HOXD3-overexpression increases integrin αvβ3 expression and deprives E-cadherin while it enhances cell motility in A549 cells

We have previously shown that transduction of HOXD3, one of homeobox genes, into human lung cancer A549 cells enhances cell motility, invasion and metastasis. In the present study, we examined the roles of integrin β3 which was up-regulated by HOXD3-overexpression in the HOXD3-induced motility of A549 cells. We first established integrin β3-transfectants and compared their motile activity to those of the HOXD3-transfected, control-transfected and parental cells by three different assays. The integrin β3-transfectants as well as the HOXD3-transfectants formed heterodimer with integrin αv subunit, and showed highly motile activities assessed by haptotaxis or phagokinetic track assay compared to the control transfectants or parental cells. In vitro wound-healing assay revealed that migratory activities were graded as the HOXD3-transfectants > the integrin β3-transfectants > the control transfectants or parental cells. E-cadherin was expressed in the integrin β3-transfectants but not expressed in the HOXD3-transfectants. An addition of function-blocking antibody to E-cadherin into the wound-healing assay promoted the migratory activity of the integrin β3-transfectants, suggesting that E-cadherin prevented the cells from dissociating from the wound edges. These results indicate that increased expression of integrin αv β3 and loss of E-cadherin by HOXD3-overexpression are responsible for the enhanced motility and dissociation.     

Upregulated expression and function of the α4β1 integrin in multiple myeloma cells resistant to bortezomib

The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment promotes MM cell retention, survival, and resistance to different anti-MM agents, including proteasome inhibitors (PIs) such as bortezomib (BTZ). The α4β1 integrin is a main adhesion receptor mediating MM cell–stroma interactions and MM cell survival, and its expression and function are downregulated by BTZ, leading to inhibition of cell adhesion-mediated drug resistance (CAM-DR) and MM cell apoptosis. Whether decreased α4β1 expression and activity are maintained or recovered upon development of resistance to BTZ represents an important question, as a potential rescue of α4β1 function could boost MM cell survival and disease progression. Using BTZ-resistant MM cells, we found that they not only rescue their α4β1 expression, but its levels were higher than in parental cells. Increased α4β1 expression in resistant cells correlated with enhanced α4β1-mediated cell lodging in the BM, and with disease progression. BTZ-resistant MM cells displayed enhanced NF-κB pathway activation relative to parental counterparts, which contributed to upregulated α4 expression and to α4β1-dependent MM cell adhesion. These data emphasize the upregulation of α4β1 expression and function as a key event during resistance to BTZ in MM, which might indirectly contribute to stabilize this resistance, as stronger MM cell attachment to BM stroma will regain CAM-DR and MM cell growth and survival. Finally, we found a strong correlation between high ITGB1 (integrin β1) expression in MM and poor progression-free survival (PFS) and overall survival (OS) during treatment of MM patients with BTZ and IMIDs, and combination of high ITGB1 levels and presence of the high-risk genetic factor amp1q causes low PFS and OS. These results unravel a novel prognostic value for ITGB1 in myeloma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Is there a role for the tumor cell integrin αIIbβ3 and cytoskeleton in tumor cell-platelet interaction?

In vitro tumor cell-platelet interaction was examined using B16 amelanotic (B16a) melanoma cells. These tumor cells express the _IIb3-type cytoadhesin. Aggregation studies demonstrated that tumor cell surface _IIb3 mediates the recognition of platelets since pretreatment of tumor cells with antibody against _IIb3 prevents platelet-tumor cell interaction as well as platelet activation measured by aggregometry, platelet eicosanoid metabolism and ultrastructural analysis. In B16a cells, disruption of the microfilaments and intermediate filaments inhibits mobility of _IIb3 on the cell surface. Microtubules do not play a role in receptor mobility, because B16a cells do not possess well-defined microtubules in interphase and colchicine does not affect receptor mobility. Disruption of microfilaments or intermediate filaments results in an inhibition of tumor cell-platelet interaction as evidenced by aggregometry studies and ultrastructural analysis. We suggest that platelet interaction with tumor cells begins with _IIb3-mediated receptor recognition followed by not only platelet activation but also microfilament- and vimentin intermediate filament-dependent tumor cell activation.     

