The opportunistic pathogen Pseudomonas aeruginosa can infect almost any site in the body but most often targets epithelial cell-lined tissues such as the airways, skin, and the cornea of the eye. A common predisposing factor is cystic fibrosis (CF), caused by defects in the cystic fibrosis transmembrane-conductance regulator (CFTR). Previously, we showed that when P. aeruginosa enters epithelial cells it replicates intracellularly and occupies plasma membrane blebs. This phenotype is dependent on the type 3 secretion system (T3SS) effector ExoS, shown by others to induce host cell apoptosis. Here, we examined mechanisms for P. aeruginosa-induced bleb formation, focusing on its relationship to apoptosis and the CFTR. The data showed that P. aeruginosa-induced blebbing in epithelial cells is independent of actin contraction and is inhibited by hyperosmotic media (400 to 600 mOsM), distinguishing bacterially induced blebs from apoptotic blebs. Cells with defective CFTR displayed enhanced bleb formation upon infection, as demonstrated using bronchial epithelial cells from a patient with cystic fibrosis and a CFTR inhibitor, CFTR(Inh)-172. The defect was found to be correctable either by incubation in hyperosmotic media or by complementation with CFTR (pGFP-CFTR), suggesting that the osmoregulatory function of CFTR counters P. aeruginosa-induced bleb-niche formation. Accordingly, and despite their reduced capacity for bacterial internalization, CFTR-deficient cells showed greater bacterial occupation of blebs and enhanced intracellular replication. Together, these data suggest that P. aeruginosa bleb niches are distinct from apoptotic blebs, are driven by osmotic forces countered by CFTR, and could provide a novel mechanism for bacterial persistence in the host.

In alveolar epithelial cells (AECs), the membrane-anchored proteoglycan dystroglycan (DG) is a mechanoreceptor that transmits mechanical stretch forces to activate independently the ERK1/2 and the adenosine 5-monophosphate-activated protein kinase (AMPK) signaling cascades in a process called pathway bifurcation. We tested the hypothesis that the cytoskeleton cross-linker plectin, known to bind both DG and AMPK in muscle cells, acts as a scaffold to regulate DG-mediated mechanical stimulation and pathway bifurcation. We demonstrate that plectin and DG form a complex in AECs and that this complex interacts with ERK1/2 and AMPK. Plectin knockdown reduces DG interaction with AMPK but not with ERK1/2. Despite this, mechanoactivation of both signaling pathways is significantly attenuated in AECs deficient in plectin. Thus, DG has the dual role of mechanical receptor and scaffold for ERK1/2, whereas plectin acts as a scaffold for AMPK signaling but is also required for DG-mediated ERK1/2 activation. We conclude that the DG-plectin complex plays a central role in transmitting mechanical stress from the extracellular matrix to the cytoplasm.In the lung, alveolar epithelial cells (AECs) 3 not only mediate the exchange of gases between the circulation system of the host and its external environment but are also highly responsive to a number of mechanical forces (1). These forces include deformation and strain that occur during lung expansion and relaxation from breathing movements, and shear stress during the distension of the airway walls and blood vessels from bulk air and blood flow (2). Over the past few years, there has been increasing interest in identifying molecules that "sense" physical forces on the cell surface and in defining the signaling pathways activated by mechanical stimulation (1, 3-6). Stretch-activated ion channels, integrins, cell-cell adhesion molecules, cytoskeleton elements, and the extracellular matrix (ECM) have all been implicated in transducing mechanical signals in a manner that is detectable as chemical signals (e.g. protein phosphorylation) in the cytoplasm of the stimulated cell (1, 7).We are interested in investigating the molecular underpinnings of cellular responses to physical force in rat AECs. In particular, we have previously tested the hypothesis that matrix molecules secreted by cultured AECs and transmembrane matrix receptors on the substratum surface of these cells are crucial molecular links in the process of "converting" a mechanical stimulus in the form of cyclic stretching into a cytoplasmic signal (8). Specifically, in prior studies we demonstrated that rat AECs assemble an ECM rich in fibers composed of the ␣3, ␤1, and ␥1 subunits of laminin (laminin-311), complexed with perlecan and nidogen (8). This complex transmits mechanosignals in the form of stretch, via the matrix receptor dystroglycan (DG), to activate ERK1/2 (8). Moreover, we have also shown that DG is required for stretch-induced activation of the adenosine 5Ј-monophosphate-activated protein kinase (AMPK) sign...

