Inhalation vs. aspiration of single-walled carbon nanotubes in C57BL/6 mice: inflammation, fibrosis, oxidative stress, and mutagenesis

Nanomaterials are frontier technological products used in different manufactured goods. Because of their unique physicochemical, electrical, mechanical, and thermal properties, single-walled carbon nanotubes (SWCNT) are finding numerous applications in electronics, aerospace devices, computers, and chemical, polymer, and pharmaceutical industries. SWCNT are relatively recently discovered members of the carbon allotropes that are similar in structure to fullerenes and graphite. Previously, we (47) have reported that pharyngeal aspiration of purified SWCNT by C57BL/6 mice caused dose-dependent granulomatous pneumonia, oxidative stress, acute inflammatory/cytokine responses, fibrosis, and decrease in pulmonary function. To avoid potential artifactual effects due to instillation/agglomeration associated with SWCNT, we conducted inhalation exposures using stable and uniform SWCNT dispersions obtained by a newly developed aerosolization technique (2). The inhalation of nonpurified SWCNT (iron content of 17.7% by weight) at 5 mg/m(3), 5 h/day for 4 days was compared with pharyngeal aspiration of varying doses (5-20 microg per mouse) of the same SWCNT. The chain of pathological events in both exposure routes was realized through synergized interactions of early inflammatory response and oxidative stress culminating in the development of multifocal granulomatous pneumonia and interstitial fibrosis. SWCNT inhalation was more effective than aspiration in causing inflammatory response, oxidative stress, collagen deposition, and fibrosis as well as mutations of K-ras gene locus in the lung of C57BL/6 mice.     

A Conserved residue in the yeast Bem1p SH3 domain maintains the high level of binding specificity required for function

The yeast Bem1p SH3b and Nbp2p SH3 domains are unusual because they bind to peptides containing the same consensus sequence, yet they perform different functions and display low sequence similarity. In this work, by analyzing the interactions of these domains with six biologically relevant peptides containing the consensus sequence, they are shown to possess finely tuned and distinct binding specificities. We also identify a residue in the Bem1p SH3b domain that inhibits binding, yet is highly conserved for the purpose of preventing nonspecific interactions. Substitution of this residue results in a marked reduction of in vivo function that is caused by titration of the domain away from its proper targets through nonspecific interactions with other proteins. This work provides a clear illustration of the importance of intrinsic binding specificity for the function of protein-protein interaction modules, and the key role of "negative" interactions in determining the specificity of a domain.     

The structure and function of cardiac t-tubules in health and disease

The transverse tubules (t-tubules) are invaginations of the cell membrane rich in several ion channels and other proteins devoted to the critical task of excitation-contraction coupling in cardiac muscle cells (cardiomyocytes). They are thought to promote the synchronous activation of the whole depth of the cell despite the fact that the signal to contract is relayed across the external membrane. However, recent work has shown that t-tubule structure and function are complex and tightly regulated in healthy cardiomyocytes. In this review, we outline the rapidly accumulating knowledge of its novel roles and discuss the emerging evidence of t-tubule dysfunction in cardiac disease, especially heart failure. Controversy surrounds the t-tubules' regulatory elements, and we draw attention to work that is defining these elements from the genetic and the physiological levels. More generally, this field illustrates the challenges in the dissection of the complex relationship between cellular structure and function.     

Receptor desensitization and blockade of the suppressive effects of prostaglandin E<Subscript>2</Subscript> and adenosine on the cytotoxic activity of human melanoma-infiltrating T lymphocytes

Previous studies document that PGE₂ and adenosine suppress production of inflammatory cytokines. The present study demonstrates for the first time that (1) PGE₂ and 2-chloroadenosine (CADO; a stable analog of adenosine) directly inhibit the cytolytic function of human tumor-infiltrating lymphocytes (TILs); (2) the combination PGE₂ and CADO have additive suppressive effects; and (3) the cooperative immunosuppressive actions of PGE₂ and CADO are mediated via EP2 receptors (EP2Rs) and A_2A receptors (A_2ARs) and are due to amplification of cAMP production, activation of protein kinase A (PKA) and T cell receptor (TCR) inhibitor Csk leading to inhibition of Lck, ZAP-70 and Akt phosphorylation. (4) During ex vivo expansion, TILs undergo three stages of differentiation converting from TILs with high cytotoxic activity and relative resistance to combined EP2R/A_2AR suppression (stage I) to TILs retaining high cytotoxicity and gaining sensitivity to combined suppression (stage II) and then to TILS that are less cytotoxic and very sensitive to combined suppression (stage III). (5) Finally, we find that pretreatment of TILs with non-inhibitory concentrations of EP2R agonists (such as PGE₂ or butaprost) or A_2AR agonists (such as CADO or CGS21680) increases their cytotoxic activity and induces resistance to EP2R and A_2AR inhibitory signaling (cross-resistance) due to homologous and heterologous desensitization and internalization of EP2Rs and A_2ARs, thus preventing their inhibitory signaling. We conclude that inducing resistance of TILs to the suppressive effects of PGE₂ and adenosine in the tumor microenvironment could represent a novel strategy for improving the efficacy of adoptive immunotherapy.     

Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains

The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1-FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.     

Shape and compliance of endothelial cells after shear stress in vitro or from different aortic regions: scanning ion conductance microscopy study

Objective

To measure the elongation and compliance of endothelial cells subjected to different patterns of shear stress in vitro, and to compare these parameters with the elongation and compliance of endothelial cells from different regions of the intact aorta.

Materials and Methods

Porcine aortic endothelial cells were cultured for 6 days under static conditions or on an orbital shaker. The shaker generated a wave of medium, inducing pulsatile shear stress with a preferred orientation at the edge of the well or steadier shear stress with changing orientation at its centre. The topography and compliance of these cells and cells from the inner and outer curvature of ex vivo porcine aortic arches were measured by scanning ion conductance microscopy (SICM).

Results

Cells cultured under oriented shear stress were more elongated and less compliant than cells grown under static conditions or under shear stress with no preferred orientation. Cells from the outer curvature of the aorta were more elongated and less compliant than cells from the inner curvature.

Conclusion

The elongation and compliance of cultured endothelial cells vary according to the pattern of applied shear stress, and are inversely correlated. A similar inverse correlation occurs in the aortic arch, with variation between regions thought to experience different haemodynamic stresses.     

Yeast adaptor protein, Nbp2p, is conserved regulator of fungal Ptc1p phosphatases and is involved in multiple signaling pathways

Nbp2p is an Src homology 3 (SH3) domain-containing yeast protein that is involved in a variety of cellular processes. This small adaptor protein binds to a number of different proteins through its SH3 domain, and a region N-terminal to the SH3 domain binds to the protein phosphatase, Ptc1p. Despite its involvement in a large number of physical and genetic interactions, the only well characterized function of Nbp2p is to recruit Ptc1p to the high osmolarity glycerol pathway, which results in down-regulation of this pathway. In this study, we have discovered that Nbp2p orthologues exist in all Ascomycete and Basidiomycete fungal genomes and that all possess an SH3 domain and a conserved novel Ptc1p binding motif. The ubiquitous occurrence of these two features, which we have shown are both critical for Nbp2p function in Saccharomyces cerevisiae, implies that a conserved role of Nbp2p in all of these fungal species is the targeting of Ptc1p to proteins recognized by the SH3 domain. We also show that in a manner analogous to its role in the high osmolarity glycerol pathway, Nbp2p functions in the down-regulation of the cell wall integrity pathway through SH3 domain-mediated interaction with Bck1p, a component kinase of this pathway. Based on functional studies on the Schizosaccharomyces pombe and Neurospora crassa Nbp2p orthologues and the high conservation of the Nbp2p binding site in Bck1p orthologues, this function of Nbp2p appears to be conserved across Ascomycetes. Our results also clearly imply a function for the Nbp2p-Ptc1p complex other cellular processes.     

An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy

Current knowledge of the structural changes taking place during clathrin-mediated endocytosis is largely based on electron microscopy images of fixed preparations and x-ray crystallography data of purified proteins. In this paper, we describe a study of clathrin-coated pit dynamics in living cells using ion conductance microscopy to directly image the changes in pit shape, combined with simultaneous confocal microscopy to follow molecule-specific fluorescence. We find that 70% of pits closed with the formation of a protrusion that grew on one side of the pit, covered the entire pit, and then disappeared together with pit-associated clathrin-enhanced green fluorescent protein (EGFP) and actin-binding protein-EGFP (Abp1-EGFP) fluorescence. This was in contrast to conventionally closing pits that closed and cleaved from flat membrane sheets and lacked accompanying Abp1-EGFP fluorescence. Scission of both types of pits was found to be dynamin-2 dependent. This technique now enables direct spatial and temporal correlation between functional molecule-specific fluorescence and structural information to follow key biological processes at cell surfaces.     

In vivo iron and zinc deficiency diminished T- and B-selective mitogen stimulation of murine lymphoid cells through protein kinase C-mediated mechanism

Zinc and iron are crucial mineral components of human diet, because their deficiency leads to several disorders, including alterations of the immune function. It has been demonstrated, in both humans and rodents, that a diminished number of lymphoid cells and a loss of lymphocyte activity accompany deprivation of these essential minerals. The aim of this work was to analyze if iron and/or zinc imbalances regulate lymphocyte activity and the intracellular signals involved in the effect. Mice from the BALB/c strain were fed with iron- and/or zinc-deficient or mineral-supplemented diets, according to the American Institute of Nutrition Rodent Diets. Levels of iron and zinc were assessed in blood, liver, or bone samples. Selective mitogen stimulation of T- and B-lymphocytes were performed. We found a diminished proliferative response in T- and B-lymphocytes from zinc- and/or iron-deficient animals with respect to controls. These effects were related to decreased mitogen-induced translocation of protein kinase C (PKC) activity to cell membranes on both cell types from all animals fed with deficient diets. Our results demonstrate that iron and zinc deficiencies affect both T- and B-lymphocyte function by PKC-dependent mechanisms.