Membrane Partitioning: ��Classical�� and ��Nonclassical�� Hydrophobic Effects

The free energy of transfer of nonpolar solutes from water to lipid bilayers is often dominated by a large negative enthalpy rather than the large positive entropy expected from the hydrophobic effect. This common observation has led to the idea that membrane partitioning is driven by the "nonclassical" hydrophobic effect. We examined this phenomenon by characterizing the partitioning of the well-studied peptide melittin using isothermal titration calorimetry (ITC) and circular dichroism (CD). We studied the temperature dependence of the entropic (-TΔS) and enthalpic (ΔH) components of free energy (ΔG) of partitioning of melittin into lipid membranes made of various mixtures of zwitterionic and anionic lipids. We found significant variations of the entropic and enthalpic components with temperature, lipid composition and vesicle size but only small changes in ΔG (entropy-enthalpy compensation). The heat capacity associated with partitioning had a large negative value of about -0.5 kcal mol(-1) K(-1). This hallmark of the hydrophobic effect was found to be independent of lipid composition. The measured heat capacity values were used to calculate the hydrophobic-effect free energy ΔG (hΦ), which we found to dominate melittin partitioning regardless of lipid composition. In the case of anionic membranes, additional free energy comes from coulombic attraction, which is characterized by a small effective peptide charge due to the lack of additivity of hydrophobic and electrostatic interactions in membrane interfaces [Ladokhin and White J Mol Biol 309:543-552, 2001]. Our results suggest that there is no need for a special effect-the nonclassical hydrophobic effect-to describe partitioning into lipid bilayers.     

Cell Adhesion, the Backbone of the Synapse: ��Vertebrate�� and ��Invertebrate�� Perspectives

