ANO5 ensures trafficking of annexins in wounded myofibers

Mutations in ANO5 (TMEM16E) cause limb-girdle muscular dystrophy R12. Defective plasma membrane repair is a likely mechanism. Using myofibers from Ano5 knockout mice, we show that trafficking of several annexin proteins, which together form a cap at the site of injury, is altered upon loss of ANO5. Annexin A2 accumulates at the wound to nearly twice the level observed in WT fibers, while annexin A6 accumulation is substantially inhibited in the absence of ANO5. Appearance of annexins A1 and A5 at the cap is likewise diminished in the Ano5 knockout. These changes are correlated with an alteration in annexin repair cap fine structure and shedding of annexin-positive vesicles. We conclude that loss of annexin coordination during repair is disrupted in Ano5 knockout mice and underlies the defective repair phenotype. Although ANO5 is a phospholipid scramblase, abnormal repair is rescued by overexpression of a scramblase-defective ANO5 mutant, suggesting a novel, scramblase-independent role of ANO5 in repair.     

The effects of interleukin-1 therapy on peripheral blood granulocyte function in humans

During a phase I trial of interleukin-1 (IL-1) in patients with ovarian carcinomas, the effects of this treatment on blood granulocyte respiratory burst and locomotive responses were examined. Differences in baseline granulocyte function in patients as well as dose-related effects of IL-1 treatment were observed. Patients enrolled early in the trial (low-dose patients) had significantly lower locomotive responses before treatment than their paired controls; these low responses normalized after 5 days of continuous-infusion IL-1 treatment. Patients enrolled later (high-dose patients) had normal locomotive responses before treatment and IL-1 treatment was associated with suppression of responses to selected stimuli at the end of treatment. Pretreatment respiratory burst responses in both low-and high-dose patient groups were essentially normal, but the rates of granulocyte H₂O₂ production following phorbol myristate acetate stimulation became significantly less than control values at the end of treatment. In vitro exposure of either patient or control cells to 150 U/ml IL-1 did not alter their locomotive or respiratory burst responses, suggesting the observed in vivo effects were not mediated directly by IL-1. Treatment with IL-1 is associated with changes in ex vivo granulocyte function that are related to the IL-1 dose. Treatment with low doses of IL-1 may provide a means of normalizing abnormal polymorphonuclear leukocyte function in some patients with ovarian malignancies.     

Use of the maize transposonsActivator andDissociation to show that phosphinothricin and spectinomycin resistance genes act non-cell-autonomously in tobacco and tomato seedlings

Cell-autonomous genes have been used to monitor the excision of both endogenous transposons in maize andAntirrhinum, and transposons introduced into transgenic plants. In tobacco andArabidopsis, the streptomycin phosphotransferase (SPT) gene reveals somatic excision of the maize transposonActivator (Ac) as green sectors on a white background in cotyledons of seedlings germinated in the presence of streptomycin. Cotyledons of tomato seedlings germinated on streptomycin-containing medium do not bleach, suggesting that a different assay for transposon excision in tomato is desirable. We have tested the use of the spectinomycin resistance (SPEC) gene (aadA) and a Basta resistance (BAR) gene (phosphinothricin acetyltransferase, or PAT) for monitoring somatic excision ofAc in tobacco and tomato. Both genetic and molecular studies demonstrate that genotypically variegated individuals that carry clones of cells from whichAc orDs have excised from either SPEC or BAR genes, can be phenotypically completely resistant to the corresponding antibiotic. This demonstrates that these genes act non-cell-autonomously, in contrast to the SPT gene in tobacco. Possible reasons for this difference are discussed.     

