Silencing of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor. (57/485)

Insecticidal crystal proteins of Bacillus thuringiensis bind to receptors in the midgut of susceptible insects leading to pore formation and death of the insect. The identity of the receptor is not clearly established. Recently a direct interaction between a cloned and heterologously expressed aminopeptidase (slapn) from Spodoptera litura and the Cry1C protein was demonstrated by immunofluorescence and in vitro ligand blot interaction. Here we show that administration of slapn double-stranded RNA to S. litura larvae reduces its expression. As a consequence of the reduced expression, a corresponding decrease in the sensitivity of these larvae to Cry1C toxin was observed. The gene silencing was retained during the insect's moulting and development and transmitted to the subsequent generation albeit with a reduced effect. These results directly implicate larval midgut aminopeptidase N as receptor for Bacillus thuringiensis insecticidal proteins.  (+info)

The angiogenic regulator CD13/APN is a transcriptional target of Ras signaling pathways in endothelial morphogenesis. (58/485)

Angiogenesis, the formation of new blood vessels, is a critical step for tumor growth and metastasis and an integral component of the pathologic inflammatory response in arthritis and the proliferative retinopathies. The CD13/aminopeptidase N (CD13/APN) metalloprotease is an important regulator of angiogenesis where its expression on activated blood vessels is induced by angiogenic signals. Here, we show that cytokine induction of CD13/APN in endothelial cells is regulated by distinct Ras effector pathways involving Ras/mitogen-activated protein kinase (MAPK) or PI-3K. Signals transduced by activated Ras, Raf, and mitogen-induced extracellular kinase (MEK) stimulate transcription from the CD13/APN proximal promoter. Inhibition of these pathways and extracellular signal-regulated serine/threonine kinase (ERK-2) and PI-3K by expression of dominant-negative proteins or chemical inhibitors prevented induction of CD13/APN transcription in response to basic fibroblast growth factor (bFGF). We show that Ras-induced signal transduction is required for growth factor-induced angiogenesis, because inhibition of downstream mediators of Ras signaling (MEK or PI-3K) abrogated endothelial cell migration, invasion, and morphogenesis in vitro. Reintroduction of CD13/APN, a shared downstream target of these pathways, overrode the suppressive effect of these inhibitors and restored the function of endothelial cells in migration/invasion and capillary morphogenesis assays. Similarly, inhibition of MEK abrogated cell invasion and the formation of endothelial-lined capillaries in vivo, which was effectively rescued by addition of exogenous CD13/APN protein. These studies provide strong evidence that CD13/APN is an important target of Ras signaling in angiogenesis and is a limiting factor in angiogenic progression.  (+info)

Soluble aminopeptidase N/CD13 in malignant and nonmalignant effusions and intratumoral fluid. (59/485)

PURPOSE: On the basis of the finding of marked overexpression in angiogenic microvessels, aminopeptidase N/CD13 has recently been suggested to play a prominent role in tumor angiogenesis. A soluble form of CD13 (sCD13) is present in human plasma, but its role in cancer has not been addressed. We hypothesized that sCD13 would be shed by tumor cells and/or endothelial cells lining tumor vessels, giving high levels of sCD13 in intratumoral fluid (TF) deposits and in malignant effusions. If so, sCD13 could be a convenient potential marker for tumor load and/or activated tumor endothelium. EXPERIMENTAL DESIGN: We have measured the specific sCD13 activity in effusions from 90 cancer patients and 12 patients with a nonmalignant condition, and studied its relationship with other major (anti-)angiogenic factors. In a separate group of patients (n = 41), the relationship of sCD13 activity in plasma with tumor load was studied. RESULTS: The sCD13 activity was highest in plasma from cancer patients 71.9 (fmol/ml/s hydrolyzed substrate) versus 42.4 for healthy subjects. In TF, malignant effusions, and nonmalignant effusions, the activities were 52.8, 33.5, and 18.6, respectively. We further studied the relationship of sCD13 with tumor load as well as with vascular endothelial growth factor (VEGF), endostatin, matrix metalloproteinase (MMP)-2, MMP-9, urokinase-type plasminogen activator, and plasmin. A significant correlation of sCD13 activity in plasma was found with tumor load (r = 0.68; P = 0.01), suggesting that plasma sCD13 is, at least, partly originating from tumor(-endothelium). The concentrations of VEGF and endostatin and the activities of urokinase-type plasminogen activator and MMP-9, but not MMP-2, were significantly higher in TF compared with all other effusions. In TF, a correlation between sCD13 and VEGF was found (r = 0.67; P = 0.03). No correlation of sCD13 with the other protease activities was found. CONCLUSION: The sCD13 activity is elevated in plasma and effusions of cancer patients. A strong correlation of plasma sCD13 with tumor load was found. On the basis of these results, the potential of sCD13 activity as a tumor and/or angiogenesis marker warrants further investigation.  (+info)