Synapses are asymmetric intercellular junctions that mediate neuronal communication. The number, type, and connectivity patterns of synapses determine the formation, maintenance, and function of neural circuitries. The complexity and specificity of synaptogenesis relies upon modulation of adhesive properties, which regulate contact initiation, synapse formation, maturation, and functional plasticity. Disruption of adhesion may result in structural and functional imbalance that may lead to neurodevelopmental diseases, such as autism, or neurodegeneration, such as Alzheimer''s disease. Therefore, understanding the roles of different adhesion protein families in synapse formation is crucial for unraveling the biology of neuronal circuit formation, as well as the pathogenesis of some brain disorders. The present review summarizes some of the knowledge that has been acquired in vertebrate and invertebrate genetic model organisms.Synapses are asymmetric, intercellular junctions that are the basic structural units of neuronal transmission. The correct development of synaptic specializations and the establishment of appropriate connectivity patterns are crucial for the assembly of functional neuronal circuits. Improper synapse formation and function may cause neurodevelopmental disorders, such as mental retardation (MsR) and autism spectrum disorders (ASD) (McAllister 2007; Sudhof 2008), and likely play a role in neurodegenerative disorders, such as Alzheimer''s disease (AD) (Haass and Selkoe 2007).At chemical synapses (reviewed in Sudhof 2004; Zhai and Bellen 2004; Waites et al. 2005; McAllister 2007; Jin and Garner 2008), the presynaptic compartment contains synaptic vesicles (SV), organized in functionally distinct subcellular pools. A subset of SVs docks to the presynaptic membrane around protein-dense release sites, named active zones (AZ). Upon the arrival of an action potential at the terminal, the docked and “primed” SVs fuse with the plasma membrane and release neurotransmitter molecules into the synaptic cleft. Depending on the type of synapse (i.e., excitatory vs. inhibitory synapses), neurotransmitters ultimately activate an appropriate set of postsynaptic receptors that are accurately apposed to the AZ.Synapse formation occurs in several steps (Fig. 1) (reviewed in Eaton and Davis 2003; Goda and Davis 2003; Waites et al. 2005; Garner et al. 2006; Gerrow and El-Husseini 2006; McAllister 2007). Spatiotemporal signals guide axons through heterogeneous cellular environments to contact appropriate postsynaptic targets. At their destination, axonal growth cones initiate synaptogenesis through adhesive interactions with target cells. In the mammalian central nervous system (CNS), immature postsynaptic dendritic spines initially protrude as thin, actin-rich filopodia on the surface of dendrites. Similarly, at the Drosophila neuromuscular junction (NMJ), myopodia develop from the muscles (Ritzenthaler et al. 2000). The stabilization of intercellular contacts and their elaboration into mature, functional synapses involves cytoskeletal arrangements and recruitment of pre- and postsynaptic components to contact sites in spines and boutons. Conversely, retraction of contacts results in synaptic elimination. Both stabilization and retraction sculpt a functional neuronal circuitry.Open in a separate windowFigure 1.(A–C) Different stages of synapse formation. (A) Target selection, (B) Synapse assembly, (C) Synapse maturation and stabilization. (D–F) The role of cell adhesion molecules in synapse formation is exemplified by the paradigm of N-cadherin and catenins in regulation of the morphology and strength of dendritic spine heads. (D) At an early stage the dendritic spines are elongated from motile structures “seeking” their synaptic partners. (E) The contacts between the presynaptic and postsynaptic compartments are stabilized by recruitment of additional cell adhesion molecules. Adhesional interactions activate downstream pathways that remodel the cytoskeleton and organize pre- and postsynaptic apparatuses. (F) Cell adhesion complexes, stabilized by increased synaptic activity, promote the expansion of the dendritic spine head and the maturation/ stabilization of the synapse. Retraction and expansion is dependent on synaptic plasticity.In addition to the plastic nature of synapse formation, the vast heterogeneity of synapses (in terms of target selection, morphology, and type of neurotransmitter released) greatly enhances the complexity of synaptogenesis (reviewed in Craig and Boudin 2001; Craig et al. 2006; Gerrow and El-Husseini 2006). The complexity and specificity of synaptogenesis relies upon the modulation of adhesion between the pre- and postsynaptic components (reviewed in Craig et al. 2006; Gerrow and El-Husseini 2006; Piechotta et al. 2006; Dalva et al. 2007; Shapiro et al. 2007; Yamada and Nelson 2007; Gottmann 2008). Cell adhesive interactions enable cell–cell recognition via extracellular domains and also mediate intracellular signaling cascades that affect synapse morphology and organize scaffolding complexes. Thus, cell adhesion molecules (CAMs) coordinate multiple synaptogenic steps.However, in vitro and in vivo studies of vertebrate CAMs are often at odds with each other. Indeed, there are no examples of mutants for synaptic CAMs that exhibit prominent defects in synapse formation. This apparent “resilience” of synapses is probably caused by functional redundancy or compensatory effects among different CAMs (Piechotta et al. 2006). Hence, studies using simpler organisms less riddled by redundancy, such as Caenorhabditis elegans and Drosophila, have aided in our understanding of the role that these molecules play in organizing synapses.In this survey, we discuss the roles of the best characterized CAM families of proteins involved in synaptogenesis. Our focus is to highlight the complex principles that govern the molecular basis of synapse formation and function from a comparative perspective. We will present results from cell culture studies as well as in vivo analyses in vertebrate systems and refer to invertebrate studies, mainly performed in Drosophila and C. elegans, when they have provided important insights into the role of particular CAM protein families. However, we do not discuss secreted factors, for which we refer the reader to numerous excellent reviews (as for example Washbourne et al. 2004; Salinas 2005; Piechotta et al. 2006; Shapiro et al. 2006; Dalva 2007; Yamada and Nelson 2007; Biederer and Stagi 2008; Salinas and Zou 2008).     

Expression of Integrin ��v��6 and TGF-�� in Scarless vs Scar-forming Wound Healing

