Cross-Talk between Adherens Junctions and Desmosomes Depends on Plakoglobin

Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immediately obvious why formation of an adherens junction is a prerequisite for the formation of a desmosome. The adherens junction is composed of a transmembrane classical cadherin (E-cadherin and/or P-cadherin in squamous epithelial cells) linked to either β-catenin or plakoglobin, which is linked to α-catenin, which is linked to the actin cytoskeleton. The desmosome is composed of transmembrane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the intermediate filament cytoskeleton, presumably through plakoglobin and desmoplakin. To begin to study the role of adherens junctions in the assembly of desmosomes, we produced an epithelial cell line that does not express classical cadherins and hence is unable to organize desmosomes, even though it retains the requisite desmosomal components. Transfection of E-cadherin and/or P-cadherin into this cell line did not restore the ability to organize desmosomes; however, overexpression of plakoglobin, along with E-cadherin, did permit desmosome organization. These data suggest that plakoglobin, which is the only known common component to both adherens junctions and desmosomes, must be linked to E-cadherin in the adherens junction before the cell can begin to assemble desmosomal components at regions of cell–cell contact. Although adherens junctions can form in the absence of plakoglobin, making use only of β-catenin, such junctions cannot support the formation of desmosomes. Thus, we speculate that plakoglobin plays a signaling role in desmosome organization.Squamous epithelial cells typically contain two prominent types of cell–cell junctions: the adherens junction and the desmosome. The adherens junction is an intercellular adhesion complex that is composed of a transmembrane protein (a classical cadherin) and numerous cytoplasmic proteins (α-catenin, β-catenin and plakoglobin, vinculin and α-actinin; for reviews see Takeichi, 1990; Geiger and Ayalon, 1992). The cadherins are directly responsible for adhesive interactions via a Ca²⁺-dependent, homotypic mechanism, i.e., in the presence of sufficient Ca²⁺, cadherin on one cell binds to an identical molecule on an adjacent cell. The desmosome, also an intercellular adhesion complex, is composed of at least two different transmembrane proteins (desmoglein and desmocollin) as well as several cytoplasmic proteins, including desmoplakins and plakoglobin (Koch and Franke, 1994). The transmembrane components of the desmosome are members of the broadly defined cadherin family and also require Ca²⁺ for adhesive activity. However, decisive experimental evidence for homophilic or heterophilic interactions between desmosomal cadherins via their extracellular domains has not yet been presented (Koch and Franke, 1994; Kowalczyk et al., 1996). While members of the cadherin family constitute the transmembrane portion of both adherens junctions and desmosomes, the different classes of cadherins are linked to different cytoskeletal elements by the cytoplasmic components of each junction. Specifically, the classical cadherins are linked to actin filaments and the desmosomal cadherins to intermediate filaments.The organization of the proteins within the adherens junction is well understood (for reviews see Kemler, 1993; Cowin, 1994; Wheelock et al., 1996). Specifically, the intracellular domain of cadherin interacts directly with either plakoglobin or β-catenin, which in turns binds to α-catenin (Jou et al., 1995; Sacco et al., 1995). α-Catenin interacts with α-actinin and actin filaments, thereby linking the cadherin/ catenin complex to the cytoskeleton (Knudsen et al., 1995; Rimm et al., 1995). Cadherin/catenin complexes include either plakoglobin or β-catenin but not both (Näthke et al., 1994). The importance of the classical cadherins to the formation of adherens junctions and desmosomes has been demonstrated. Keratinocytes maintained in medium with low Ca²⁺ (i.e., 30 μM) grow as a monolayer and do not exhibit adherens junctions or desmosomes; however, elevation of Ca²⁺ concentration induces the rapid formation of adherens junctions followed by the formation of desmosomes (Hennings et al., 1980; Tsao et al., 1982; Boyce and Ham, 1983; Hennings and Holbrook, 1983; O''Keefe et al., 1987; Wheelock and Jensen, 1992; Hodivala and Watt, 1994; Lewis et al., 1994). Simultaneous blocking with functionperturbing antibodies against the two classical cadherins (E- and P-cadherin) found in keratinocytes inhibits not only Ca²⁺-induced adherens junction formation but also severely limits desmosome formation (Lewis et al., 1994; Jensen et al., 1996). Consistent with these findings, expression of a dominant-negative cadherin by keratinocytes results in decreased E-cadherin expression and delayed assembly of desmosomes (Fujimori and Takeicki, 1993; Amagai, et al., 1995). These data suggest some form of cross-talk between the proteins of the adherens junction and those of the desmosome. One candidate protein that might mediate such cross-talk is plakoglobin, since it is the only known common component of both junctions.Plakoglobin is found to be associated with the cytoplasmic domains of both the classical cadherins and the desmosomal cadherins. Despite the high degree of identity between plakoglobin and β-catenin (65% at the amino acid level; Fouquet et al., 1992), β-catenin only associates with the classical cadherins and not with the desmosomal cadherins. In the adherens junction, plakoglobin and β-catenin have at least one common function, i.e., the linking of cadherin to α-catenin and thus to actin. However, there is emerging evidence that other functions of these two proteins are not identical. For example, in a study by Navarro et al. (1993), E-cadherin transfected into a spindle cell carcinoma was shown to associate with α- and β-catenin, but not with the low levels of endogenous plakoglobin. The transfected cells did not revert to a more epithelial morphology in spite of the presence of functional E-cadherin, and the authors suggested that the lack of plakoglobin may have prevented such morphological reversion.In the present study, we have tested the hypothesis that plakoglobin, through its interaction with E- or P-cadherin, serves as a regulatory molecule for desmosome organization. Even though plakoglobin is not an essential structural component of the adherens junction (Sacco et al., 1995), our data indicate that plakoglobin can function as a regulator of desmosome formation only when it is associated with a classical cadherin. Thus, we propose that plakoglobin has at least two functions: (a) as a structural component of the adherens junction and the desmosome and (b) as a signaling molecule that regulates communication between the adherens junction and the desmosome.     

