An odorant receptor mediates the attractiveness of cis-jasmone to Campoletis chlorideae, the endoparasitoid of Helicoverpa armigera.

Parasitic wasps rely on olfaction to locate their hosts in complex chemical environments. Odorant receptors (ORs) function together with well-conserved odorant coreceptors (ORcos) to determine the sensitivity and specificity of olfactory reception. Campoletis chlorideae (Hymenoptera: Ichneunmonidae) is a solitary larval endoparasitoid of the cotton bollworm, Helicoverpa armigera, and some other noctuid species. To understand the molecular basis of C. chlorideae's olfactory reception, we sequenced the transcriptome of adult male and female heads (including antennae) and identified 211 OR transcripts, with 95 being putatively full length. The tissue expression profiles, as assessed by reverse-transcription PCR, showed that seven ORs were expressed only or more highly in female antennae. Their functions were analysed using the Xenopu slaevis oocyte expression system and two-electrode voltage-clamp recordings. CchlOR62 was tuned to cis-jasmone, which was attractive to female C. chlorideae adults and H. armigera larvae in the subsequent behavioural assays. Further bioassays using caged plants showed that the parasitism rate of H. armigera larvae by C. chlorideae on cis-jasmone-treated tobacco plants was higher than on the control plants. Thus, cis-jasmone appears to be an important infochemical involved in the interactions of plants, H. armigera and C. chlorideae, and CchlOR62 mediates the attractiveness of cis-jasmone to C. chlorideae.

Complexity and diversity of F8 genetic variations in the 1000 genomes.

BACKGROUND: Hemophilia A (HA) is an X-linked bleeding disorder caused by deleterious mutations in the coagulation factor VIII gene (F8). To date, F8 mutations have been documented predominantly in European subjects and in American subjects of European descent. Information on F8 variants in individuals of more diverse ethnic backgrounds is limited. OBJECTIVES: To discover novel and rare F8 variants, and to characterize F8 variants in diverse population backgrounds. PATIENTS/METHODS: We analyzed 2535 subjects, including 26 different ethnicities, whose data were available from the 1000 Genomes Project (1000G) phase 3 dataset, for F8 variants and their potential functional impact. RESULTS: We identified 3030 single nucleotide variants, 31 short deletions and insertions (Indels) and a large, 497 kb, deletion. Among all variants, 86.4% were rare variants and 55.6% were novel. Eighteen variants previously associated with HA were found in our study. Most of these 'HA variants' were ethnic-specific with low allele frequency; however, one variant (p.M2257V) was present in 27% of African subjects. The p.E132D, p.T281A, p.A303V and p.D422H 'HA variants' were identified only in males. Twelve novel missense variants were predicted to be deleterious. The large deletion was discovered in eight female subjects without affecting F8 transcription and the transcription of genes on the X chromosome. CONCLUSION: Characterizing F8 in the 1000G project highlighted the complexity of F8 variants and the importance of interrogating genetic variants on multiple ethnic backgrounds for associations with bleeding and thrombosis. The haplotype analysis and the orientation of duplicons that flank the large deletion suggested that the deletion was recurrent and originated by homologous recombination.

Regulation of MEIS1 by distal enhancer elements in acute leukemia.

Aberrant activation of the three-amino-acid-loop extension homeobox gene MEIS1 shortens the latency and accelerates the onset and progression of acute leukemia, yet the molecular mechanism underlying persistent activation of the MEIS1 gene in leukemia remains poorly understood. Here we used a combined comparative genomics analysis and an in vivo transgenic zebrafish assay to identify six regulatory DNA elements that are able to direct green fluorescent protein expression in a spatiotemporal manner during zebrafish embryonic hematopoiesis. Analysis of chromatin characteristics and regulatory signatures suggests that many of these predicted elements are potential enhancers in mammalian hematopoiesis. Strikingly, one of the enhancer elements (E9) is a frequent integration site in retroviral-induced mouse acute leukemia. The genomic region corresponding to enhancer E9 is differentially marked by H3K4 monomethylation and H3K27 acetylation, hallmarks of active enhancers, in multiple leukemia cell lines. Decreased enrichment of these histone marks is associated with downregulation of MEIS1 expression during hematopoietic differentiation. Further, MEIS1/HOXA9 transactivate this enhancer via a conserved binding motif in vitro, and participate in an autoregulatory loop that modulates MEIS1 expression in vivo. Our results suggest that an intronic enhancer regulates the expression of MEIS1 in hematopoiesis and contributes to its aberrant expression in acute leukemia.