Oral mucosal wounds heal with reduced scar formation compared with skin. The epithelial integrin αvβ6 is induced during wound healing, and it can activate fibrogenic transforming growth factor β1 (TGF-β1) and anti-fibrogenic TGF-β3 that play key roles in scar formation. In this study, expression of β6 integrin and members of the TGF-β pathway were studied in experimental wounds of human gingiva and both gingiva and skin of red Duroc pigs using real-time PCR, gene microarrays, and immunostaining. Similar to human wounds, the expression of β6 integrin was induced in the pig wounds 7 days after wounding and remained upregulated >49 days. The αvβ6 integrin was colocalized with both TGF-β isoforms in the wound epithelium. Significantly higher expression levels of β6 integrin and TGF-β1 were observed in the pig gingival wounds compared with skin. Early gingival wounds also expressed higher levels of TGF-β3 compared with skin. The spatio-temporal colocalization of αvβ6 integrin with TGF-β1 and TGF-β3 in the wound epithelium suggests that αvβ6 integrin may activate both isoforms during wound healing. Prolonged expression of αvβ6 integrin along with TGF-β3 in the gingival wound epithelium may be important in protection of gingiva from scar formation. (J Histochem Cytochem 57:543–557, 2009)     

Integrins ��2��1 and ��11��1 regulate the survival of mesenchymal stem cells on collagen I

Although mesenchymal stem cells (MSCs) are the natural source for bone regeneration, the exact mechanisms governing MSC crosstalk with collagen I have not yet been uncovered. Cell adhesion to collagen I is mostly mediated by three integrin receptors – α1β1, α2β1 and α11β1. Using human MSC (hMSC), we show that α11 subunit exhibited the highest basal expression levels but on osteogenic stimulation, both α2 and α11 integrins were significantly upregulated. To elucidate the possible roles of collagen-binding integrins, we applied short hairpin RNA (shRNA)-mediated knockdown in hMSC and found that α2 or α11 deficiency, but not α1, results in a tremendous reduction of hMSC numbers owing to mitochondrial leakage accompanied by Bcl-2-associated X protein upregulation. In order to clarify the signaling conveyed by the collagen-binding integrins in hMSC, we analyzed the activation of focal adhesion kinase, extracellular signal-regulated protein kinase and serine/threonine protein kinase B (PKB/Akt) kinases and detected significantly reduced Akt phosphorylation only in α2- and α11-shRNA hMSC. Finally, experiments with hMSC from osteoporotic patients revealed a significant downregulation of α2 integrin concomitant with an augmented mitochondrial permeability. In conclusion, our study describes for the first time that disturbance of α2β1- or α11β1-mediated interactions to collagen I results in the cell death of MSCs and urges for further investigations examining the impact of MSCs in bone conditions with abnormal collagen I.     

Targeting of Integrin ��1 and Kinesin 2�� by MicroRNA 183

MicroRNA 183 (miR-183) has been reported to inhibit tumor invasiveness and is believed to be involved in the development and function of ciliated neurosensory organs. We have recently found that expression of miR-183 increased after the induction of cellular senescence by exposure to H₂O₂. To gain insight into the biological roles of miR-183 we investigated two potential novel targets: integrin β1 (ITGB1) and kinesin 2α (KIF2A). miR-183 significantly decreased the expression of ITGB1 and KIF2A measured by Western blot. Targeting of the 3′-untranslated region (3′-UTR) of ITGB1 and KIF2A by miR-183 was confirmed by luciferase assay. Transfection with miR-183 led to a significant decrease in cell invasion and migration capacities of HeLa cells that could be rescued by expression of ITGB1 lacking the 3′-UTR. Although miR-183 had no effects on cell adhesion in HeLa cells, it significantly decreased adhesion to laminin, gelatin, and collagen type I in normal human diploid fibroblasts and human trabecular meshwork cells. These effects were also rescued by expression of ITGB1 lacking the 3′-UTR. Targeting of KIF2A by miR-183 resulted in some increase in the formation of cells with monopolar spindles in HeLa cells but not in human diploid fibroblast or human trabecular meshwork cells. The regulation of ITGB1 expression by miR-183 provides a new mechanism for the anti-metastatic role of miR-183 and suggests that this miRNA could influence the development and function in neurosensory organs, and contribute to functional alterations associated with cellular senescence in human diploid fibroblasts and human trabecular meshwork cells.     

Efficacy and safety of the ��mother��s kiss�� technique: a systematic review of case reports and case series

Background:

Foreign bodies lodged in the nasal cavity are a common problem in children, and their removal can be challenging. The published studies relating to the “mother’s kiss” all take the form of case reports and case series. We sought to assess the efficacy and safety of this technique.