Scale- and taxon-dependent patterns of plant diversity in steppes of Khakassia,South Siberia (Russia)

The drivers of plant richness at fine spatial scales in steppe ecosystems are still not sufficiently understood. Our main research questions were: (i) How rich in plant species are the natural steppes of Southern Siberia compared to natural and semi-natural grasslands in other regions of the Palaearctic? (ii) What are the main environmental drivers of the diversity patterns in these steppes? (iii) What are the diversity–environment relationships and do they vary between spatial scales and among different taxonomic groups? We sampled the steppe vegetation (vascular plants, bryophytes and lichens) in Khakassia (Russia) with 39 nested-plot series (0.0001–100-m² plot size) and 54 additional 10-m² quadrats across the regional range of steppe types and measured various environmental variables. We measured β-diversity using z-values of power-law species–area relationships. GLM analyses were performed to assess the importance of environmental variables as predictors of species richness and z-value. Khakassian steppes showed both high α- and β-diversity. We found significant scale dependence for the z-values, which had their highest values at small spatial scales and then decreased exponentially. Total species richness was controlled predominantly by heat load index, mean annual precipitation, humus content and soil skeleton content. The positive role of soil pH was evident only for vascular plant species richness. Similar to other studies, we found that the importance of environmental factors strongly differed among taxonomic groups and across spatial scales, thus highlighting the need to study more than one taxon and more than one plot size to get a reliable picture.     