Expression and function of the high affinity αIibβ3 integrin in murine melanoma cells

In resting platelets integrin alphaIIbbeta3 is constitutively expressed in an inactive state and it does not recognize soluble proteins. Platelet activation results in a conformational change of the low-affinity alphaIIbbeta3 to a high-affinity state which then recognizes plasma fibrinogen. The ectopic expression of alphaIIbbeta3 integrin in rodent and human cells derived from solid tumors is well documented, although little is known about its affinity state in these tumor cells. In this study we analysed expression and function of high-affinity alphaIIbbeta3 in murine metastatic melanoma B16a cells by using a mAb that specifically recognizes high-affinity alphaIIbbeta3 (PAC-1). These tumor cells while in suspension bound PAC-1 and fibrinogen. Immunofluorescent studies of B16a cells indicated that high-affinity alphaIIbbeta3 is associated with the Golgi complex and the cell surface. Stimulation of B16a cells with a PKC-activator, 12(S)-HETE, induced translocation of the high-affinity integrin from an intracellular pool to the plasma membrane, which resulted in increased tumor cell adhesion to fibronectin. In addition to participating in 12(S)-HETE-stimulated adhesion of B16a cells, the high-affinity alphaIIbbeta3 integrin is also involved in tumor cell invasion through a reconstituted basement membrane. In conclusion, results from this study suggest that in non-megakaryocytic lineage B16a cells alphaIIbbeta3 is constitutively expressed in a high-affinity state, and that this conformation participates in tumor cell adhesion and invasion.     

Fibroblast expression of α-smooth muscle actin, α2β1 integrin and αvβ3 integrin: influence of surface rigidity

Open wound contraction necessitates cell and connective tissue interactions, that produce tension. Investigating fibroblast responses to tension utilizes collagen coated polyacrylamide gels with differences in stiffness. Human foreskin fibroblasts were plated on native type I collagen-coated polyacrylamide gel cover slips with different rigidities, which were controlled by bis-acrylamide concentrations. Changes in alpha smooth muscle actin (αSMA), α2β1 integrin (CD49B) and αvβ3 integrin (CD-51) were documented by immuno-histology and Western blot analysis. Cells plated on rigid gels were longer, and expressed αvβ3 integrin and αSMA within cytoplasmic stress fibers. In contrast, cells on flexible gels were shorter, expressed α2β1 integrin and had fine cytoskeletal microfilaments without αSMA. Increased tension changed the actin makeup of the cytoskeleton and the integrin expressed on the cell's surface. These in vitro findings are in agreement with the tension buildup as an open wound closes by wound contraction. It supports the notion that cells under minimal tension in early granulation tissue express α2β1 integrin, required for organizing fine collagen fibrils into thick collagen fibers. Thicker fibers create a rigid matrix, generating more tension. With increased tension cytoskeletal stress fibers develop that contain αSMA and αvβ3 integrin that replaces α2β1 integrin, consistent with cell switching from collagen to non-collagen proteins interactions.     

Microgravity inhibition of lipopolysaccharide-induced tumor necrosis factor-α expression in macrophage cells

Objective and design

Microgravity environments in space can cause major abnormalities in human physiology, including decreased immunity. The underlying mechanisms of microgravity-induced inflammatory defects in macrophages are unclear.

Material or subjects

RAW264.7 cells and primary mouse macrophages were used in the present study. Lipopolysaccharide (LPS)-induced cytokine expression in mouse macrophages was detected under either simulated microgravity or 1g control.

Methods

Freshly isolated primary mouse macrophages and RAW264.7 cells were cultured in a standard simulated microgravity situation using a rotary cell culture system (RCCS-1) and 1g control conditions. The cytokine expression was determined by real-time PCR and ELISA assays. Western blots were used to investigate the related intracellular signals.

Results

LPS-induced tumor necrosis factor-α (TNF-α) expression, but not interleukin-1β expression, in mouse macrophages was significantly suppressed under simulated microgravity. The molecular mechanism studies showed that LPS-induced intracellular signal transduction including phosphorylation of IKK and JNK and nuclear translocation of NF-κB in macrophages was identical under normal gravity and simulated microgravity. Furthermore, TNF-α mRNA stability did not decrease under simulated microgravity. Finally, we found that heat shock factor-1 (HSF1), a known repressor of TNF-α promoter, was markedly activated under simulated microgravity.