Methods:

We performed a comprehensive search of the Cochrane library, MEDLINE, CINAHL, Embase, AMED Complementary and Allied Medicine and the British Nursing Index for relevant articles. We restricted the results to only those studies involving humans. In addition, we checked the references of relevant studies to identify further possibly relevant studies. We also checked current controlled trials registers and the World Health Organization search portal. Our primary outcome measures were the successful extraction of the foreign object from the nasal cavity and any reported adverse effects. We assessed the included studies for factors that might predict the chance of success of the technique. We assessed the validity of each study using the Newcastle–Ottawa scale.

Results:

Eight relevant published articles met our inclusion criteria. The overall success rate for all of the case series was 59.9% (91/152). No adverse effects were reported.

Interpretation:

Evidence from case reports and case series suggests that the mother’s kiss technique is a useful and safe first-line option for the removal of foreign bodies from the nasal cavities of children.Nasal foreign bodies are a common problem in children, most frequently occurring between the ages of 2 and 5 years, and their removal can be challenging.^1,2 Children in this age group have a natural fear of the unknown, and providing care to them can be difficult, especially if previous attempts to remove the foreign body have been painful.Potential complications, most notably the risk of aspiration of the foreign body, mean that objects should be removed from the nasal cavity in a timely fashion. Various techniques have been described: instrumental extraction (using a hook or nasal forceps), suction, balloon catheters,³ cyanoacrylate glue⁴ and various positive-pressure techniques, the simplest of which is to ask the child to blow his or her nose while occluding the unaffected nostril. However, this technique is only possible for older children.⁵ Alternatively, a bag valve mask can be applied over the child’s face, the bag then squeezed to apply a puff of air into the child’s mouth;⁶ a male–male tube adaptor can be attached to an oxygen or air outlet via oxygen tubing placed in the unaffected nostril;⁷ or the “mother’s kiss” or “parent’s kiss” technique can be used.The mother’s kiss was first described in 1965 by Vladimir Ctibor, a general practitioner from New Jersey.⁸ The mother, or other trusted adult, places her mouth over the child’s open mouth, forming a firm seal as if about to perform mouth-to-mouth resuscitation. While occluding the unaffected nostril with a finger, the adult then blows until they feel the resistance caused by closure of the child’s glottis, at which point the adult gives a sharp exhalation to deliver a short puff of air into the child’s mouth. This puff of air passes through the nasopharynx, out through the unoccluded nostril and, if successful, results in the expulsion of the foreign body. The procedure is fully explained to the adult before starting, and the child is told that the parent will give him or her a “big kiss” so that minimal distress is caused to the child. The procedure can be repeated a number of times if not initially successful. A modified mother’s kiss technique has been described,⁹ which involves the adult blowing into a straw in the child’s mouth. We did not include this technique in our review.Although the mother’s kiss technique has been sporadically mentioned in the literature in case reports and case series, it has yet to gain widespread acceptance. It is not a suitable intervention for evaluation using a randomized controlled trial, because there is no appropriate control group: nontreatment is unacceptable, and there is no gold standard for comparison.Randomized controlled trials are considered to be the best trial design, but some treatments result in a dramatic effect that may not require randomized trials.¹⁰ The mother’s kiss technique falls into this category, because the foreign body will not usually move without intervention. Hence, case reports are sufficient to show that the technique sometimes works. However, a systematic review is needed to clarify how often it works and under what circumstances.We sought to examine the existing evidence for the efficacy and safety of the mother’s kiss technique, to help clinicians understand this evidence and to confirm or refute the appropriateness of current practice.Although systematic reviews of randomized controlled clinical trials are now common, it is rare to see a report of a systematic review of case reports or case series, and the methods for performing such a review are less clearly defined and tested. The principal elements of a systematic review are the location, appraisal and synthesis of individual studies; however, there are pitfalls to traditional systematic reviews of clinical trials that can introduce bias and inaccuracy in the results, which must be avoided. For this systematic review of case reports and case series, we were ever mindful of the rationale behind the stages in systematic reviews of clinical trials and endeavoured to apply the same principles to reduce bias and improve accuracy.     