Characterizing polymorphisms and allelic diversity of von Willebrand factor gene in the 1000 Genomes.

BACKGROUND: The von Willebrand factor (VWF) gene is highly polymorphic, with variants correlated with VWF antigen levels, adhesion activity, clearance and factor VIII binding. VWF mutations are detected in patients with von Willebrand disease (VWD), whereas polymorphic variants could be associated with thrombosis. However, information on the ethnic diversity of VWF variants and their association with diseases is limited. OBJECTIVES: To characterize novel VWF variants from different ethnicities in the general population. PATIENTS/METHODS: We analyzed samples from 1092 subjects of 14 ethnicities available in the 1000 Genomes database for VWF variants and their potential functional impacts. RESULTS: We identified 2728 SNPs and 91 insertions and deletions that had a high level of ethnic diversity, with Africans having the highest number of variants. The highest level of diversity was found in the D' and D2 domains. Among 94 non-synonymous variants, 31 were predicted to be deleterious, including 19 that were previously associated with VWD. Most of these 'VWD variants' had allele frequencies consistent with disease incidence in European subjects, but some had a significantly higher frequency in other ethnicities. The mutations R2185Q, H817Q and M740I associated with type 1 and type 2N VWD were present in more than 13% of African subjects. CONCLUSIONS: These results highlight the complexity of VWF variations in different ethnic groups and emphasize the importance of interrogating variations on multiple ethnic backgrounds for associations with bleeding and thrombosis.

Multilayer vascular grafts based on collagen-mimetic proteins.

A major roadblock in the development of an off-the-shelf, small-caliber vascular graft is achieving rapid endothelialization of the conduit while minimizing the risk of thrombosis, intimal hyperplasia, and mechanical failure. To address this need, a collagen-mimetic protein derived from group A Streptococcus, Scl2.28 (Scl2), was conjugated into a poly(ethylene glycol) (PEG) hydrogel to generate bioactive hydrogels that bind to endothelial cells (ECs) and resist platelet adhesion. The PEG-Scl2 hydrogel was then reinforced with an electrospun polyurethane mesh to achieve suitable biomechanical properties. In the current study, initial evaluation of this multilayer design as a potential off-the-shelf graft was conducted. First, electrospinning parameters were varied to achieve composite burst pressure, compliance, and suture retention strength that matched reported values of saphenous vein autografts. Composite stability following drying, sterilization, and physiological conditioning under pulsatile flow was then demonstrated. Scl2 bioactivity was also maintained after drying and sterilization as indicated by EC adhesion and spreading. Evaluation of platelet adhesion, aggregation, and activation indicated that PEG-Scl2 hydrogels had minimal platelet interactions and thus appear to provide a thromboresistant blood contacting layer. Finally, evaluation of EC migration speed demonstrated that PEG-Scl2 hydrogels promoted higher migration speeds than PEG-collagen analogs and that migration speed was readily tuned by altering protein concentration. Collectively, these results indicate that this multilayer design warrants further investigation and may have the potential to improve on current synthetic options.

The influence of corticosterone on antizyme gene expression in early regenerating rat liver.