Conclusions

Short-term treatment with microgravity caused significantly decreased TNF-α production. Microgravity-activated HSF1 may contribute to the decreased TNF-α expression in macrophages directly caused by microgravity, while the LPS-induced NF-κB pathway is resistant to microgravity.     

Osteopontin is associated with decreased apoptosis and αv integrin expression in lung adenocarcinoma

Osteopontin (OPN) is a glycoprotein involved in invasion, progression and metastasis of many carcinomas. It contains several functional domains including binding sites for αv integrins, cell surface molecules playing a major role in mediating cell migration and adhesion. The aim of the study was to evaluate the expression of osteopontin in human non-small cell lung cancer (NSCLC) and to determine its possible prognostic significance as well as relation to apoptosis and αv integrin expression. We analyzed 111 surgically resected NSCLC for immunohistochemical expression of OPN and αv integrin. OPN expression was compared to apoptotic rate and clinicopathological parameters such as tumor size, histological grade, lymph node status, pT, and TNM stage. Apoptotic rate was measured by TUNEL staining method. OPN expression in NSCLC was significantly higher in lung adenocarcinomas (AC) then in squamous cell carcinomas (p < 0.001). There was no correlation between OPN expression and clinicopathological parameters. The level of OPN expression in AC was associated with decreased apoptotic activity of tumor cells (p = 0.006), and correlated with αv integrin expression (p = 0.048), particularly in low stage tumors (p = 0.013). Prolonged tumor cell survival in lung AC due to OPN and αv integrin overexpression may have an impact on tumor progression and resistance to therapy.     

IL-4 and TNF-α induce changes in integrin expression and adhesive properties and decrease the lung-colonizing potential of HT-29 colon carcinoma cells

The colon carcinoma cell line HT-29 was used to explore the potential of interleukin-4 (IL-4) and tumor necrosis factor (TNF-) to modify integrin expression and adhesive functions of tumor cells in vitro and to examine corresponding metastatic effects in vivo. Preincubation of HT-29 cells with 100 U/ml of IL-4 for 48 h downregulated the surface expression of the integrin subunits 2, 3, l and 4 after 48 h, whereas the 1 subunit was upregulated. In contrast, 100 U/ml of TNF-a selectively upmodulated the expression of av. Attachment to fibronectin of cells treated with IL-4 increased twofold (63.5% vs 32.4%). Adhesion to fibronectin (54.0% vs 32.4%) and vitronectin (37.9% vs 16.4%) was elevated in the case of TNF-a stimulation. Using an experimental metastasis model, HT-29 cells showed a significant reduction of their lung-colonizing potential in nude mice when preincubated with IL-4 for 48 h before intravenous injection. The decrease also observed for TNF--treated cells was less pronounced. The data indicate that the cytokines IL-4 and TNF- can act as direct regulators of adhesive mechanisms of tumor cells bearing adequate receptors, thus influencing lung-colony formation.     

Contribution of sialic acids to integrin α5β1 functioning in melanoma cells

PurposeTo establish the relationship between sialylation of integrin α5β1 and possible alteration in the function of α5β1 receptor in melanoma cells.Materials and methodsIntegrin α5β1 was isolated from primary WM115 (RGP/VGP-like phenotype) and metastatic WM266-4 (lymph node metastasis) cells via affinity chromatography. Integrin α5β1 sialylation and the shift in relative masses of the enzymatically desialylated subunits were confirmed by confocal microscopy and SDS-PAGE, respectively. The ELISA assay was performed to evaluate sialic acid (SA) influence on integrin α5β1 binding to fibronectin (FN). Cell invasion was investigated by the Transwell invasion assay. The effect of neuraminidases treatment on melanoma cells was assessed by flow cytometry using Maackia amurensis and Sambucus nigra lectins.ResultsBoth subunits of integrin α5β1 were found to be more abundantly sialylated in primary than in metastatic cells. The removal of SA had no effect on the purified integrin α5β1 binding to FN. Although metastatic cells underwent more pronounced desialylation than primary cells, invasion of primary WM115 cells was more dependent on the presence of α2-3 linked SA than it was in the case of metastatic WM266-4 cells. In both melanoma cell lines not only integrin α5β1 was involved in invasion, however simultaneous desialylation and usage of anti-integrin α5β1 antibodies resulted in lower invasion abilities of primary WM115 cells.ConclusionsOur data suggest that in primary melanoma cells integrin α5β1 action is more likely dependent on its glycosylation profile, i.e. the presence of SA residues, which influence (decreased) their invasion properties and may facilitate malignant melanoma progression.     