Characterization of Erg K+ Channels in ��- and ��-Cells of Mouse and Human Islets

Voltage-gated eag-related gene (Erg) K⁺ channels regulate the electrical activity of many cell types. Data regarding Erg channel expression and function in electrically excitable glucagon and insulin producing cells of the pancreas is limited. In the present study Erg1 mRNA and protein were shown to be highly expressed in human and mouse islets and in α-TC6 and Min6 cells α- and β-cell lines, respectively. Whole cell patch clamp recordings demonstrated the functional expression of Erg1 in α- and β-cells, with rBeKm1, an Erg1 antagonist, blocking inward tail currents elicited by a double pulse protocol. Additionally, a small interference RNA approach targeting the kcnh2 gene (Erg1) induced a significant decrease of Erg1 inward tail current in Min6 cells. To investigate further the role of Erg channels in mouse and human islets, ratiometric Fura-2 AM Ca²⁺-imaging experiments were performed on isolated α- and β-cells. Blocking Erg channels with rBeKm1 induced a transient cytoplasmic Ca²⁺ increase in both α- and β-cells. This resulted in an increased glucose-dependent insulin secretion, but conversely impaired glucagon secretion under low glucose conditions. Together, these data present Erg1 channels as new mediators of α- and β-cell repolarization. However, antagonism of Erg1 has divergent effects in these cells; to augment glucose-dependent insulin secretion and inhibit low glucose stimulated glucagon secretion.Voltage-gated eag-related gene (Erg)² potassium (K⁺) channels are part of the larger family of voltage dependent K⁺ (Kv) channels (1). Three channel isoforms Erg1, Erg2, and Erg3 have been discovered (2, 3), and they differ by their activation and inactivation voltage dependence, gating properties, and pharmacological profile (4–7). Erg channels control cellular activity by controlling the repolarization of the action potential (AP). In atrial cells and ventricular myocytes, Erg regulates plateau formation and AP repolarization, as blocking Erg channels increases AP length (8, 9). These channels are also strongly involved in the pacemaking activity of cardiac cells (10, 11). Interestingly, a rare congenital heart condition, the inherited form of long QT syndrome is caused by mutations of Erg channel genes (9, 12). Erg channels also control the resting membrane potential in various cell types. For example, in neurons of the medial vestibular nucleus, blocking Erg channels produce an increase in AP discharge or in smooth muscle cells, blocking Erg channels mediates depolarization up to 20 mV (13–15). Hormone secretion studies also demonstrated the involvement of Erg channels in the secretion of prolactin from neurons of the anterior pituitary. Thyrotropin-releasing factor decreases Erg current, which depolarizes neurons and thereby stimulates prolactin secretion (16, 17).In the pancreas, Kv channels and more specifically Kv2.1, regulate insulin secretion by controlling the repolarization of β-cell membrane potential (18–20), although the contribution of this isoform in humans has recently been questioned (21). In α-cells, Kv2.1 and Kv1.4 channels repolarize the membrane potential (22, 23); however, the involvement of Kv channels in the secretion of glucagon is yet to be investigated. One study showed that Erg1, -2, and -3 are expressed in rat α- and β-cells and the rat insulinoma cell line, INS-1, and that they are involved in decreasing membrane potential. Blocking Erg channels with the channel antagonist E4031 increases insulin secretion from INS1 cells (24); however, definitive data regarding the role of Erg channels in insulin and glucagon secretion is limited.Therefore this study aimed to define the functions of Erg channels in α- and β-cells. We found that Erg1 channels are strongly expressed in pancreatic α- and β-cells. Pharmacological and genetic manipulation combined with whole cell recordings in pancreatic cell lines and primary islet cells determined that Erg1 produces a functional current in α- and β-cells. Blocking Erg1 increased intracellular calcium ([Ca²⁺]_i) in mouse β-cells, but only in a minority of mouse and human α-cells. Secretion studies using isolated mouse islets demonstrated that Erg1 are negative regulators of insulin secretion, but positive regulators of glucagon secretion, suggesting distinct roles for Erg1 in β- and α-cells.     