Physiological and molecular effects of brassinosteroids on Arabidopsis thaliana

We examined the effects of brassinosteroids on Arabidopsis thaliana (L.) Henyh. ecotype Columbia in order to develop a model system for studying gene regulation by plant steroids. Submicromolar concentrations of two brassinosteroids, brassinolide and 24-epibrassinolide, stimulated elongation of Arabidopsis peduncles and inhibited root elongation, respectively. Furthermore, brassinolide altered the abundance of specific in vitro translatable mRNAs from peduncles and whole plants of Arabidopsis. Root elongation in the auxin-insensitive Arabidopsis mutant axr1 was inhibited by 24-epibrassinolide but not by 2,4-D, indicating an independent mode of action for these growth regulators in this physiological response.Abbreviations BR brassinolide - EBR 24-epibrassinolide; 2.4-D,2,4-dichlorophenoxyacetic acid - KPSC 10 mM potassium phosphate, pH 6.0, 2% sucrose, 50 g/ml chloramphenicol - PAGE polyacrylamide gel electrophoresis     

Airway epithelial cells suppress T cell proliferation by an IFNγ/STAT1/TGFβ-dependent mechanism

Organ-specific regulation of immune responses relies on the exchange of information between nonimmune and immune cells. In a primary culture model of the lung airway, we demonstrate that T cell proliferation is potently inhibited by airway epithelial cells (ECs). This is mediated by activation of the IFNγ/STAT1 pathway in the EC and transforming growth factor-β (TGFβ)-dependent suppression of T cell proliferation. In this way, the EC can restrict the expansion of T cells. Given the constant exposure of the airway to inhaled antigen, this may be important in setting a threshold for the initiation of T cell-dependent immune responses and preventing unwanted, chronic inflammation.     

The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins providing both biophysical and biochemical cues that are important regulators of cell proliferation, survival, differentiation, and migration. We present here a proteomic strategy developed to characterize the in vivo ECM composition of normal tissues and tumors using enrichment of protein extracts for ECM components and subsequent analysis by mass spectrometry. In parallel, we have developed a bioinformatic approach to predict the in silico "matrisome" defined as the ensemble of ECM proteins and associated factors. We report the characterization of the extracellular matrices of murine lung and colon, each comprising more than 100 ECM proteins and each presenting a characteristic signature. Moreover, using human tumor xenografts in mice, we show that both tumor cells and stromal cells contribute to the production of the tumor matrix and that tumors of differing metastatic potential differ in both the tumor- and the stroma-derived ECM components. The strategy we describe and illustrate here can be broadly applied and, to facilitate application of these methods by others, we provide resources including laboratory protocols, inventories of ECM domains and proteins, and instructions for bioinformatically deriving the human and mouse matrisome.     

Immunoelectron microscopy studies with a monoclonal antibody directed against a receptor recognition site epitope in human α2-macroglobulin

α₂-Macroglobulin (α₂M) is a plasma proteinase inhibitor that binds up to 2 mole of proteinase per mole of inhibitor. Proteinase binding or reaction with small primary amines causes a major conformational change in α₂M. As a result of this conformational change, a new epitope recognized by monoclonal antibody 7H11D6 is exposed. The association of α₂M-proteinase or α₂M-methylamine with α₂M cellular receptors is prevented by 7H11D6. In this investigation, the binding of 7H11D6 to α₂M was studied by electron microscopy. 7H11D6 bound to α₂M-methylamine and α₂M-trypsin but not to native α₂M. The structure of α₂M after conformational change resembled the letter “H.” 7H11D6 epitopes were identified near the apices of the four arms in the α₂M “H” structure. 7H11D6 that was adducted to colloidal gold (7HAu) retained the specificity of the free antibody (binding to α₂M-trypsin but not to native α₂M). α₂M conformational change intermediates prepared by sequential reaction with a protein crosslinker and trypsin also bound 7HAu. These results suggest that a complete α₂M conformational change is not necessary for 7H11D6 epitope exposure and may not be required for receptor recognition. 7HAu was used to isolate a preparation consisting primarily of binary α₂M-trypsin (1 mole trypsin per mole α₂M instead of 2). Structures resembling the letter “H” were most common; however, each field showed some atypical molecules with arms that were compacted instead of thin and elongated. These incompletely transformed structures were similar to the α₂M conformational intermediates described previously (S. L. Gonias and N. L. Figler (1989) J. Biol. Chem. 264, 9565–9570). We propose that lateral arm extension is a critical step in α₂M conformational change. Failure of lateral arm extension is probably a common property of different α₂M conformational intermediates.     

