The LAR protein tyrosine phosphatase enables PDGF β-receptor activation through attenuation of the c-Abl kinase activity

The receptor tyrosine phosphatase (RPTP) LAR negatively regulates the activity of several receptor tyrosine kinases. To investigate if LAR affects the platelet-derived growth factor (PDGF) receptor signaling, mouse embryonic fibroblasts (MEFs) from mice where the LAR phosphatase domains were deleted (LARΔP), and wt littermates, were stimulated with 20 ng/ml PDGF-BB. In LAR phosphatase deficient MEFs, the phosphorylation of the PDGF β-receptor was surprisingly reduced, an event that was rescued by re-expression of wt LAR. The decreased phosphorylation of the PDGF β-receptor was observed independent of ligand concentration and occurred on all tyrosine residues, as determined by immunoblotting analysis using site-selective phosphotyrosine antibodies. This suggests that LAR is required for full PDGF β-receptor kinase activation. Downstream of receptor activation, phosphorylation of Akt and PLCγ were decreased in LARΔP MEFs, whereas Src and Erk MAP kinase pathways were less affected. The proliferation of LARΔP MEFs in response to PDGF-BB was also reduced. The inhibitory effect on the PDGF β-receptor in LARΔP cells was exerted via increased basal activity of c-Abl, since inhibition of c-Abl, by AG957 or siRNA, restored PDGF β-receptor phosphorylation. These observations suggest that LAR reduces the basal c-Abl activity thereby allowing for PDGF β-receptor kinase activation.     

Genistein inhibits PDGF-stimulated proteoglycan synthesis in vascular smooth muscle without blocking PDGFβ receptor phosphorylation

The signaling pathways that regulate the synthesis and structure of proteoglycans secreted by vascular smooth muscle cells are potential therapeutic targets for preventing lipid deposition in the early stage of atherosclerosis. PDGF stimulates both core protein expression and elongation of glycosaminoglycan (GAG) chains on proteoglycans. In this study we investigated the effects of the tyrosine kinase inhibitor genistein on PDGF mediated receptor phosphorylation and proteoglycan synthesis in human vascular smooth muscle cells. We demonstrate that genistein does not block phosphorylation of the activation site of the PDGF receptor at Tyr(857) and two other downstream sites Tyr(751) and Tyr(1021). Genistein blocked PDGF-mediated proteoglycan core protein synthesis however it had no effect on GAG chain elongation. These results differ markedly to two other tyrosine kinase inhibitors, imatinib and Ki11502, that block PDGF receptor phosphorylation and PDGF mediated GAG elongation. We conclude that the action of genistein on core protein synthesis does not involve the PDGF receptor and that PDGF mediates GAG elongation via the PDGF receptor.     

Emerging targets and novel strategies in the treatment of AIDS-related Kaposi's sarcoma: bidirectional translational science

Through the mentorship process, Dr. Arthur Pardee emphasized the critical importance of bidirectional translational research-not only advancing drug development from bench to bedside, but also bringing back precious clinical material to the laboratory to assess the biologic effects of therapeutic agents on their targets. This mini-review focuses on the signal transduction pathways of Kaposi's sarcoma (KS) and on how the knowledge of such pathways has led to the rational development of molecularly targeted pathogenesis-driven therapies. Acquired immune deficiency syndrome (AIDS) related-KS results from co-infection with human immunodeficiency virus and KS herpesvirus/human herpesvirus-8 (KSHV/HHV8), which leads to the development of an angiogenic-inflammatory state that is critical in the pathogenesis of KS. KS is driven by KSHV/HHV8-specific pathways, which include viral G protein-coupled receptor (vGPCR), viral interleukin-6 (vIL-6), and viral chemokine homologues. In addition, cellular growth/angiogenic pathways, such as vascular endothelial growth factor (VEGF), insulin-like growth factor, platelet-derived growth factor (PDGF), angiopoietin and matrix metalloproteinases (MMPs) are "pirated" by KSHV/HHV8. As a very tangible example of how translational research has led to a marked improvement in patient outcome, the signal transduction inhibitor imatinib (a tyrosine kinase inhibitor of c-kit and PDGF) was administered to patients with KS whose tumors were serially biopsied. Not only did the patients' tumors regress, but also the regression was correlated with the inhibition of PDGF receptor (PDGFR) in the biopsy samples. Recent and future clinical trials of molecularly targeted therapy for the treatment of KS are a prelude to a shift in the paradigm of how KS is managed.     

