Osmotic stress-induced remodeling of the cortical cytoskeleton

Osmoticstress is known to affect the cytoskeleton; however, this adaptiveresponse has remained poorly characterized, and the underlyingsignaling pathways are unexplored. Here we show that hypertonicityinduces submembranous de novo F-actin assembly concomitant with theperipheral translocation and colocalization of cortactin and theactin-related protein 2/3 (Arp2/3) complex, which are key components ofthe actin nucleation machinery. Additionally, hyperosmolarity promotesthe association of cortactin with Arp2/3 as revealed bycoimmunoprecipitation. Using various truncation orphosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, butthe process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. Thisappears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active smallGTPases translocate cortactin, 2) Rac and cortactincolocalize at the periphery of hypertonically challenged cells, and3) dominant-negative Rac and Cdc42 inhibit thehypertonicity-provoked cortactin and Arp3 translocation. The Rhofamily-dependent cytoskeleton remodeling may be an importantosmoprotective response that reinforces the cell cortex.

     

Intracellular localization of the matrix enzyme lysyl oxidase in polarized epithelial cells.

Considerable evidence supports novel functions for lysyl oxidase (LOX) beyond its traditional role in initiating cross-linkages in collagen and elastin within the extracellular matrix. These novel roles are particularly relevant during the transition of malignant epithelial cells towards a migratory and invasive phenotype. However, knowledge on cellular and matrix functions of LOX has been generated almost exclusively in mesenchymal cell types. But it is becoming increasingly evident that these cell types are not adequate to address these novel and highly significant roles for LOX in epithelial tissues. In this initial report, we demonstrate that active LOX is expressed by polarized MDCK II kidney and MCF-10A breast epithelial cells. Furthermore, we show evidence for the presence of mature LOX in the cytoplasm and establish these cell lines as models for epithelial LOX studies.     

Indicators for the on-farm assessment of crop cultivar and livestock breed diversity: a survey-based participatory approach

Agrobiodiversity plays a fundamental role in guaranteeing food security. However, still little is known about the diversity within crop and livestock species: the genetic diversity. In this paper we present a set of indicators of crop accession and breed diversity for different farm types at farm-level, which may potentially supply a useful tool to assess and monitor farming system agrobiodiversity in a feasible and relatively affordable way. A generic questionnaire was developed to capture the information on crops and livestock in 12 European case study regions and in Uganda by 203 on-farm interviews. Through a participatory approach, which involved a number of stakeholders, eight potential indicators were selected and tested. Five of them are recommended as potentially useful indicators for agrobiodiversity monitoring per farm: (1) crop-species richness (up to 16 crop species), (2) crop-cultivar diversity (up to 15 crop cultivars, 1–2 on average), (3) type of crop accessions (landraces accounted for 3 % of all crop cultivars in Europe, 31 % in Uganda), (4) livestock-species diversity (up to 5 livestock species), and (5) breed diversity (up to five cattle and eight sheep breeds, on average 1–2).We demonstrated that the selected indicators are able to detect differences between farms, regions and dominant farm types. Given the present rate of agrobiodiversity loss and the dramatic effects that this may have on food production and food security, extensive monitoring is urgent. A consistent survey of crop cultivars and livestock breeds on-farm will detect losses and help to improve strategies for the management and conservation of on-farm genetic resources.     

Mutating a critical lysine in ShK toxin alters its binding configuration in the pore-vestibule region of the voltage-gated potassium channel,Kv1.3