Bacterial genome replication at subzero temperatures in permafrost

Microbial metabolic activity occurs at subzero temperatures in permafrost, an environment representing ∼25% of the global soil organic matter. Although much of the observed subzero microbial activity may be due to basal metabolism or macromolecular repair, there is also ample evidence for cellular growth. Unfortunately, most metabolic measurements or culture-based laboratory experiments cannot elucidate the specific microorganisms responsible for metabolic activities in native permafrost, nor, can bulk approaches determine whether different members of the microbial community modulate their responses as a function of changing subzero temperatures. Here, we report on the use of stable isotope probing with ¹³C-acetate to demonstrate bacterial genome replication in Alaskan permafrost at temperatures of 0 to −20 °C. We found that the majority (80%) of operational taxonomic units detected in permafrost microcosms were active and could synthesize ¹³C-labeled DNA when supplemented with ¹³C-acetate at temperatures of 0 to −20 °C during a 6-month incubation. The data indicated that some members of the bacterial community were active across all of the experimental temperatures, whereas many others only synthesized DNA within a narrow subzero temperature range. Phylogenetic analysis of ¹³C-labeled 16S rRNA genes revealed that the subzero active bacteria were members of the Acidobacteria, Actinobacteria, Chloroflexi, Gemmatimonadetes and Proteobacteria phyla and were distantly related to currently cultivated psychrophiles. These results imply that small subzero temperature changes may lead to changes in the active microbial community, which could have consequences for biogeochemical cycling in permanently frozen systems.     

Rings and ribbons in protein structures: Characterization using helical parameters and Ramachandran plots for repeating dipeptides

Helical parameters displayed on a Ramachandran plot allow peptide structures with successive residues having identical main chain conformations to be studied. We investigate repeating dipeptide main chain conformations and present Ramachandran plots encompassing the range of possible structures. Repeating dipeptides fall into the categories: rings, ribbons, and helices. Partial rings occur in the form of “nests” and “catgrips”; many nests are bridged by an oxygen atom hydrogen bonding to the main chain NH groups of alternate residues, an interaction optimized by the ring structure of the nest. A novel recurring feature is identified that we name unpleated β, often situated at the ends of a β‐sheet strand. Some are partial rings causing the polypeptide to curve gently away from the sheet; some are straight. They lack β‐pleat and almost all incorporate a glycine. An example is the first glycine in the GxxxxGK motif of P‐loop proteins. Ribbons in repeating dipeptides can be either flat, as seen in repeated type II and type II′ β‐turns, or twisted, as in multiple type I and type I′ β‐turns. Hexa‐ and octa‐peptides in such twisted ribbons occur frequently in proteins, predominantly with type I β‐turns, and are the same as the “β‐bend ribbons” hitherto identified only in short peptides. One is seen in the GTPase‐activating protein for Rho in the active, but not the inactive, form of the enzyme. It forms a β‐bend ribbon, which incorporates the catalytic arginine, allowing its side chain guanidino group to approach the active site and enhance enzyme activity. Proteins 2014; 82:230–239. © 2013 Wiley Periodicals, Inc.     

C‐terminal β‐strand swapping in a consensus‐derived fibronectin Type III scaffold

The crystal structures of six different fibronectin Type III consensus‐derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C‐terminal β‐strand resulting in the formation of Tencon dimers in which the target‐binding surface is blocked. All of the variants differ in sequence in the FG loop, which is the hinge loop in the β‐strand‐swapped dimers. The six tencon variants have between 0 and 5 residues inserted between positions 77 and 78 in the FG loop. Analysis of the structures suggests that a non‐glycine residue at position 77 and insertions of <4 residues may destabilize the β‐turn in the FG loop promoting β‐strand swapping. Swapped dimers with an odd number of inserted residues may be less stable, particularly if they contain proline residues, because they cannot form perfect β‐bridges in the FG regions that link the swapped dimers. The Tencon β‐swapped variants with the longest FG sequences are observed to form higher order hexameric or helical oligomeric structures in the crystal correlating well with the aggregation properties of these domains observed in solution. Understanding the structural basis for domain‐swapped dimerization and oligomerization will support engineering efforts of the Tencon domain to produce variants with desired biophysical properties. Proteins 2014; 82:1359–1369. © 2013 Wiley Periodicals, Inc.