Phosphorylation of protein kinase Cdelta on distinct tyrosine residues regulates specific cellular functions

Protein kinase Cdelta (PKCdelta) inhibits proliferation and decreases expression of the differentiation marker glutamine synthetase (GS) in C6 glioma cells. Here, we report that distinct, specific tyrosine residues on PKCdelta are involved in these two responses. Transfection of cells with PKCdelta mutated at tyrosine 155 to phenylalanine caused enhanced proliferation in response to 12-phorbol 12-myristate 13-acetate, whereas GS expression resembled that for the PKCdelta wild-type transfectant. Conversely, transfection with PKCdelta mutated at tyrosine 187 to phenylalanine resulted in increased expression of GS, whereas the rate of proliferation resembled that of the PKCdelta wild-type transfectant. The tyrosine phosphorylation of PKCdelta and the decrease in GS expression induced by platelet-derived growth factor (PDGF) were abolished by the Src kinase inhibitors PP1 and PP2. In response to PDGF, Fyn associated with PKCdelta via tyrosine 187. Finally, overexpression of dominant negative Fyn abrogated the decrease in GS expression and reduced the tyrosine phosphorylation of PKCdelta induced by PDGF. We conclude that the tyrosine phosphorylation of PKCdelta and its association with tyrosine kinases may be an important point of divergence in PKC signaling.     

PDGF-induced vascular smooth muscle cell proliferation is associated with dysregulation of insulin receptor substrates

In vascular smooth muscle cells (VSMCs), platelet-derived growth factor (PDGF) plays a major role in inducing phenotypic switching from contractile to proliferative state. Importantly, VSMC phenotypic switching is also determined by the phosphorylation state/expression levels of insulin receptor substrate (IRS), an intermediary signaling component that is shared by insulin and IGF-I. To date, the roles of PDGF-induced key proliferative signaling components including Akt, p70S6kinase, and ERK1/2 on the serine phosphorylation/expression of IRS-1 and IRS-2 isoforms remain unclear in VSMCs. We hypothesize that PDGF-induced VSMC proliferation is associated with dysregulation of insulin receptor substrates. Using human aortic VSMCs, we demonstrate that prolonged PDGF treatment led to sustained increases in the phosphorylation of protein kinases such as Akt, p70S6kinase, and ERK1/2, which mediate VSMC proliferation. In addition, PDGF enhanced IRS-1/IRS-2 serine phosphorylation and downregulated IRS-2 expression in a time- and concentration-dependent manner. Notably, phosphoinositide 3-kinase (PI 3-kinase) inhibitor (PI-103) and mammalian target of rapamycin inhibitor (rapamycin), which abolished PDGF-induced Akt and p70S6kinase phosphorylation, respectively, blocked PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. In contrast, MEK1/ERK inhibitor (U0126) failed to block PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. PDGF-induced IRS-2 downregulation was prevented by lactacystin, an inhibitor of proteasomal degradation. Functionally, PDGF-mediated IRS-1/IRS-2 dysregulation resulted in the attenuation of insulin-induced IRS-1/IRS-2-associated PI 3-kinase activity. Pharmacological inhibition of PDGF receptor tyrosine kinase with imatinib prevented IRS-1/IRS-2 dysregulation and restored insulin receptor signaling. In conclusion, strategies to inhibit PDGF receptors would not only inhibit neointimal growth but may provide new therapeutic options to prevent dysregulated insulin receptor signaling in VSMCs in nondiabetic and diabetic states.     

Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C-deficient human fibroblasts

We have tested the hypothesis that the mechanism of platelet-derived growth factor (PDGF) and phorbol diester action to decrease the apparent affinity of the epidermal growth factor (EGF) receptor is the phosphorylation of the EGF receptor at the Ca2+/phospholipid-dependent protein kinase (protein kinase C) phosphorylation site, threonine 654. Protein kinase C-deficient cells were prepared by prolonged incubation of human fibroblasts with phorbol diester. Addition of phorbol diesters to these cells fails to regulate EGF receptor affinity or threonine 654 phosphorylation. In contrast, PDGF treatment of both control and protein kinase C-deficient fibroblasts causes a decrease in the apparent affinity of the EGF receptor and an increase in threonine 654 phosphorylation. Thus, the ability of PDGF or phorbol diester to modulate EGF receptor affinity occurs only when threonine 654 phosphorylation is increased. The stoichiometry of threonine 654 phosphorylation associated with a 50% decrease in the binding of 125I-EGF to high affinity sites was 0.15 versus 0.3 mol of phosphate per mole of EGF receptor when 32P-labeled fibroblasts are treated with PDGF or phorbol diester, respectively. It is concluded that EGF receptor phosphorylation at threonine 654 can be regulated by PDGF independently of protein kinase C, substoichiometric phosphorylation of the total EGF receptor pool at threonine 654 is caused by maximally effective concentrations of PDGF, and different extents of phosphorylation of EGF receptors at threonine 654 are observed for maximally effective concentrations of PDGF and phorbol diester, respectively. The data are consistent with the hypothesis that a specific subpopulation of EGF receptors that exhibit high affinity for EGF are regulated by threonine 654 phosphorylation.     

Differential tyrosine phosphorylation of leukemic cells during apoptosis as a result of treatment with imatinib mesylate

Bcr-Abl fusion tyrosine kinase contributes to leukemic transformation. Imatinib mesylate inhibits Bcr-Abl tyrosine kinase, resulting in a blockage of tyrosine phosphorylation in its downstream pathways. We analyzed the alteration of tyrosine phosphorylation, on BCR/ABL+ chronic myelogenous leukemia cells, after treatment with imatinib mesylate. Data were collected using a two-dimensional gel electrophoresis followed by Western blot and mass spectrometry. The inhibition of Bcr-Abl tyrosine kinase by 2.5 microM imatinib mesylate caused both cell cycle arrest in the G0/G1 phase and increased the portion of apoptotic cells. As a result, the population of leukemic cells decreased by 30% and 70% compared to controls at 24 and 72 h, respectively. Furthermore, treatment with imatinib mesylate altered tyrosine phosphorylation of 24 protein spots as the incubation time proceeded from 0 to 24 and 72 h. Ten of the 24 protein spots are visible at all three times. Four are detectable at both the 0 and 24 h points in time. Eight were detectable only at time 0.     

Heparin amplifies platelet-derived growth factor (PDGF)- BB-induced PDGF alpha -receptor but not PDGF beta -receptor tyrosine phosphorylation in heparan sulfate-deficient cells. Effects on signal transduction and biological responses

Platelet-derived growth factor (PDGF) induces mitogenic and migratory responses in a wide variety of cells, by activating specific receptor tyrosine kinases denoted the PDGF alpha- and beta-receptors. Different PDGF isoforms bind in a distinct manner to glycosaminoglycans, particularly heparan sulfate. In the present study, we show potentiation by exogenous heparin of PDGF-BB-induced PDGF alpha-receptor tyrosine phosphorylation in heparan sulfate-deficient Chinese hamster ovary (CHO) 677 cells. This effect was not seen for PDGF-AA treatment, and heparin lacked a potentiating effect on PDGF-BB stimulation of the PDGF beta-receptor. Heparin did not affect the affinity of PDGF-BB binding for the PDGF receptors on CHO 677 cells. The PDGF-BB-stimulated PDGF alpha-receptor phosphorylation was enhanced in a dose-dependent fashion by heparin at low concentration. The effect was modulated by 2-O- and 6-O-desulfation of the polysaccharide. Maximal induction of PDGF alpha-receptor tyrosine phosphorylation (6-fold) in CHO 677 cells was achieved by treatment with a heparin decasaccharide, but shorter oligosaccharides consisting of four or more monosaccharide units were also able to augment PDGF alpha-receptor phosphorylation, albeit at higher concentrations. Heparin potentiated PDGF-BB-induced activation of mitogen-activated protein kinase and protein kinase B (Akt) and allowed increased chemotaxis of the CHO 677 cells toward PDGF-BB. In conclusion, heparin modulates PDGF-BB-induced PDGF alpha-receptor phosphorylation and downstream signaling, with consequences for cellular responsiveness to the growth factor.     

Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity

The phosphorylation of proteins on tyrosine in vivo and in vitro was examined in 3T3 cells stimulated by platelet-derived growth factor (PDGF) and transformed by polyoma middle T antigen (MTAg) by using an antibody directed against phosphotyrosine (P-tyr). Two common events were observed upon PDGF stimulation or MTAg transformation of cells: the appearance in the immunoprecipitates of an 85 kd phosphoprotein, and increased phosphatidylinositol (PI) kinase activity. In PDGF-stimulated cells, the 85 kd phosphoprotein and PI kinase activity appeared rapidly, within 1 min of growth factor addition. The PI kinase activity and 85 kd phosphorylation were also increased in anti-P-tyr immunoprecipitates from cells transformed by v-fms and v-sis, but not by SV40 T antigen. The presence of the tyrosine-phosphorylated 85 kd protein correlated with PI kinase activity during several purification steps. These results suggest that the 85 kd phosphoprotein, a putative PI kinase, is a substrate for both the PDGF receptor and MTAg/pp60c-src tyrosine kinase activities.     

Cross-talk between PDGF and S1P signalling elucidates the inhibitory effect and potential antifibrotic action of the immunomodulator FTY720 in activated HSC-cultures

Platelet-derived growth factor (PDGF) has been shown to be essential in the activation of hepatic stellate cells (HSCs), contributing to the onset and development of hepatic fibrosis. Recently, sphingosine-1-phosphate (S1P) has been shown to be a mitogen and stimulator of chemotaxis also for HSCs. Since it has been demonstrated in several cell types that cross-talk between PDGF and S1P signalling pathways occurs, our aim was to investigate the potential antifibrotic effect of FTY720, whose phosphorylated form acts as a potent S1P receptor (S1PR) modulator, on HSCs. FTY720 inhibits cell proliferation and migration after PDGF stimulation on HSCs in a concentration range between 0.1 and 1 muM. By using compounds that block S1P signalling (PTX and VPC23019), we assessed that FTY720 also acts in an S1P receptor-independent way by decreasing the level of tyrosine phosphorylation of PDGF receptor, with subsequent inhibition of the PDGF signalling pathway. In addition, inhibition of sphingosine kinase2 (SphK2), which is responsible for FTY720 phosphorylation, by DMS/siRNA unveils a mechanism of action irrespective of its phosphorylation, in particular decreasing the level of S1P(1) on the plasma membrane. These findings led us to hypothesize a potential use of FTY720 as a potential antifibrotic drug for further clinical application.     

c-Src binds to the cancer drug imatinib with an inactive Abl/c-Kit conformation and a distributed thermodynamic penalty

The cancer drug imatinib inhibits the tyrosine kinases c-Abl, c-Kit, and the PDGF receptor. Imatinib is less effective against c-Src, which is difficult to understand because residues interacting with imatinib in crystal structures of Abl and c-Kit are conserved in c-Src. The crystal structure of the c-Src kinase domain in complex with imatinib closely resembles that of Abl*imatinib and c-Kit*imatinib, and differs significantly from the inactive "Src/CDK" conformation of the Src family kinases. Attempts to increase the affinity of c-Src for imatinib by swapping residues with the corresponding residues in Abl have not been successful, suggesting that the thermodynamic penalty for adoption of the imatinib-binding conformation by c-Src is distributed over a broad region of the structure. Two mutations that are expected to destabilize the inactive Src/CDK conformation increase drug sensitivity 15-fold, suggesting that the free-energy balance between different inactive states is a key to imatinib binding.     

Rapid detection of phosphotyrosine proteins by flow cytometric analysis in Bcr-Abl-positive cells.