Darwin��s Busts and Public Evolutionary Outreach and Education

For the 1909 Darwin Centennial, the New York Academy of Sciences gave a large bronze bust of Charles Darwin to the American Museum of Natural History. Created by the well-known sculptor, William Couper, the bust was placed on its tall granite pedestal at the entrance at the newly designated exhibition hall, the Charles Darwin Hall of Invertebrate Zoology. Later that year, the American Museum ordered a bronze copy of the bust and presented it to Christ’s College, in Cambridge, England at the British Darwinian celebration. In 1935, Victor Von Hagen requested a plaster copy of the bust for a monument he was erecting on San Cristóbal in the Galapagos Islands to celebrate Darwin’s arrival in the Galapagos. During 1960, the American Museum of Natural History returned the original bronze bust to the New York Academy of Science, where it is now on display at its headquarters in New York City. To celebrate the Darwin bicentennial, the National Academy of Sciences recreated the bust in a computer-generated copy for display at their Washington, DC headquarters.     

Synergistic Induction of Endothelin-1 by Tumor Necrosis Factor �� and Interferon �� Is due to Enhanced NF-��B Binding and Histone Acetylation at Specific ��B Sites

Endothelin-1 (ET-1) is a potent vasoconstrictor and co-mitogen for vascular smooth muscle and is implicated in pulmonary vascular remodeling and the development of pulmonary arterial hypertension. Vascular smooth muscle is an important source of ET-1. Here we demonstrate synergistic induction of preproET-1 message RNA and release of mature peptide by a combination of tumor necrosis factor α (TNFα) and interferon γ (IFNγ) in primary human pulmonary artery smooth muscle cells. This induction was prevented by pretreatment with the histone acetyltransferase inhibitor anacardic acid. TNFα induced a rapid and prolonged pattern of nuclear factor (NF)-κB p65 subunit activation and binding to the native preproET-1 promoter. In contrast, IFNγ induced a delayed activation of interferon regulatory factor-1 without any effect on NF-κB p65 nuclear localization or consensus DNA binding. However, we found cooperative p65 binding and histone H4 acetylation at distinct κB sites in the preproET-1 promoter after stimulation with both TNFα and IFNγ. This was associated with enhanced recruitment of RNA polymerase II to the ATG start site and read-through of the ET-1 coding region. Understanding such mechanisms is crucial in determining the key control points in ET-1 release. This has particular relevance to developing novel treatments targeted at the inflammatory component of pulmonary vascular remodeling.Endothelin-1 is a 21-amino acid peptide which is known to be both a potent vasoconstrictor and mitogen for vascular smooth muscle (1, 2). It is released as a 38-amino acid precursor (Big ET-1²) before cleavage to the mature ET-1 form. As such it has been implicated in the pathogenesis of vascular disease and is particularly associated with pulmonary arterial hypertension (3). Indeed, several endothelin receptor antagonists are now approved for the treatment of pulmonary arterial hypertension (4). However, endothelin receptor antagonists as a class are associated with potentially serious side effects (4), making new treatments aimed at blocking ET-1 synthesis an attractive alternative.Although endothelial cells are thought to be the main source of ET-1 release, several groups including our own have shown that ET-1 can be released from the more numerous vascular smooth muscle cells (5–10). The vascular pathology observed in pulmonary arterial hypertension is propagated by inflammation, and circulating levels of cytokines including tumor necrosis factor α (TNFα) are elevated in patients with pulmonary arterial hypertension (11–15). In many cell types cytokines mediate their biological effects at least in part by the activation of the nuclear factor κB (NF-κB) pathway (16), and a role for NF-κB in pulmonary arterial hypertension has been proposed (17). In addition, we have shown previously that a combination of TNFα and interferon γ (IFNγ) stimulates human pulmonary artery smooth muscle (HPASM) cells to release ET-1 (18). However, the mechanisms underlying this effect are unknown.The preproET-1 promoter region has been shown experimentally to possess binding sites for nuclear factor (NF)-1 and phorbol ester-sensitive c-Fos and c-Jun complexes (19), acute phase reactant regulatory proteins, and binding sites for AP-1 and GATA-2 (20–22). In addition, binding sites for interferon regulatory factor-1 (IRF-1) and NF-κB are predicted by Transfac analysis (23). The close proximity of the IRF-1 site and one of the NF-κB sites is characteristic of genes that are regulated by the synergistic action of TNFα and IFNγ, such as interleukin-6 (IL-6) and intercellular adhesion molecule-1 (24, 25), although ET-1 has not previously been recognized in this group.Our aims were, therefore, to investigate the role of NF-κB in ET-1 release by primary HPASM cells. In addition, we were interested in the role of histone acetylation in the epigenetic control of the ET-1 production. Understanding these novel mechanisms will allow a greater understanding of the pathogenesis of vascular remodeling in pulmonary vessels and aid in the development of new treatment strategies aimed at blocking synthesis of ET-1.     