Action of three ectopeptidases on corticotropin-releasing factor: metabolism and functional aspects. (60/485)

Using purified enzyme preparations, we investigated the actions of angiotensin-converting enzyme, aminopeptidase N, and endopeptidase 24.11 on corticotropin-releasing factor (CRF). The effects of inhibition of these enzymes on CRF action in rat anterior pituitary cultures were also determined. Finally, specific inhibitors were used to evaluate ectopeptidase action on the regional brain metabolism of CRF. K(m) values for CRF were 165, 90, and 42 microM for angiotensin-converting enzyme, aminopeptidase N, and endopeptidase 24.11, respectively. A CRF metabolite profile for each enzyme was determined. In pituitary cultures, inhibition of endopeptidase 24.11 and aminopeptidase N potentiated CRF-stimulated release of adrenocorticotropic hormone (ACTH). In rat pituitary and hypothalamus membrane preparations, specific inhibitor experiments indicated that CRF hydrolysis involved members of the neutral endopeptidase and aminopeptidase enzyme families. In cortex membranes, similar peptidase inhibition was without effect. These data support the hypothesis that ectopeptidases play a major role in CRF metabolism and biological function.  (+info)

Direct evidence that Bcr-Abl tyrosine kinase activity disrupts normal synergistic interactions between Kit ligand and cytokines in primary primitive progenitor cells. (61/485)

We previously reported that chronic myelogenous leukemia (CML) primitive granulocyte-monocyte (GM) progenitors have a greatly reduced requirement for kit ligand (KL) to achieve optimal growth with granulocyte colony-stimulating factor (G-CSF) + granulocyte-monocyte colony-stimulating factor (GM-CSF). Conversely, others have demonstrated that unlike normal, CML CD34+ progenitors can proliferate in response to KL as a sole stimulus. To address these seemingly paradoxical findings, we examined the growth responses of CML CD34+ GM progenitors to various cytokines with and without a potent inhibitor of Bcr-Abl tyrosine kinase activity, PD173955. The heightened growth responses of CML GM progenitors to KL alone and to G-CSF + GM-CSF were abrogated by 10 nM PD173955 while having no effect on normal GM progenitors. While normal GM progenitors exhibited the expected synergistic response when KL was added to G-CSF + GM-CSF, CML GM progenitors had a minimal response; however, some synergism was restored by 10 nM PD173955. Normal erythroid progenitors require the synergistic interaction between KL and a saturating amount of erythropoietin (EPO, 1 unit) for optimal growth. In contrast, CML erythroid progenitors had up to 50% of optimal growth in KL alone, and, only a subthreshold amount of EPO (0.1 unit) was needed with KL to achieve 85% of the optimal response; these heightened growth responses were largely abrogated by 10 nM PD173955. Thus, direct evidence is provided that constitutively activated Bcr-Abl kinase pathways in primitive CML progenitors cooperate with single growth factors producing a heightened growth response, and, in so doing, disrupt the normally required synergistic interactions between KL and other cytokines to achieve activation and optimal growth of primitive progenitors. Coupled with our previous findings that a larger than normal proportion of CML primitive progenitors are at a later stage of maturation, we propose that this disruption of normal synergistic responses leads to increased progenitor recruitment into a committed pool by a process of accelerated maturation.  (+info)