BACKGROUND AND STUDY AIMS: Antizyme 1, a specific inhibitor of Ornithine decarboxylase (ODC), plays a critical role in cell proliferation. Little is known about the impact of glucocorticoid on antizyme expression in the regenerating liver. In this paper, the effect of corticosterone on the gene expression of antizyme 1 in early regenerating rat liver induced by partial hepatectomy (PH) was investigated. MATERIALS AND METHODS: Bilateral adrenalectomies (ADX) were performed 3 days before PH. Corticosterone in sesame oil or sesame oil was injected sub-cutaneously to ADX rats. Antizyme 1 mRNA and protein levels as well as polyamine contents in the regenerating liver were determined by RT-PCR, Western blotting and HPLC, respectively. RESULTS: Antizyme 1 protein content in the oil-treated ADX group decreased significantly at 5, 7 and 9 h after PH compared to control. Following corticosterone administration the content rose dose-dependently during the whole experiment. At 5 h post-PH, the protein levels in 10 and 40 mg/kg corticosterone-treated ADX rats increased by 66% and 148%, respectively, when compared with the control group. However, no significant changes in antizyme 1 mRNA levels were observed in oil-treated ADX rats or corticosterone-treated groups compared to control. Polyamine contents in oil-treated ADX rats were the highest among all groups at 5 and 9 h. Corticosterone treatment resulted in a dramatic decrease of polyamine contents at most of the time points investigated when compared with those in control rats. CONCLUSIONS: Corticosterone treatment induces antizyme 1 protein synthesis in early regenerating rat liver. However, it has little effect on antizyme 1 gene transcription. (Acta gastroenterol. belg., 2011, 74, 289-294).

Erythropoietin increases circulating endothelial progenitor cells and reduces the formation and progression of cerebral aneurysm in rats.

Endothelial dysfunction triggers early pathological changes in vessel walls, potentially leading to the formation of cerebral aneurysm (CA). Endothelial progenitor cells (EPCs) are critical in repairing damaged endothelium and could prevent or slow CA formation. We hypothesize that erythropoietin (EPO) stimulates EPCs mobilization, could alter the rate of CA formation and progression. The hypothesis was tested in a rat model of CA. CAs were induced in male Sprague-Dawley rats and treated with s.c. administration of EPO. Circulating EPCs and serum vascular endothelial grow factor (VEGF) were measured be flow cytometry and ELISA, respectively. mRNAs for inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), matrix metalloproteinase-2 (MMP-2), and MMP-9 in aneurysm tissue were quantified by Real-time PCR. The size, internal elastic lamina (IEL), and media thickness of CAs were evaluated 1 and 3 months after aneurysm induction. Circulating EPCs were significantly lower in CA rats as compared to non-surgical controls. EPO increased levels of circulating EPCs and VEGF. It also decreased iNOS, MMP-2, and MMP-9 mRNA levels, while increased eNOS mRNA in aneurysm tissue. The changes in EPCs and biochemical markers are associated with suppression of new CA formation and prevention of preexisting CA progression. We have shown a close association among circulating EPCs, biochemical markers related to vascular remodeling, and the rate of CA formation and progression. Changes in patterns of cerebral blood flow and hypertension induced by surgical ligations of selected arteries exert significant hemodynamic stress to weaken vessel walls, primarily at sites of basilar bifurcation. The surgical stress also reduced circulating EPCs and slowed vascular repairs. EPO mobilizes EPCs from the bone marrow and promotes their homing. These results suggest that EPCs may serve as a marker for CA progression and EPO a promising candidate for the clinical management of CA.

Disulfide bond reduction of von Willebrand factor by ADAMTS-13.

BACKGROUND: von Willebrand factor (VWF) released from endothelial cells is rich in ultra-large (UL) multimers that are intrinsically active in binding platelets, whereas plasma-type VWF multimers require shear stress to be activated. This functional difference may be attributed to thiols exposed on the surface of plasma-type VWF multimers, but not on ULVWF multimers. Shear stress induces the exposed thiols to form disulfide bonds between laterally apposed plasma-type VWF multimers, leading to enhanced VWF binding to platelets. OBJECTIVES: We tested a hypothesis that ADAMTS-13 has a disulfide bond reducing activity that regulates shear-induced thiol-disulfide exchange of VWF. METHODS: Thiol blocking agents and active thiol bead capturing were used to identify and locate this activity, along with truncated ADAMTS-13 mutants. RESULTS: ADAMTS-13 contains a disulfide bond reducing activity that primarily targets disulfide bonds in plasma-type VWF multimers induced by high shear stress or formed with thiol beads, but not disulfide bonds in native multimeric structures. Cysteine thiols targeted by this activity are in the VWF C-domain and are known to participate in shear-induced thiol-disulfide exchange. ADAMTS-13 contains cysteine thiols that remain exposed after being subjected to hydrodynamic forces. Blocking these active thiols eliminates this reducing activity and moderately decreases ADAMTS-13 activity in cleaving ULVWF strings anchored to endothelial cells under flow conditions, but not under static conditions. This activity is located in this C-terminal region of ADAMTS-13. CONCLUSIONS: This novel disulfide-bond-reducing activity of ADAMTS-13 may prevent covalent lateral association and increased platelet adherence of plasma-type VWF multimers induced by high fluid shear stress.

