution of Ala390 by Val in the catalytic domain

Earlier studies with factor IXBm Lake Elsinore (IXBmLE), a nonfunctional variant of factor IX, suggested that the defect in this protein may reside in the catalytic domain of the molecule (Usharani, P., Warn-Cramer, B. J., Kasper, C. K., and Bajaj, S. P. (1985) J. Clin. Invest. 75, 76-83). In this report, genomic DNA fragments from normal IX and IXBmLE alleles were cloned into phage lambda EMBL3 and the recombinant phage identified using normal IX cDNA and synthetic oligonucleotides. Exons VI, VII, and VIII of normal IX and IXBmLE gene were also amplified using a newly developed primer-directed polymerase chain reaction method. All eight exons and flanking regions of the normal IX and IXBmLE gene were sequenced by the dideoxy chain termination method. Comparison of the normal IX and IXBmLE sequences revealed a single base substitution (C----T) in the exon VIII of the BmLE variant, which results in the replacement of Ala390 by Val in the variant molecule. Although this mutation is in the catalytic domain of the molecule, purified factor IXaBmLE is indistinguishable from normal IXa in its activity toward a small synthetic substrate, L-tosylarginine methyl ester. These data, coupled with the previous data, identify a region (around residue 390) in the normal factor IXa which appears to play a major role in the extended macromolecular substrate binding site.     

Distribution of male yellow dungflies around ovipasition sites: the effect of body size

Abstract.

• 1 Spatial and temporal variation in body size of yellow dungflies, Scatophaga stercoraria, gathering on and around cow droppings was studied in an Icelandic population in order to elucidate the effect of male and female size on male mating tactics.
• 2 Males copulating on droppings were on average larger than males copulating in the grass, but of similar size to males guarding ovipositing females. Males searching on droppings were smaller than males copulating or guarding females on droppings but larger than males copulating in the grass. No such differences were found in female size.
• 3 Resource-holding power of males (RHP, i.e. male: female size ratio) differed between the three mating groups and was highest for males on the droppings. Size and RHP clearly affect the tactics of copulating males. Males with low RHP tend to copulate in the grass in spite of the cost of longer copulation duration. We argue that this is caused by risk of takeovers from large searching males.
• 4 There was no change in male size with the age of individual droppings. Contrary to what might be expected, large searching males are not predominantly found at fresh droppings when the probability of catching unpaired females is highest. We suggest instead that good prospects in taking females over from other males must make the strategy to search for females on older droppings profitable.
• 5 RHP did not change with age of dropping in the three mating groups. The size of ovipositing females increased with age of dropping, probably reflecting longer copulation and egg-laying times of large females.
• 6 We found an overall positive relationship between sizes of male and female partners. This correlation was highly significant for copulating pairs in the grass. This is probably a consequence of males with low RHP copulating in the grass and fights in which larger males take over females from smaller males. A weaker, but significant, correlation was found amongst ovipositing pairs. This must be due to take-over effects. No size correlation was found for pairs copulating on droppings.

     

Active site blockade of factor VIIa alters its intracellular distribution

Factor VIIa binding to tissue factor on cell surfaces not only triggers the coagulation cascade but also induces various intracellular responses that may contribute to many pathophysiological processes. Active site-inhibited factor VIIa, similar to factor VIIa, binds to tissue factor on cell surfaces and subsequently gets internalized and degraded. At present, it is unknown whether factor VIIa and active site-inhibited factor VIIa undergo a similar intracellular processing. The data presented herein show that although a fraction of both the internalized factor VIIa and active site-inhibited factor VIIa recycle back to the cell surface, the amount of active site-inhibited factor VIIa recycled back to the cell surface was substantially higher than that of factor VIIa. Furthermore, internalized factor VIIa and not active site-inhibited factor VIIa associates with nuclear fractions. Factor VIIa associated with the nuclear fraction was intact and functionally active. In contrast to factor VIIa, tissue factor is not found in the nuclear fraction. Additional studies show that the internalized factor VIIa specifically associates with cytoskeletal proteins, actin, and tubulin. In summary, the present data reveal that despite the common pathway of tissue factor-mediated processing, considerable differences exist in the trafficking of factor VIIa and active site-inhibited factor VIIa in fibroblasts.     