Human coronavirus 229E: receptor binding domain and neutralization by soluble receptor at 37 degrees C. (62/485)

Truncated human coronavirus HCoV-229E spike glycoproteins containing amino acids 407 to 547 bound to purified, soluble virus receptor, human aminopeptidase N (hAPN). Soluble hAPN neutralized the infectivity of HCoV-229E virions at 37 degrees C, but not 4 degrees C. Binding of hAPN may therefore trigger conformational changes in the viral spike protein at 37 degrees C that facilitate virus entry.  (+info)

Clinical significance of aminopeptidase N/CD13 expression in human pancreatic carcinoma. (63/485)

PURPOSE: We previously established a novel murine monoclonal antibody(MH8-11) that recognized aminopeptidase N (APN)/cluster of differentiation antigen 13 (CD13). This monoclonal antibody inhibited human umbilical vein endothelial cells migration and capillary-like tube formation of human umbilical vein endothelial cells on Matrigel. In this study, we investigated the expression of APN/CD13 and the intratumor microvessel density (IMD) as the number of microvessel counts in 50 patients with pancreatic carcinoma. EXPERIMENTAL DESIGN: We investigated APN/CD13 gene expression using the reverse transcriptase-PCR. We also used immunohistochemistry with MH8-11 to investigate APN/CD13 protein expression. Moreover, we investigated the relationship between APN/CD13 expression and tumor angiogenesis by measuring the IMD. RESULTS: APN/CD13 gene expression detected by reverse transcriptase-PCR was positive in 50.0% (25 of 50) of the tumors, and APN/CD13 protein positive was detected by immunohistochemistry in 48.0% (24 of 50). APN/CD13 gene expression agreed well with the immunohistochemical findings (90.0% concordance). APN/CD13 was also significantly associated with an increase of the IMD (r = 0.71, P = 0.0003). However, APN/CD13 expression was not associated with various prognostic factors. The median survival time of patients with APN/CD13 expression was significantly shorter than that of patients without APN/CD13 expression (P = 0.009), and multivariate analysis showed that the APN/CD13 status was a significant independent factor (P = 0.016). CONCLUSIONS: Our data suggest that APN/CD13 may be a new prognostic marker for patients with pancreatic carcinoma and may have a relationship with the angiogenesis for this cancer.  (+info)

Flow cytometric characterization of perfused human bone marrow cultures: identification of the major cell lineages and correlation with the CFU-GM assay. (64/485)

BACKGROUND: Prolific cultures of human bone marrow mononuclear cells (BM MNCs) were recently developed that include a full spectrum of hematopoietic and accessory cells, with the presence of autofluorescent cells indicating adequate cell expansion. However, phenotypic and functional clonogenic characterizations of the autofluorescent cells and the various other subpopulations present in these cultures have not been carried out. METHODS: Cells from a continuously perfused bioreactor inoculated with BM MNCs and cultured for 12 days in serum-containing medium with PIXY321, erythropoietin, and with or without FLT3-L were evaluated by using flow cytometry. RESULTS: Two antibodies, CD71 and CD13, allowed the separation of the autofluorescent cells into two distinct populations. The CD71+CD13++ autofluorescent population contained the colony-forming unit (CFU) fibroblast, and the CD71++CD13++ autofluorescent population contained macrophage/dendritic like cells. The CFU-granulocyte/macrophage (CFU-GM) could not be thoroughly evaluated with CD71 and CD13. However, the number of CD13+/++Lin- cells correlated with the number of CFU-GM (r = 0.83), with approximately 1 CFU-GM for every 30 CD13+/++Lin- cells. CONCLUSIONS: The data showed that CD71 and CD13 antibodies separate the autofluorescent cells into two populations but do not separate hematopoietic cells into specific phenotypic populations. The data also showed that the number of CD13+/++Lin- cells correlated with the number of CFU-GM. These data present the initial step toward detailed phenotypic analysis of ex vivo expanded human BM MNC cultures.  (+info)