VAMP8/endobrevin is overexpressed in hyperreactive human platelets: suggested role for platelet microRNA.

BACKGROUND: Variation in platelet reactivity contributes to disorders of hemostasis and thrombosis, but the molecular mechanisms are not well understood. OBJECTIVES: To discover associations between interindividual platelet variability and the responsible platelet genes, and to begin to define the molecular mechanisms altering platelet gene expression. SUBJECTS/METHODS: Two hundred and eighty-eight healthy subjects were phenotyped for platelet responsiveness. Platelet RNA from subjects demonstrating hyperreactivity (n=18) and hyporeactivity (n=11) was used to screen the human transcriptome. RESULTS: Distinctly different mRNA profiles were observed between subjects with differing platelet reactivity. Increased levels of mRNA for VAMP8/endobrevin, a critical v-SNARE involved in platelet granule secretion, were associated with platelet hyperreactivity (Q=0.0275). Validation studies of microarray results showed 4.8-fold higher mean VAMP8 mRNA levels in hyperreactive than hyporeactive platelets (P=0.0023). VAMP8 protein levels varied 13-fold among platelets from these normal subjects, and were 2.5-fold higher in hyperreactive platelets (P=0.05). Among our cohort of 288 subjects, a VAMP8 single-nucleotide polymorphism (rs1010) was associated with platelet reactivity in an age-dependent manner (P<0.003). MicroRNA-96 was predicted to bind to the 3'-untranslated regionof VAMP8 mRNA and was detected in platelets. Overexpression of microRNA-96 in VAMP8-expressing cell lines caused a dose-dependent decrease in VAMP8 protein and mRNA, suggesting a role in VAMP8 mRNA degradation. CONCLUSIONS: These findings support a role for VAMP8/endobrevin in the heterogeneity of platelet reactivity, and suggest a role for microRNA-96 in the regulation of VAMP8 expression.

Protein phosphatase 2B inhibition promotes the secretion of von Willebrand factor from endothelial cells.

BACKGROUND: Secretion of Weibel-Palade body (WPB) contents is regulated, in part, by the phosphorylation of proteins that constitute the endothelial exocytotic machinery. In comparison to protein kinases, a role for protein phosphatases in regulating endothelial exocytosis is undefined. OBJECTIVE AND METHOD: In this study, we investigated the role of protein phosphatase 2B (PP2B) in the process of endothelial exocytosis using pharmacological and gene knockdown approaches. RESULTS: We show that inhibition of protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus or a cell-permeable PP2B autoinhibitory peptide promotes the secretion of ultralarge von Willebrand factor (ULVWF) from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs mediate platelet tethering. In support of a role for PP2B in von Willebrand factor (VWF) secretion, the catalytic subunit of PP2B interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, is increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis. Furthermore, the plasma VWF antigen level is also enhanced in CsA-treated mice, and small interfering RNA-mediated knockdown of the alpha and beta isoforms of the PP2B-A subunit in HUVECs enhanced VWF secretion. CONCLUSIONS: These observations suggest that CsA promotes VWF release, in part by inhibition of PP2B activity, and are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans.

VH1-69 germline encoded antibodies directed towards ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura.