A novelin vitro assay system for transendothelial tumor cell invasion: significance of E-selectin and α3 integrin in the transendothelial invasion by HT1080 fibrosarcoma cells

The interaction of tumor cells with endothelial cells is a key event in tumor metastasis. We established anin vitro invasion assay system, in which the invasion of tumor cells after interaction with endothelial cells can be examined. Two chamber culture wells separated by porous membrane were used. Human umbilical vein endothelial cells (HUVEC) were placed on porous membranes coated with matrix components. The invasion by HT1080 fibrosarcoma cells was determined in this system by counting the number of cells that moved through the membranes from upper to lower chambers. HUVEC cells did not migrate through the membranes as judged by the staining with UEA-I. Observation by scanning electron microscopy revealed that HT1080 cells bound to HUVEC surfaces and migrated underneath the HUVEC monolayer. Effects of antibodies specific for cell surface adhesion molecules on the migration of HT1080 cells were examined. Invasion of uncoated membranes and membranes coated with HUVEC cells was compared. Antibody against E-selectin significantly suppressed an increase of HT1080 cell invasion of HUVEC monolayers stimulated by IL-1 or TNF. Antibody against integrin ₃ subunit remarkably inhibited the invasion of HUVEC cell-coated membranes, suggesting that integrins with the ₃ subunit may play an important role in the transendothelial invasion by HT1080 cells.     

Macrophages regulate expression of α1,2-fucosyltransferase genes in human endometrial epithelial cells

The epithelial cell surface of the endometrium undergoes substantial biochemical changes to allow embryo attachment and implantation in early pregnancy. We hypothesized that tissue macrophages influence these events to promote uterine receptivity. To investigate the role of macrophages in regulating epithelial cell expression of genes linked to glycan-mediated embryo adhesion, Ishikawa, RL95-2 and HEC1A endometrial epithelial cells were cultured alone or with unactivated or lipopolysaccharide-activated monocytic U937 cells, separated using transwell inserts. Expression of mRNAs encoding two α1,2-fucosyltransferases (FUT1, FUT2) was increased in all three epithelial cell lines following co-culture with U937 cells, and was associated with increased fucosylation of cell surface glycoproteins detected using lectins from Ulex europaeus (UEA-1) and Dolichos biflorus (DBA). FUT1 induction by U937 cells also occurred in primary endometrial epithelial cells collected in luteal but not proliferative phase. Activation of the interleukin-6 (IL6)/leukemia inhibitory factor (LIF) cytokine signaling pathway with phosphorylation of STAT3 and elevated SOCS3 mRNA expression was evident in epithelial cells stimulated by U937 co-culture. Several recombinant macrophage-secreted cytokines exerted stimulatory or inhibitory effects on FUT1 and FUT2 mRNA expression, and the macrophage-derived cytokine LIF partially replicated the effects of U937 cells on both FUT1 and FUT2 expression and UEA-1 and DBA lectin reactivity in Ishikawa cells. These results suggest that macrophage-derived factors including LIF might facilitate development of an implantation-receptive endometrium by regulating surface glycan structures in epithelial cells. Abnormal phenotypes or altered abundance of uterine macrophages could contribute to the pathophysiology of primary unexplained infertility in women.     

Phosphorylation of α-synuclein upregulates tyrosine hydroxylase activity in MN9D cells

Hyperphosphorylated α-synuclein is considered an important event in the pathogenesis of Parkinson's disease but its function remains elusive. In this study we provide evidence that tyrosine hydroxylase (TH) expression was unaffected by overexpression of wild-type and phospho-mimic mutant α-synuclein (S129D) in dopaminergic MN9D cells. However, α-synuclein overexpression evidently inhibited TH phosphorylation at Ser40 and dopamine synthesis, while α-synuclein (S129D) mutant enhanced TH phosphorylation and dopamine synthesis. This phospho-mimic mutant prevented wild-type α-synuclein cytotoxicity to MN9D cells, which might be due to aggregation of mutant α-synuclein in the cytoplasm and nuclei. These results demonstrated that phosphorylation at Ser129 was involved in the regulation of TH activity, as well as in eliminating the neurotoxicity of wild-type α-synuclein overexpression in MN9D cells.     