BACKGROUND: Constitutive tyrosine phosphorylation derived from Bcr-Abl kinase activity is the major characteristic of Bcr-Abl positive cells. In this study, we developed a method to detect the phosphotyrosine proteins by flow cytometry and we asked whether phosphorylation was affected by imatinib mesylate treatment. METHODS: Cells were treated or not with imatinib mesylate, fixed and permeabilized by PFA followed by saponin, then stained with anti-phosphotyrosine (p-tyr) monoclonal antibody and analyzed by flow cytometry. RESULTS: Optimal staining parameters were performed with p-tyr antibody using K562 and LAMA84 lines that displayed high levels of tyrosine phosphorylation as compared to the control line, HL60. Tyrosine phosphorylation was inhibited by imatinib in a dose-dependent manner, but not modified by other inhibitors demonstrating that the staining detected is specific to Bcr-Abl phosphorylation. The staining of imatinib-resistant cell lines such as the mutated BaF/Bcr-AblT315I cell line or resistant CML patient cells, showed that hyperphosphorylation was not affected by imatinib treatment. In one CML patient, our technique permitted us to detect a small hyperphosphorylated population resistant to imatinib that appeared hyperphosphorylated and amplified at the time of relapse. CONCLUSIONS: We have developed a flow cytometric technique presenting several advantages such as rapidity and sensitivity, which requires fewer cells than the Western blot.     

Biosynthesis of platelet-activating factor (PAF) in human polymorphonuclear leucocytes. The role of lyso-PAF disposal and free arachidonic acid.

Tumour necrosis factor (TNF) is a potent mitogen for some fibroblast cell lines. Here we have examined the TNF-mediated changes in protein phosphorylation in Swiss 3T3 and human FS-4 fibroblasts, and compared them with changes observed after the treatment of cells with other mitogens, such as platelet-derived growth factor (PDGF) and bombesin. TNF stimulated the rapid phosphorylation of two 41,000-Mr and two 43,000-Mr cytosol proteins on tyrosine, threonine and/or serine, as did PDGF, epidermal growth factor and fibroblast growth factor; the increased levels of this mitogen-induced protein-tyrosine phosphorylation correlated well with the extent of mitogen-induced DNA synthesis as determined by the percentage of labelled nuclei. In contrast, bombesin, which is an even better mitogen for Swiss 3T3 cells than TNF, stimulated the tyrosine phosphorylation of 41,000-Mr and 43,000-Mr proteins only to a limited extent. On the other hand, bombesin and PDGF stimulated the rapid serine phosphorylation of an 80,000-Mr acidic protein, a major substrate for protein kinase C; increased phosphorylation of the 80,000-Mr protein was not observed at all when cells were stimulated with TNF. These results suggest significant differences among the mitogenic signalling pathways of TNF, PDGF and bombesin as regards the involvement of protein kinases; the mitogenic signalling pathway of TNF involves the activation of tyrosine kinase, but not of protein kinase C, whereas bombesin seems to transduce its mitogenic signal mainly through the activation of protein kinase C, and the activation of both kinases seems to be involved in the mitogenic signalling pathway of PDGF.     

CeReS-18, a cell regulatory sialoglycopeptide, inhibits proliferation and migration of rat vascular smooth muscle cells

CeReS-18, a cell regulatory sialoglycopeptide, has been shown to inhibit proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on vascular smooth muscle cell (SMC) proliferation was characterized in cultured rat aorta SMCs (A7r5). More extensively, the effect of CeReS-18 on platelet-derived growth factor (PDGF)-induced SMC migration was examined using a modified Boyden's chamber assay. CeReS-18 inhibits both SMC proliferation and migration in a concentration-dependent, calcium-sensitive, and reversible manner. Furthermore, cells preincubated with the inhibitor had an increased sensitivity to CeReS-18-mediated inhibition of SMC migration. Immunoprecipitation and in vitro phosphorylation assays demonstrated that MAP kinase activity was inhibited in the CeReS-18-treated cells and pretreatment with CeReS-18 suppressed the activation of MAP kinase stimulated by PDGF. However, it is not likely that the suppression of the MAP kinase pathway was directly responsible for the ability of CeReS-18 to inhibit migration of the rat aorta smooth muscle cells since a MEK-specific inhibitor, PD98059, did not influence A7r5 cell migration.     