BACKGROUND: Autoantibodies directed towards ADAMTS13 are present in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). Analysis of a set of antibodies derived from two patients with acquired TTP revealed frequent use of the VH1-69 heavy chain gene segment for the assembly of anti-ADAMTS13 antibodies. OBJECTIVE: We explored the ability of two VH1-69 germline gene-encoded antibodies to inhibit the von Willebrand factor (VWF)-processing activity of ADAMTS13 under different experimental conditions. Furthermore, the presence of VH1-69 encoded anti-ADAMTS13 antibodies in 40 patients with acquired TTP was monitored using monoclonal antibody G8, which specifically reacts with an idiotype expressed on VH1-69 encoded antibodies. METHODS AND RESULTS: Binding of the two VH1-69 encoded monoclonal antibodies was dependent on the presence of the spacer domain. Both antibodies inhibited ADAMTS13 activity under static conditions, as measured by cleavage of FRETS-VWF73 substrate and cleavage of VWF multimers. The recombinant antibodies were also capable of inhibiting the processing of UL-VWF strings on the surface of endothelial cells. G8-reactive antibodies directed towards ADAMTS13 were present in plasma of all patients containing anti ADAMTS13 antibodies. CONCLUSIONS: These results suggest that VH1-69 derived antibodies directed towards ADAMTS13 develop in the majority of patients with acquired TTP.

The membrane-proximal intermolecular disulfide bonds in glycoprotein Ib influence receptor binding to von Willebrand factor.

BACKGROUND: In the platelet glycoprotein (GP)Ib-IX complex, the binding site for its ligand von Willebrand factor (VWF) is restricted to the N-terminal domain of the GPIbalpha subunit. How the other subunits in the complex, GPIbbeta and GPIX, regulate the GPIbalpha-VWF interaction is not clear. OBJECTIVES AND METHODS: As GPIbalpha connects with two GPIbbeta subunits via disulfide bonds, we tested whether these intersubunit covalent links were important to the proper VWF-binding activity of the GPIb-IX complex by characterizing the structure and VWF-binding activity of a mutant GPIb-IX complex that lacked the GPIbalpha-GPIbbeta disulfide bonds. RESULTS: Mutating both Cys484 and Cys485 of GPIbalpha to serine prevents GPIbalpha from forming covalent disulfide bonds with GPIbbeta, while maintaining the integrity of the complex in the membrane. The mutations cause two GPIbbeta subunits to form a disulfide bond between themselves. As compared to Chinese hamster ovary (CHO) cells stably expressing the wild-type GPIb-IX complex at a comparable level, CHO cells stably expressing the mutant GPIb-IX complex bind to significantly less soluble VWF in the presence of ristocetin and roll on the immobilized VWF under flow at a higher velocity. CONCLUSIONS: The disulfide bonds between GPIbalpha and GPIbbeta are necessary for optimal GPIbalpha binding to VWF. The structural plasticity around the disulfide bonds may also help to shed light on the inside-out mechanism underlying GPIbbeta modulation of VWF binding.

Covalent regulation of ULVWF string formation and elongation on endothelial cells under flow conditions.

BACKGROUND AND OBJECTIVES: The adhesion ligand von Willebrand factor (VWF) is a multimeric glycoprotein that mediates platelet adhesion to exposed subendothelium. On endothelial cells, freshly released ultra-large (UL) VWF multimers form long string-like structures to which platelets adhere. METHODS: The formation and elongation of ULVWF strings were studied in the presence of the thiol-blocking N-ethylmaleimide (NEM). The presence of thiols in ULVWF and plasma VWF multimers was determined by maleimide-PEO(2)-Biotin labeling and thiol-chromatography. Finally, covalent re-multimerization of ULVWF was examined in a cell- and enzyme-free system. RESULTS: We found that purified plasma VWF multimers adhere to and elongate ULVWF strings under flow conditions. The formation and propagation of ULVWF strings were dose-dependently reduced by blocking thiols on VWF with NEM, indicating that ULVWF strings are formed by the covalent association of perfused VWF to ULVWF anchored to endothelial cells. The association is made possible by the presence of free thiols in VWF multimers and by the ability of (UL) VWF to covalently re-multimerize. CONCLUSION: The data provide a mechanism by which the thrombogenic ULVWF strings are formed and elongated on endothelial cells. This mechanism suggests that the thiol-disulfide state of ULVWF regulates the adhesion properties of strings on endothelial cells.