Immunohistochemical expression of the α5 integrin subunit in the normal adult rat central nervous system

We investigated the distribution of the 5 integrin subunit in the normal adult rat CNS using immunohistochemical methods. Results indicated that the 5 integrin subunit was expressed on the vast majority of neurons throughout the brain and spinal cord. In general, neurons showed diffuse cytoplasmic labelling, although many cortical neurons in layers 4 and 5 did show punctate labelling on the cell surface. In addition, axons within the white matter of the brainstem and caudal CNS areas were labelled, with the most intense labelling seen within the white matter of the spinal cord. In addition, labelling of astrocytes was seen throughout white matter, with particularly heavy astrocyte labelling in the spinal cord. The widespread distribution of the 5 subunit suggests a general function for the 51 integrin receptor (the only integrin receptor that includes the 5 subunit) in the adult CNS. The increased expression of fibronectin, the only known ligand for the 51 integrin receptor, known to occur around the site of a CNS lesion suggests a possible role for the 51 receptor in the response of neurons in the vicinity of a CNS injury.     

Rotavirus receptor proteins Hsc70 and integrin αvβ3 are located in the lipid microdomains of animal intestinal cells

Several cell surface molecules such as integrins, sialic acid and Hsc70 have been reported to participate in the process of adsorption and penetration of rotaviruses into cells. Some of them have been found in susceptible cell lines but not in epithelial cells of natural hosts so that their real role in the infection process is unclear. In this study, we attempted to confirm the presence of Hsc70 and integrin αvβ3 in the cytoplasmic membrane of isolated intestinal epithelial cells of pig, mouse and cow. Using immunocytochemistry, immunofluorescence, co-immunoprecipitation, ELISA, Western blot analysis and flow cytometry we established that in these cells, (i) Hsc70 and integrin αvβ3 formed a complex in lipid raft microdomains of the cytoplasmatic membrane and (ii) Hsc70 levels increased after rotavirus infection. These results indicate that these molecules act as receptors of rotaviruses in susceptible cells.     

Effect of recombinant tumor necrosis factor α on in vitro amoebicidal activity of murine Kupffer cells

The present study was undertaken to determine whether recombinant tumor necrosis factor α (TNF) could induce Kupffer cells to kill Entamoeba histolytica parasite in vitro. C57BL/6 mice were used in this study. The liver was perfused and Kupffer cells harvested and treated with TNF for 6 h. It was found that Kupffer cells treated with TNF are able to kill amoebic trophozoites in vitro. These results further show that amoebicidal activity of TNF-activated Kupffer cells is dependent on the ratio of Kupffer cells to amoebic trophozoites. The maximum amoebicidal activity of Kupffer cells was observed with the ratio of one Kupffer cell to five amoebae. This study also shows that the optimal concentration of TNF is required in the induction of amoebicidal activity in Kupffer cells (10⁵ units). It seems that both oxidative-dependent and -independent mechanisms are important for the killing of amoebae by the TNF-treated Kupffer cells. It is likely that TNF-treated Kupffer cells produce endogenous TNF or other cytotoxic molecules which are capable of mediating the parasite killing. Our results indicate that the immunologic production of TNF is important in the activation of Kupffer cells to kill amoebic trophozoites.     

Inhibition of αvβ3 integrin reduces angiogenesis, bone turnover, and tumor cell proliferation in experimental prostate cancer bone metastases