The flavoprotein methylenetetrahydrofolate reductase from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH 2 -H 4 folate) by NADH via a ping-pong reaction mechanism. Structures of the reduced enzyme in complex with NADH and of the oxidized Glu28Gln enzyme in complex with CH 3 -H 4 folate [Pejchal, R., Sargeant, R., and Ludwig, M. L. (2005) Biochemistry 44, 11447-11457] have revealed Phe223 as a conformationally mobile active site residue. In the NADH complex, the NADH adopts an unusual hairpin conformation and is wedged between the isoalloxazine ring of the FAD and the side chain of Phe223. In the folate complex, Phe223 swings out from its position in the NADH complex in order to stack against the p-aminobenzoate ring of the folate. Although Phe223 contacts each substrate in E. coli MTHFR, this residue is not invariant; for example, a leucine occurs at this site in the human enzyme. To examine the role of Phe223 in substrate binding and catalysis, we have constructed mutants Phe223Ala and Phe223Leu. As predicted, our results indicate that Phe223 participates in the binding of both substrates. The Phe223Ala mutation impairs NADH and CH 2 -H 4 folate binding each 40-fold, yet slows catalysis of both half-reactions less than 2-fold. Affinity for CH 2 -H 4 folate is unaffected by the Phe223Leu mutation, and the variant catalyzes the oxidative half-reaction 3-fold faster than the wild-type enzyme. Structures of ligandfree Phe223Leu and Phe223Leu/Glu28Gln MTHFR in complex with CH 3 -H 4 folate have been determined at 1.65 Å and 1.70 Å resolution, respectively. The structures show that the folate is bound in a catalytically competent conformation, and Leu223 undergoes a conformational change similar to that observed for Phe223 in the Glu28Gln-CH 3 -H 4 folate structure. Taken together, our results suggest that Leu may be a suitable replacement for Phe223 in the oxidative half-reaction of E. coli MTHFR. † This work was supported in part by the American Chemical Society Petroleum Research Fund Grant 39599-GB4 (E.E. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 August 18. Published in final edited form as:Biochemistry. Methylenetetrahydrofolate reductase (MTHFR) 1 is a flavoprotein that catalyzes the NAD(P) H-dependent reduction of 5,10-methylenetetrahydrofolate (CH 2 -H 4 folate), as shown in eq 1.(1)The reaction provides CH 3 -H 4 folate, which is the sole methyl donor to homocysteine in the production of methionine by methionine synthase. MTHFR plays a significant role in the homeostasis of homocysteine; mutations in the enzyme lead to hyperhomocyst(e)inemia, (1, 2), which is associated with an increased risk for the development of cardiovascular disease (reviewed in (3)) and Alzheimer's disease (4-6) in adults, and of neural tube defects in the fetus (reviewed in (7)).MTHFRs from pig liver, human, E. coli, yeast, Arabidopsis, and Leishmania have been characterized (reviewed in (8); (9-12)). Whereas the mammalian and yeast MTHFRs are h...

SummaryLaminins are heterotrimeric glycoproteins of the extracellular matrix that are secreted by epithelial cells and which are crucial for the normal structure and function of the basement membrane. We have generated a mouse harboring a conditional knockout of a3 laminin (Lama3 fl/fl ), one of the main laminin subunits in the lung basement membrane. At 60 days after intratracheal treatment of adult Lama3 fl/fl mice with an adenovirus encoding Cre recombinase (Ad-Cre), the protein abundance of a3 laminin in whole lung homogenates was more than 50% lower than that in control-treated mice, suggesting a relatively long half-life for the protein in the lung. Upon exposure to an injurious ventilation strategy (tidal volume of 35 ml per kg of body weight for 2 hours), the mice with a knockdown of the a3 laminin subunit had less severe injury, as shown by lung mechanics, histology, alveolar capillary permeability and survival when compared with Ad-Null-treated mice. Knockdown of the a3 laminin subunit resulted in evidence of lung inflammation. However, this did not account for their resistance to mechanical ventilation. Rather, the loss of a3 laminin was associated with a significant increase in the collagen content of the lungs. We conclude that the loss of a3 laminin in the alveolar epithelium results in an increase in lung collagen, which confers resistance to mechanical injury.

SummaryReceptor clustering upon cell attachment to the substrate induces assembly of cytoplasmic protein complexes termed focal adhesions (FAs), which connect, albeit indirectly, the extracellular matrix to the cytoskeleton. A subset of cultured primary alveolar epithelial cells (AEC) display a unique pattern of vinculin/paxillin/talin-rich FAs in two concentric circles when cultured on glass and micropatterned substrates: one ring of FAs located at the cell periphery (pFAs), and another FA ring located centrally in the cell (cFAs). Unusually, cFAs associate with an aster-like actin array as well as keratin bundles. Moreover, cFAs show rapid paxillin turnover rates following fluorescence recovery after photobleaching and exert traction forces similar to those generated by FAs at the cell periphery. The plakin protein plectin localizes to cFAs and is normally absent from pFAs, whereas tensin, a marker of mature/fibrillar adhesions, is found in both cFAs and pFAs. In primary AEC in which plectin expression is depleted, cFAs are largely absent, with an attendant reorganization of both the keratin and actin cytoskeletons. We suggest that the mechanical environment in the lung gives rise to the assembly of unconventional FAs in AEC. These FAs not only show a distinctive arrangement, but also possess unique compositional and functional properties.

The present study combines community-based participatory research (CBPR) and peer education to create NuFit, a nutrition and fitness curriculum, adapted by community and student peer leaders for Latino and African-American high-school students in Chicago. The purpose of this pilot study was to assess the feasibility and efficacy of the NuFit curriculum to improve the knowledge, attitudes, and behaviors regarding nutrition and fitness for minority and adolescent student populations. The NuFit curriculum improved students' short-term self-reported behaviors and attitudes around nutrition and fitness. The NuFit curriculum shows promise as one mechanism to help prevent and combat childhood obesity by fostering healthy attitudes and behaviors during the critical developmental stage of adolescence. Involvement of and collaboration between community stakeholders and youth appeared to increase the likelihood of NuFit's cultural relevance and sustainability. More work is necessary to evaluate the long-term effects of NuFit.

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