The voltage-gated potassium channel in T lymphocytes, Kv1.3, an important target for immunosuppressants, is blocked by picomolar concentrations of the polypeptide ShK toxin and its analogue ShK-Dap22. ShK-Dap22 shows increased selectivity for Kv1.3, and our goal was to determine the molecular basis for this selectivity by probing the interactions of ShK and ShK-Dap22 with the pore and vestibule of Kv1.3. The free energies of interactions between toxin and channel residues were measured using mutant cycle analyses. These data, interpreted as approximate distance restraints, guided molecular dynamics simulations in which the toxins were docked with a model of Kv1.3 based on the crystal structure of the bacterial K(+)-channel KcsA. Despite the similar tertiary structures of the two ligands, the mutant cycle data imply that they make different contacts with Kv1.3, and they can be docked with the channel in configurations that are consistent with the mutant cycle data for each toxin but quite distinct from one another. ShK binds to Kv1.3 with Lys22 occupying the negatively charged pore of the channel, whereas the equivalent residue in ShK-Dap22 interacts with residues further out in the vestibule, producing a significant change in toxin orientation. The increased selectivity of ShK-Dap22 is achieved by strong interactions of Dap22 with His404 and Asp386 on Kv1.3, with only weak interactions between the channel pore and the toxin. Potent and specific blockade of Kv1.3 apparently occurs without insertion of a positively charged residue into the channel pore. Moreover, the finding that a single residue substitution alters the binding configuration emphasizes the need to obtain consistent data from multiple mutant cycle experiments in attempts to define protein interaction surfaces using these data.     

Density of DC-LAMP+ mature dendritic cells in combination with activated T lymphocytes infiltrating primary cutaneous melanoma is a strong independent prognostic factor

As the most potent antigen presenting cells, dendritic cells (DCs) play key roles in the immune response against tumors. Their density in the tumor tissue has been associated with prognosis in patients with various cancers. However, few studies have been aimed at the presence and maturation state of DCs in cutaneous melanoma, with regard to their potential clinical correlates. In this study, the density of DCs expressing CD1a and the maturation marker DC-LAMP was determined by immunohistochemistry in primary tumor samples from 82 patients with cutaneous malignant melanoma. Intratumoral and peritumoral cell densities were analyzed in relation to tumor thickness and the subsequent development of metastases, as well as to patients’ survival. CD1a⁺ DCs were found both infiltrating melanoma cell nests and in the surrounding stroma, while DC-LAMP⁺ mature DCs were generally confined to the peritumoral areas, associated with lymphocytic infiltrates. DC density values significantly correlated with the number of activated (CD25⁺ or OX40⁺) T lymphocytes (p < 0.001). The degree of infiltration by CD1a⁺ and DC-LAMP⁺ DCs showed strong inverse correlation with the thickness of melanomas (p < 0.001). High peritumoral density of mature DCs was associated with significantly longer survival (p = 0.0195), while density of CD1a⁺ cells had a prognostic impact of borderline significance (p = 0.0610). Moreover, combination of high peritumoral CD1a⁺ or DC-LAMP⁺ cell density with high number of CD25⁺ or OX40⁺ lymphocytes identified patient subgroups with more favorable survival compared to other subgroups. A multivariate survival analysis involving DC and activated T-cell densities alone and in combinations, as well as traditional prognostic factors, identified high DC-LAMP⁺ cell/high OX40⁺ cell density and Breslow index as independent predictors of good prognosis. These results suggest that the presence of CD1a⁺ DCs primarily depends on the thickness of melanomas, without direct relationship with the patients’ survival. On the other hand, the density of mature DCs, especially in association with that of activated T cells, proved of prognostic importance, suggesting that these parameters could be considered as signs of a functional immune response associated with better outcome of the disease.     

Construction of a basic genetic map for alfalfa using RFLP, RAPD, isozyme and morphological markers

The genetic map for alfalfa presented here has eight linkage groups representing the haploid chromosome set of the Medicago species. The genetic map was constructed by ordering the linkage values of 89 RFLP, RAPD, isozyme and morphological markers collected from a segregating population of 138 individuals. The segregating population is self-mated progeny of an F1 hybrid plant deriving from a cross between the diploid (2n=2x=16) yellow-flowered Medicago sativa ssp. quasifalcata and the diploid (2n=2x=16) blue-flowered M. sativa ssp. coerulea. The inheritance of many traits displayed distorted segregation, indicating the presence of lethal loci in the heterozygotic parent plants. In spite of the lack of uniform segregation, linkage groups could be assigned and the order of the markers spanning > 659 centimorgans could be unambiguously determined. This value and the calculated haploid genome size for Medicago (1n=1x=1.0 x 10⁹ bp) gives a ratio of < 1500 kb per centimorgan.