The growth of metastatic prostate cancer cells in the bone involves an intimate interaction between the tumor cells and various elements of the bone microenvironment, resulting in increased rate of bone turnover and rapid tumor growth. The αvβ3 integrin has been shown to play an important role in tumor growth and angiogenesis, and is known to be critical to osteoclast formation and activity. This study was designed to examine the role of αvβ3 expressed by cells native to the bone in the growth and pathogenesis of prostate cancer bone metastases. Human prostate cancer cells which do not express αvβ3 or αIIbβ3 integrins were injected directly into human bone fragments previously implanted subcutaneously in SCID mice (SCID-human-bone model). At the same time treatment with anti-β3 antibody fragment (m7E3 F(ab′)₂) i.p. at 300 μg/dose 3× per week was initiated and continued for 2 weeks. In this system, m7E3 F(ab′)₂ only recognizes human bone-derived αvβ3. Antibody inhibition of αvβ3 integrin in vivo resulted in a specific reduction in the proportion of antigenically-human blood vessels within tumor-bearing bone implants (from 73.5% ± 3.93 in controls to 17.74% ± 5.64 in treated animals). Proliferation of the αvβ3-negative tumor cells was also reduced, although the overall vessel density was maintained by compensating mouse vasculature. Blockage of human bone-derived αvβ3 also significantly reduced the recruitment of osteoclasts in response to tumor cells, as well as degradation of calcified bone tissue. Together these observations confirm the importance of αvβ3 in bone metabolism and angiogenesis, and point to the role of these processes in controlling growth of metastatic prostate cancer cells in the bone. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phoyunnanin E inhibits migration of non-small cell lung cancer cells via suppression of epithelial-to-mesenchymal transition and integrin αv and integrin β3

Background

The conversion of the epithelial phenotype of cancer cells into cells with a mesenchymal phenotype-so-called epithelial–mesenchymal transition (EMT)-has been shown to enhance the capacity of the cells to disseminate throughout the body. EMT is therefore becoming a potential target for anti-cancer drug discovery. Here, we showed that phoyunnanin E, a compound isolated from Dendrobium venustum, possesses anti-migration activity and addressed its mechanism of action.

Methods

The cytotoxic and proliferative effects of phoyunnanin E on human non-small cell lung cancer-derived H460, H292, and A549 cells and human keratinocyte HaCaT cells were investigated by MTT assay. The effect of phoyunnanin E on EMT was evaluated by determining the colony formation and EMT markers. The migration and invasion of H460, H292, A549 and HaCaT cells was evaluated by wound healing assay and transwell invasion assay, respectively. EMT markers, integrins and migration-associated proteins were examined by western blot analysis.

Results

Phoyunnanin E at the concentrations of 5 and 10 μM, which are non-toxic to H460, H292, A549 and HaCaT cells showed good potential to inhibit the migratory activity of three types of human lung cancer cells. The anti-migration effect of phoyunnanin E was shown to relate to the suppressed EMT phenotypes, including growth in anchorage-independent condition, cell motility, and EMT-specific protein markers (N-cadherin, vimentin, slug, and snail). In addition to EMT suppression, we found that phoyunnanin E treatment with 5 and 10 μM could decrease the cellular level of integrin αv and integrin β3, these integrins are frequently up-regulated in highly metastatic tumor cells. We further characterized the regulatory proteins in cell migration and found that the cells treated with phoyunnanin E exhibited a significantly lower level of phosphorylated focal adhesion kinase (p-FAK) and phosphorylated ATP-dependent tyrosine kinase (p-AKT), and their downstream effectors (including Ras-related C3 botulinum (Rac-GTP); Cell division cycle 42 (Cdc42); and Ras homolog gene family, member A (Rho-GTP)) in comparison to those of the non-treated control.

Conclusions

We have determined for the first time that phoyunnanin E could inhibit the motility of lung cancer cells via the suppression of EMT and metastasis-related integrins. This new information could support further development of this compound for anti-metastasis approaches.

     

hMSH6 deficiency and inactivation of the αE-catenin invasion-suppressor gene in HCT-8 colon cancer cells

Transition from an epithelioid (E) to a round (R) morphotype, in the human colon cancer cell line HCT-8, is associated with loss or truncation of αE-catenin and acquisition of invasiveness in organ culture. In E clones, like in parental HCT-8 cells, one allele of the αE-catenin gene (CTNNA1) is mutated. HCT-8 cells have also a “Microsatelite Instability-High” (MSI-H) phenotype presumably due to a mutated hMSH6 gene. Fusion of E type cells doubles the wild type CTNNA1 alleles and prevents the loss of αE-catenin. Introduction of an extra chromosome 2, carrying a wild type hMSH6 gene, restores post-replicative mismatch repair and also prevents the frequent inactivation of the remaining wild type CTNNA1 allele. This revised version was published online in July 2006 with corrections to the Cover Date.