Inactivation of Factor VIIa by Antithrombin In Vitro,Ex Vivo and In Vivo: Role of Tissue Factor and Endothelial Cell Protein C Receptor

Recent studies have suggested that antithrombin (AT) could act as a significant physiologic regulator of FVIIa. However, in vitro studies showed that AT could inhibit FVIIa effectively only when it was bound to tissue factor (TF). Circulating blood is known to contain only traces of TF, at best. FVIIa also binds endothelial cell protein C receptor (EPCR), but the role of EPCR on FVIIa inactivation by AT is unknown. The present study was designed to investigate the role of TF and EPCR in inactivation of FVIIa by AT in vivo. Low human TF mice (low TF, ∼1% expression of the mouse TF level) and high human TF mice (HTF, ∼100% of the mouse TF level) were injected with human rFVIIa (120 µg kg⁻¹ body weight) via the tail vein. At varying time intervals following rFVIIa administration, blood was collected to measure FVIIa-AT complex and rFVIIa antigen levels in the plasma. Despite the large difference in TF expression in the mice, HTF mice generated only 40–50% more of FVIIa-AT complex as compared to low TF mice. Increasing the concentration of TF in vivo in HTF mice by LPS injection increased the levels of FVIIa-AT complexes by about 25%. No significant differences were found in FVIIa-AT levels among wild-type, EPCR-deficient, and EPCR-overexpressing mice. The levels of FVIIa-AT complex formed in vitro and ex vivo were much lower than that was found in vivo. In summary, our results suggest that traces of TF that may be present in circulating blood or extravascular TF that is transiently exposed during normal vessel damage contributes to inactivation of FVIIa by AT in circulation. However, TF’s role in AT inactivation of FVIIa appears to be minor and other factor(s) present in plasma, on blood cells or vascular endothelium may play a predominant role in this process.     

Secondary metabolite from Nostoc XPORK14A inhibits photosynthesis and growth of Synechocystis PCC 6803

Screening of 55 different cyanobacterial strains revealed that an extract from Nostoc XPORK14A drastically modifies the amplitude and kinetics of chlorophyll a fluorescence induction of Synechocystis PCC 6803 cells. After 2 d exposure to the Nostoc XPORK14A extract, Synechocystis PCC 6803 cells displayed reduced net photosynthetic activity and significantly modified electron transport properties of photosystem II under both light and dark conditions. However, the maximum oxidizable amount of P700 was not strongly affected. The extract also induced strong oxidative stress in Synechocystis PCC 6803 cells in both light and darkness. We identified the secondary metabolite of Nostoc XPORK14A causing these pronounced effects on Synechocystis cells. Mass spectrometry and nuclear magnetic resonance analyses revealed that this compound, designated as M22, has a non‐peptide structure. We propose that M22 possesses a dual‐action mechanism: firstly, by photogeneration of reactive oxygen species in the presence of light, which in turn affects the photosynthetic machinery of Synechocystis PCC 6803; and secondly, by altering the in vivo redox status of cells, possibly through inhibition of protein kinases.     

Parallels,nonparallels, and plasticity in population differentiation of threespine stickleback within a lake

The frequent occurrence of parallel phenotypic divergence in similar habitats is often evoked when emphasizing the role of ecology in adaptive radiation and speciation. However, because phenotypic plasticity can contribute to the observed pattern of divergence, confirmation of divergence at loci underlying phenotypic traits is important for confirming adaptive divergence. In the present study, we examine parallel morphological, neutral, and potentially adaptive genetic divergence of threespine stickleback inhabiting different habitats within a lake. Three genetic clusters best explained the neutral genetic structure within the lake; however, morphological differences were only weakly connected to genetic clusters and there was considerable phenotypic variation within clusters. Among the factors that could contribute to the observed pattern of morphological and genetic divergence are phenotypic plasticity, selective mortality of hybrids, and habitat choice based on morphology. Several loci are identified as outliers indicating divergent selection between the morphs and some parallels in morphological and adaptive genetic divergence are found in stickleback spawning at two lava sites. However, neutral genetic structure indicates considerable genetic connectivity among the two lava sites, and the parallels in morphology may therefore represent selective distribution of phenotypes rather than parallel divergence. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 803–813.     

Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor

Vascular injury leads to the exposure of blood to fibroblasts and smooth muscle cells within the vessel wall. These cells constitutively express tissue factor (TF), the cellular receptor for plasma clotting factor VIIa (FVIIa). Formation of TF.FVIIa complexes on cell surfaces triggers the blood coagulation cascade. In the present study, we have investigated the fate of TF.FVIIa complexes formed on the cell surface of fibroblasts in the presence and absence of plasma inhibitor, tissue factor pathway inhibitor (TFPI). FVIIa bound to TF on the cell surface was internalized and degraded without depleting the cell surface TF antigen and activity. TFPI significantly enhanced the TF-specific internalization and degradation of FVIIa. TFPI-enhanced internalization and degradation of FVIIa requires the C-terminal domain of TFPI and factor Xa. TFPI. Xa-mediated internalization of FVIIa was associated with the depletion of TF from the cell surface. A majority of the internalized FVIIa was degraded, but a small portion of the internalized FVIIa recycles back to the cell surface as an intact protein. In addition to TF, other cell surface components, such as low density lipoprotein receptor-related protein (LRP) and heparan sulfates, are essential for TFPI.Xa-induced internalization of FVIIa. Acidification of cytosol, which selectively inhibits the endocytotic pathway via coated pits, inhibited TFPI.Xa-mediated internalization but not the basal internalization of FVIIa. Overall, our data support the concept that FVIIa bound to cell surface TF was endocytosed by two different pathways. FVIIa complexed with TF in the absence of the inhibitor was internalized via a LRP-independent and probably noncoated pit pathway, whereas FVIIa complexed with TF along with the inhibitor was internalized via LRP-dependent coated pit pathway.     

Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIa

Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa.     

Role of Tissue Factor in Mycobacterium tuberculosis-Induced Inflammation and Disease Pathogenesis

Tuberculosis (TB) is a chronic lung infectious disease characterized by severe inflammation and lung granulomatous lesion formation. Clinical manifestations of TB include hypercoagulable states and thrombotic complications. We previously showed that Mycobacterium tuberculosis (M.tb) infection induces tissue factor (TF) expression in macrophages in vitro. TF plays a key role in coagulation and inflammation. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth in the lung and dissemination to other organs. Wild-type C57BL/6 and transgenic mice expressing human TF, either very low levels (low TF) or near to the level of wild-type (HTF), in place of murine TF were infected with M.tb via aerosol exposure. Levels of TF expression, proinflammatory cytokines and thrombin-antithrombin complexes were measured post M.tb infection and mycobacterial burden in the tissue homogenates were evaluated. Our results showed that M.tb infection did not increase the overall TF expression in lungs. However, macrophages in the granulomatous lung lesions in all M.tb-infected mice, including low TF mice, showed increased levels of TF expression. Conspicuous fibrin deposition in the granuloma was detected in wild-type and HTF mice but not in low TF mice. M.tb infection significantly increased expression levels of cytokines IFN-γ, TNF-α, IL-6 and IL-1ß in lung tissues. However, no significant differences were found in proinflammatory cytokines among the three experimental groups. Mycobacterial burden in lungs and dissemination into spleen and liver were essentially similar in all three genotypes. Our data indicate, in contrast to that observed in acute bacterial infections, that TF-mediated coagulation and/or signaling does not appear to contribute to the host-defense in experimental tuberculosis.