Towards understanding the organisation of metacommunities in highly dynamic ecological systems

Community ecology recognises today that local biological communities are not only affected by local biotic interactions and abiotic environmental conditions, but also by regional processes (e.g. dispersal). While much is known about how metacommunities are organised in space in terrestrial, marine and freshwater ecological systems, their temporal variations remain poorly studied. Here, we address the question of the dynamics of metacommunities in highly variable systems, using intermittent rivers (IRs), those rivers which temporarily stop flowing or dry up, as a model system. We first review how habitat heterogeneity in space and time influences metacommunity organisation. Second, we compare the metacommunities in IRs to those in perennial rivers (PRs) and develop the idea that IRs could undergo highly dynamic shifts due to the temporal variability in local and regional community processes. Third, we develop the idea that in IRs, metacommunities of the wet and dry phases of IRs are closely intertwined, thereby increasing even more their respective temporal dynamics. Last, we provide a roadmap to stimulate further conceptual and empirical developments of metacommunity research and identify possible applications for improving the management of IRs and other highly dynamic ecological systems. Synthesis Extensive research has examined the importance of local biotic interactions, environmental filtering, and regional processes on community assembly. Movement of organisms between sites, i.e. dispersal, is a major set of processes within this framework. However, subsequent metacommunity organisation also varies over time in ecosystems because habitat characteristics such as configuration and composition continuously shift. Intermittent rivers are an ideal set of systems to examine these ideas because these freshwater systems temporarily cease flowing thereby limiting dispersal events. We proposed the hypothesis that metacommunities in dynamic ecosystems will undergo frequent shifts in structure and composition in response to the temporal variability in environmental filtering and dispersal. In addition to providing a roadmap for developing a more dynamic perspective for community ecology, these framework provides direct insights for the management of intermittent rivers.     

Length measurement accuracy of adaptive resolution imaging sonar and a predictive model to assess adult Atlantic salmon (Salmo salar) into two size categories with long-range data in a river

Imaging sonars are used around the world for fish population monitoring. The accuracy of the length measurements has been reported in multiple studies for relatively short (<15 m) ranges and high image resolution. However, imaging sonars are often used at longer ranges (i.e., >15 m) where the images produced from sonar returns become less detailed. The accuracy of the length measurements from the Adaptive Resolution Imaging Sonar (ARIS) was tested by releasing n = 69 known-sized adult Atlantic salmon (Salmo salar) directly into the sonar field at ranges between 15 and 29 m, and measuring their echoes manually by four users and semi-automatically using a computer workflow in Echoview software. Overall, the length measurements were very variable: compared to true (fork) lengths, the mean of differences varied between −9.9 cm and 7.8 cm in the human-generated datasets, and between −42.8 cm and −20 cm in the computer-generated dataset. In addition, the length measurements in different datasets were only in poor or moderate agreement with each other (intraclass correlation <0.61). Contrary to our expectations, the distance from the transducer or the subjectively assessed echo quality did not have an effect on the measurement accuracy in most of the datasets and when it did, the effect was not systematic between the datasets. Therefore, a size class and length prediction model was implemented in a Bayesian framework to group salmon into two size categories: One-Sea-Winter (<63 cm) and Multi-Sea-Winter (≥63 cm) groups. The model correctly predicted the size category in 83% of the fish in the computer-generated dataset and ranged from 68% to 74% in the human-generated datasets. We conclude that fish length measurements derived from long-range imaging sonar data should be used with caution, but post-processing can improve the usefulness of the data for specific purposes, such as adult Atlantic salmon population monitoring.     