Plasminogen Activator Inhibitor-1 Regulates Integrin ��v��3 Expression and Autocrine Transforming Growth Factor �� Signaling

Fibrosis is characterized by elevated transforming growth factor β (TGFβ) signaling, resulting in extracellular matrix accumulation and increased PAI-1 (plasminogen activator inhibitor) expression. PAI-1 induces the internalization of urokinase plasminogen activator/receptor and integrin αvβ3 from the cell surface. Since increased αvβ3 expression correlates with increased TGFβ signaling, we hypothesized that aberrant PAI-1-mediated αvβ3 endocytosis could initiate an autocrine loop of TGFβ activity. We found that in PAI-1 knock-out (KO) mouse embryonic fibroblasts), αvβ3 endocytosis was reduced by ∼75%, leaving αvβ3 in enlarged focal adhesions, similar to wild type cells transfected with PAI-1 small interfering RNA. TGFβ signaling was significantly enhanced in PAI-1 KO cells, as demonstrated by a 3-fold increase in SMAD2/3-containing nuclei and a 2.9-fold increase in TGFβ activity that correlated with an increase in αvβ3 and TGFβ receptor II expression. As expected, PAI-1 KO cells had unregulated plasmin activity, which was only partially responsible for TGFβ activation, as evidenced by a mere 25% reduction in TGFβ activity when plasmin was inhibited. Treatment of cells with an αvβ3-specific cyclic RGD peptide (GpenGRGD) led to a more profound (59%) TGFβ inhibition; a nonspecific RGD peptide (GRGDNP) inhibited TGFβ by only 23%. Human primary fibroblasts were used to confirm that PAI-1 inhibition and β3 overexpression led to an increase in TGFβ activity. Consistent with a fibrotic phenotype, PAI-1 KO cells were constitutively myofibroblasts that had a 1.6-fold increase in collagen deposition over wild type cells. These data suggest that PAI-1-mediated regulation of αvβ3 integrin is critical for the control of TGFβ signaling and the prevention of fibrotic disease.Fibrotic disorders can result from environmental toxins, persistent infection, autoimmune disease, or mechanical injury, leading to the hardening and scarring of tissues. In fibrotic diseases, such as liver cirrhosis, renal fibrosis, and idiopathic lung fibrosis, or in pathological wound healing, such as hypertrophic scarring, scleroderma, and Dupuytren disease, the persistence of myofibroblasts contributes to disease progression by overproduction of extracellular matrix (ECM)² and by excessive contraction (1–3). A shift in the balance of growth factors and cytokines that promote ECM deposition and proteases that degrade matrix often contributes to fibrotic disease (4, 5). Plasmin, a broad spectrum protease that is generated from plasminogen by uPA, is one of the proteases that degrades matrix and activates growth factors and other proteases (6). Since uPA activity is inhibited by PAI-1, the overexpression of PAI-1 results in matrix accumulation. For this reason, PAI-1 is a key prognostic marker for fibrotic disease. PAI-1 exerts its inhibitory activity on uPA by stimulating the endocytosis of the cell surface uPA·uPAR complex through the low density lipoprotein receptor-related protein (7). Integrin αvβ3 is also internalized with the uPA·uPAR·low density lipoprotein receptor-related protein complex (8). After endocytosis, uPAR and integrins are recycled back to the cell surface for another round of binding (8, 9). uPAR and αvβ3 promote cellular attachment and spreading, since they are receptors for the extracellular matrix molecule, vitronectin (10). Thus, cycling of the complex is thought to stimulate the attachment and detachment that is necessary for cell migration (8). Consequently, a shift in the expression of any of these components (PAI-1/uPA/uPAR/αvβ3) can result in either aggressive migration, as seen in cancer invasion, or a persistent increase in cell adhesion and cell tension, as seen in myofibroblasts in fibrotic tissue.The family of TGFβ growth factors has been intensively studied for their role in fibrotic wound healing. Up-regulation of TGFβ results in amplified and persistent overproduction of molecules, such as integrins and PAI-1 and other protease inhibitors (e.g. TIMPs) (2, 3). Up-regulated integrins continue the cycle of TGFβ signaling by participating in the sustained activation of TGFβ from its latent form. To date, studies have found that various αv integrins participate in the activation of TGFβ (αvβ3, αvβ5, αvβ6, and αvβ8), but the mechanism differs (11–15). Integrins can serve as docking proteins to localize proteases that cleave and activate latent TGFβ in the ECM, or they can directly activate latent TGFβ in a protease-independent manner. Recently, it was discovered that latent TGFβ is also activated by mechanical stress generated from an integrin-mediated interaction between myofibroblasts and the ECM, primarily involving αvβ5. The mechanical stress promotes a conformational change that activates the latent TGFβ complex (15). αv integrins also modulate TGFβ signaling through the binding of αvβ3 to TGFβ receptor II (TGFβRII) in the presence of TGFβ. This interaction was shown to promote a dramatic increase in the proliferation of lung fibroblasts and induce invasion of epithelial breast cancer cells (16, 17).Our data establish a role for the PAI-1-mediated control of αvβ3 expression and support a significant role for αvβ3 in TGFβ signaling. Using PAI-1 KO cells, we tested the hypothesis that the absence of PAI-1 would result in the accumulation of αvβ3 on the cell surface, since PAI-1 promotes the endocytosis of uPA·uPAR·αvβ3. PAI-1-mediated endocytosis of β3 was significantly reduced in the PAI-1 KO cells. Correspondingly, we report that β3 accumulated at the cell surface in enlarged β3-containing focal adhesions. Thus, we explored whether the accumulation of αvβ3 on the cell surface had fibrogenic effects even in the absence of profibrotic PAI-1. Our results demonstrate dramatically increased TGFβ activity and an increase in collagen expression in PAI-1 KO cells. Together, these findings suggest that PAI-1 modulates β3 expression and localization and, in turn, TGFβ signaling. Our data reveal that maintaining precise levels of PAI-1 is a key to preventing fibrosis. Understanding the consequence of regulating PAI-1 activity is critical in light of the many clinical therapies currently under development that target PAI-1 (18, 19).     