Inactivation of platelet-derived growth factor receptor by the tumor suppressor PTEN provides a novel mechanism of action of the phosphatase

PTEN, mutated in a variety of human cancers, is a dual specificity protein phosphatase and also possesses D3-phosphoinositide phosphatase activity on phosphatidylinositol 3,4,5-tris-phosphate (PIP(3)), a product of phosphatidylinositol 3-kinase. This PIP(3) phosphatase activity of PTEN contributes to its tumor suppressor function by inhibition of Akt kinase, a direct target of PIP(3). We have recently shown that Akt regulates PDGF-induced DNA synthesis in mesangial cells. In this study, we demonstrate that expression of PTEN in mesangial cells inhibits PDGF-induced Akt activation leading to reduction in PDGF-induced DNA synthesis. As a potential mechanism, we show that PTEN inhibits PDGF-induced protein tyrosine phosphorylation with concomitant dephosphorylation and inactivation of tyrosine phosphorylated and activated PDGF receptor. Recombinant as well as immunopurified PTEN dephosphorylates autophosphorylated PDGF receptor in vitro. Expression of phosphatase deficient mutant of PTEN does not dephosphorylate PDGF-induced tyrosine phosphorylated PDGF receptor. Rather its expression increases tyrosine phosphorylation of PDGF receptor. Furthermore, expression of PTEN attenuated PDGF-induced signal transduction including phosphatidylinositol 3-kinase and Erk1/2 MAPK activities. Our data provide the first evidence that PTEN is physically associated with platelet-derived growth factor (PDGF) receptor and that PDGF causes its dissociation from the receptor. Finally, we show that both the C2 and tail domains of PTEN contribute to binding to the PDGF receptor. These data demonstrate a novel aspect of PTEN function where it acts as an effector for the PDGF receptor function and negatively regulates PDGF receptor activation.     

Suppression of KSHV-induced angiopoietin-2 inhibits angiogenesis,infiltration of inflammatory cells,and tumor growth

Kaposi's sarcoma (KS) is a highly angiogenic and inflammatory neoplasia. The angiogenic and inflammatory cytokine angiopoietin-2 (Ang-2) is strongly expressed in KS due to Kaposi's sarcoma-associated herpesvirus (KSHV) infection. In the present study, we determined how Ang-2 contributes to development of KS by using telomerase-immortalized human umbilical vein endothelial cells (TIVE) as a model, which become malignantly transformed and express increased levels of Ang-2 following KSHV infection. Ang-2 released from TIVE-KSHV cells induces tyrosine phosphorylation of Tie-2 receptor from both human and mouse endothelial cells and promotes angiogenesis in nude mice. Functional inhibition or expressional “knock-down” of Ang-2 in these cells blocks angiogenesis and inhibits tumor growth. Ang-2 suppression also reduces the numbers of infiltrating monocytes/macrophages in tumors. In transwell-based cell migration assays, Ang-2 indeed enhances migration of human monocytes in a dose-dependent manner. These results underscore a pivotal role of KSHV-induced Ang-2 in KS tumor development by promoting both angiogenesis and inflammation. Our data also suggest that selective drug targeting of Ang-2 may be used for treatment of KS.     

The characteristics of the internalization of normal and mutant receptors for the growth factor released by thrombocytes]

The platelet-derived growth factor (PDGF) is a major mitogen in serum for connective tissue derived cells in culture. The influence of receptor structure on the ability of PDGF receptor to be internalized was studied using cell lines transfected with different PDGF-receptor constructions. CHO cell lines expressing either normal PDGF receptor (CHO wt cells) or truncated PDGF receptor, lacking all but 19 amino acids of the intracellular domain (CHO-ECTM cells) were stained according to the method of indirect immunofluorescence with monoclonal antibody B2 to PDGF receptor. It has been found that after a 30-min incubation of the CHO wt cells at 37 degrees C in the presence of PDGF-BB a typical process of endocytosis is observed, while after a 2 h incubation in the same conditions the staining of the cells is absent which suggested the existence of the down-regulation and the process of degradation of PDGF receptor in CHO wt cells. In contrast, in the case of CHO-ECTM cells after both 30-min and 2-h incubation of cells in the presence of PDGF-BB a bright staining of margins and a low staining of the cytoplasm are observed. When the cells where incubated in the presence of lysosomotropic drug chloroquine (0.1 mM), that inhibits degradation of the receptor, the immunostaining of CHO wt cells is changed, with bright compact spots appearing. But in CHO-ECTM cells the fluorescence pattern is not changed. To examine the rate of endocytosis of PDGF-BB, both the types of cells were incubated in the presence of 2 ng/ml of 125I-PDGF-BB.(ABSTRACT TRUNCATED AT 250 WORDS)