Reticulated platelets and uninhibited COX-1 and COX-2 decrease the antiplatelet effects of aspirin.

BACKGROUND: The mechanisms for the variability in antiplatelet effects of aspirin are unclear. Immature (reticulated) platelets may modulate the antiplatelet effects of aspirin through uninhibited cyclooxygenase (COX)-1 and COX-2. OBJECTIVES: To evaluate the role of reticulated platelets in the antiplatelet effects of aspirin. METHODS: Sixty healthy volunteers had platelet studies performed before and 24 h after a single 325-mg dose of aspirin. Platelet studies included light transmission aggregometry; P-selectin and integrin alpha(IIb)beta(3) expression, and serum thromboxane B(2) (TxB(2)) levels. Reticulated platelets and platelet COX-2 expression were measured using flow cytometry. RESULTS: Subjects were divided into tertiles based on the percentage of reticulated platelets in whole blood. Baseline platelet aggregation to 1 microg mL(-1) collagen, and postaspirin aggregations to 5 microm and 20 microm ADP and collagen, were greater in the upper than in the lower tertile of reticulated platelets. Stimulated P-selectin and integrin alpha(IIb)beta(3) expression were also higher in the upper tertile both before and after aspirin. Platelet COX-2 expression was detected in 12 +/- 7% (n = 10) of platelets in the upper tertile, and in 7 +/- 3% (n = 12) of platelets in the lower two tertiles (P = 0.03). Postaspirin serum TxB(2) levels were higher in the upper (5.5 +/- 4 ng mL(-1)) than in the lower tertile (3.2 +/- 2.5 ng mL(-1), P = 0.03), and decreased even further with ex vivo additional COX-1 and COX-2 inhibition. The incidence of aspirin resistance (>or= 70% platelet aggregation to 5 microm ADP) was significantly higher in the upper tertile (45%) than in the lower tertile (5%, P < 0.0001). CONCLUSIONS: Reticulated platelets are associated with diminished antiplatelet effects of aspirin and increased aspirin resistance, possibly because of increased reactivity, and uninhibited COX-1 and COX-2 activity.

Platelet hyperreactivity generalizes to multiple forms of stimulation.

BACKGROUND: Although platelet hyperreactivity constitutes an important cardiovascular risk factor, standardized methods for its measurement are lacking. We recently reported that aggregometry using a submaximal concentration of epinephrine identifies individuals with in vitro platelet hyperreactivity; this hyperreactivity was reproducible on multiple occasions over long periods of time. OBJECTIVE AND METHODS: To better understand this aberrant reactivity, we studied in a large group of subjects (n = 386) the relationship between healthy individuals' platelet reactivity to epinephrine and their platelet phenotype as measured by other functional assays. RESULTS: Subjects with hyperreactivity to epinephrine were more likely to exhibit hyperfunction in each major aspect of platelet activity, including adhesion (response to low-dose ristocetin; P < 0.001), activation (surface P-selectin expression and PAC-1 binding after stimulation; P

Novel ADAMTS-13 mutations in an adult with delayed onset thrombotic thrombocytopenic purpura.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is associated with congenital and acquired deficiency of ADAMTS-13, a metalloprotease that cleaves von Willebrand factor (VWF) and reduces its adhesive activity. Mutations throughout the ADAMTS13 gene have been identified in congenital TTP patients, most of whom have initial episodes during infancy or in early childhood. PATIENTS AND METHODS: We report the case of an adult male who was diagnosed with idiopathic thrombocytopenic purpura at age 34, and with TTP 14 years later. The patient was compound heterozygous for an 18 bp in-frame deletion (C365del) in the disintegrin domain and a point mutation of R1060W in the seventh thrombospondin domain of the ADAMTS-13 gene. CONCLUSIONS: In vitro studies found that C365del and R1060W severely impair ADAMTS-13 synthesis in transfected Hela cells, whereas the deletion mutant also failed to cleave VWF under static and flow conditions.