Investigations of �� Initiator Protein-mediated Interaction between Replication Origins �� and �� of the Plasmid R6K

A typical plasmid replicon of Escherichia coli, such as ori γ of R6K, contains tandem iterons (iterated initiator protein binding sites), an AT-rich region that melts upon initiator-iteron interaction, two binding sites for the bacterial initiator protein DnaA, and a binding site for the DNA-bending protein IHF. R6K also contains two structurally atypical origins called α and β that are located on either side of γ and contain a single and a half-iteron, respectively. Individually, these sites do not bind to initiator protein π but access it by DNA looping-mediated interaction with the seven π-bound γ iterons. The π protein exists in 2 interconvertible forms: inert dimers and active monomers. Initiator dimers generally function as negative regulators of replication by promoting iteron pairing (“handcuffing”) between pairs of replicons that turn off both origins. Contrary to this existing paradigm, here we show that both the dimeric and the monomeric π are necessary for ori α-driven plasmid maintenance. Furthermore, efficient looping interaction between α and γ or between 2 γ iterons in vitro also required both forms of π. Why does α-γ iteron pairing promote α activation rather than repression? We show that a weak, transitory α-γ interaction at the iteron pairs was essential for α-driven plasmid maintenance. Swapping the α iteron with one of γ without changing the original sequence context that caused enhanced looping in vitro caused a significant inhibition of α-mediated plasmid maintenance. Therefore, the affinity of α iteron for π-bound γ and not the sequence context determined whether the origin was activated or repressed.