Human endothelial cells synthesize and release ADAMTS-13.

Hepatic stellate cells have been considered to be a primary source for human plasma ADAMTS-13, the von Willebrand factor (VWF)-cleaving metalloprotease. In this study, ADAMTS-13 antigen was detected by immunofluorescence in both venous (HUVECs) and arterial endothelial cells (HUAECs) using both polyclonal antibodies made against peptides found in various domains of human ADAMTS-13, as well as by a monoclonal antibody against the ADAMTS-13 metalloprotease domain. ADAMTS-13 antigen had an intra-cellular distribution in endothelial cells distinct from the Weibel-Palade body location of VWF, and was released from the cells during 48 h in culture. The mRNA for ADAMTS13 was detected in HUVECs and HUAECs using reverse transcription-polymerase chain reaction (RT-PCR), indicating that the enzyme is synthesized in these cells. The ADAMTS-13 protein was immunoprecipitated from HUVECs and had an approximate M(r) of 170 kDa, similar to the molecular mass of recombinant ADAMTS-13. The ADAMTS-13 in HUVEC and HUAEC lysates had enzymatic activity using both static and flow assays. We conclude that ADAMTS-13 is synthesized in human endothelial cells, and released constitutively. The vast number of endothelial cells in the body may be an important source of ADAMTS-13.

Hybridization between Helicoverpa armigera and Helicoverpa assulta (Lepidoptera: Noctuidae): development and morphological characterization of F1 hybrids.

Reciprocal hybridizations between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) were studied. The cross between females of H. armigera and males of H. assulta yielded only fertile males and sterile individuals lacking an aedeagus, valva or ostium bursae. A total of 492 larvae of the F1 generation were obtained and 374 of these completed larval development and pupated. Only 203 pupae were morphologically normal males, the remaining 171 pupae were malformed. Larvae and pupae that gave rise to morphologically abnormal adults exhibited longer development times. Sterility was not only associated with malformed external sex organs, but also a range of abnormalities of the internal reproductive system: (i) loss of internal reproductive organs, (ii) with one to three copies of an undeveloped bursa copulatrix; or (iii) with one or two undeveloped testes. Normal male hybrid adults showed higher flight activity in comparison with males of both species. In contrast, the cross between females of H. assulta and males of H. armigera yielded morphologically normal offspring (80 males and 83 females). The interaction of the Z-chromosome from H. assulta with autosomes from H. armigera might result in morphological abnormalities found in hybrids and backcrosses, and maternal-zygotic incompatibilities might contribute to sex bias attributed to hybrid inviability.

Platelet-derived VWF-cleaving metalloprotease ADAMTS-13.

The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. As in endothelial cells, platelet VWF also contains ultra-large (UL) multimers that are hyperactive in aggregating platelets. ULVWF in platelet lysates of thrombin-stimulated platelets was only detected in the presence of EDTA, suggesting that ULVWF is cleaved by a divalent cation-dependent protease. A recent study shows that platelets contain the VWF-cleaving metalloprotease ADAMTS-13, but its activity remains unknown. In this study, we show that platelet lysates cleave endothelial cell-derived ULVWF under static and flow conditions. This activity is inhibited by EDTA and by an ADAMTS-13 antibody from the plasma of a patient with acquired TTP. ADAMTS-13 was detected in platelet lysates and on the platelet surface by four antibodies that bind to different domains of the metalloprotease. Expression of ADAMTS-13 on the platelet surface increases significantly upon platelet activation by the thrombin receptor-activating peptide, but not by ADP. These results demonstrate that platelets contain functionally active ADAMTS-13. This intrinsic activity may be physiologically important to prevent the sudden release of hyperactive ULVWF from platelets and serves as the second pool of ADAMTS-13 to encounter the increase in ULVWF release from endothelial cells.