Studies on the immunogenic potential of plant-expressed cholera toxin B subunit

Nicotiana tabacum var. Samsun was transformed via Agrobacterium-mediated transformation with a gene encoding the cholera toxin B subunit (CTB) of Vibrio cholerae, modified to contain a sequence coding for an endoplasmic reticulum retention signal (SEKDEL), under the control of the cauliflower mosaic virus 35S promoter. Total protein from the transgenic leaf tissue was isolated and an aliquot containing 5 g recombinant CTB was injected intradermally into Balb/c (H2K^d) mice. CTB-specific serum IgG was detected in animals that had been administered plant-expressed or native purified CTB. A T-cell proliferation study using splenocytes and cytokine estimations in supernatants generated by in vitro stimulation of macrophages isolated from the immuno-primed animals was carried out. Inhibition of proliferation of T lymphocytes was observed in splenic T lymphocytes isolated from animals injected with either native or plant-expressed CTB. Macrophages isolated from mice immunised with native or plant-expressed CTB showed enhanced secretion of interleukin-10 but secretion of lipopolysaccharide-induced interleukin-12 and tumor necrosis factor alpha was inhibited. These studies suggest that plant-expressed protein behaved like native CTB with regards to effects on T-cell proliferation and cytokine levels, indicating the suitability of plant expression systems for the production of bacterial antigens, which could be used as edible vaccine. The transgene was found to be inherited in the progeny and was expressed to yield a pentameric form of CTB as evident by its interaction with G_M1 ganglioside.Abbreviations BAP 6-Benzylaminopurine - Con A Concanavilin A - CTB Cholera toxin B subunit - ctxB Gene encoding cholera toxin B subunit - ELISA Enzyme-linked immunosorbent assay - HRP Horseradish peroxidase - IL-10 Interleukin-10 - IL-12 Interleukin-12 - LPS Lipopolysaccharide - NAA Naphthaleneacetic acid - PBS Phosphate-buffered saline - TNF Tumour necrosis factor alphaCommunicated by H. Uchimiya     

Surveying biodiversity in protected and managed areas: Algae, macrophytes and macroinvertebrates in boreal forest streams

Extensive surveys of biodiversity in protected and managed areas have not been conducted for a majority of taxonomic groups and ecosystem types, which makes it difficult to assess how large a portion of biodiversity is at least potentially under protection. The situation is the same in boreal regions, and only preliminary analyses of the biodiversity patterns of less well-known organism groups, including many freshwater taxa, within the protected area network have been conducted. We studied patterns of species richness and community composition of algae, macrophytes (bryophytes and vascular plants), and macroinvertebrates of headwater streams draining protected areas and managed forests in a boreal drainage basin in Finland. We found no significant differences in the species richness and community composition of these organism groups between the protected and managed streams. Gamma- and beta-diversity varied strongly among the protected and managed stream groups, yet this variation was contingent on the organism group and the beta-diversity measure used. In general, there was much species turnover within both protected and managed stream groups, masking any between-group differences. However, we found a number of redlisted and rare species in our surveys. Of these species, several macrophyte species occurred more frequently in the protected streams. By contrast, rare species of algae and macrophytes did not generally show such inclinations to the protected streams. We found no strong congruence in species richness or community dissimilarity between algae, macrophytes, and macroinvertebrates, suggesting that the main anthropogenic gradient in terms of forestry is not strong enough to modify stream environmental conditions and thereby shape biodiversity in the focal drainage basin. This finding also suggests that surveys of aquatic biodiversity across protected and managed landscapes should not rely too heavily on the surrogate taxon approach, but instead should consider patterns shown by multiple taxonomic groups that represent biologically and ecologically disparate organisms. Our results indeed suggest that the levels of alpha-, beta-, and gamma-diversity show differing among-taxon responses to forest management and naturalness of headwater streams.     

Comparative Genomics and Characterization of Hybrid Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains

Background

Shigatoxigenic Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) cause serious foodborne infections in humans. These two pathogroups are defined based on the pathogroup-associated virulence genes: stx encoding Shiga toxin (Stx) for STEC and elt encoding heat-labile and/or est encoding heat-stable enterotoxin (ST) for ETEC. The study investigated the genomics of STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli and to define the virulence genes they harbor.

Methods

The whole genomes of three STEC/ETEC strains possessing both stx and est genes were sequenced using PacBio RS sequencer. Two of the strains were isolated from the patients, one with hemolytic uremic syndrome, and one with diarrhea. The third strain was of bovine origin. Core genome analysis of the shared chromosomal genes and comparison with E. coli and Shigella spp. reference genomes was performed to determine the phylogenetic position of the STEC/ETEC strains. In addition, a set of virulence genes and ETEC colonization factors were extracted from the genomes. The production of Stx and ST were studied.

Results

The human STEC/ETEC strains clustered with strains representing ETEC, STEC, enteroaggregative E. coli, and commensal and laboratory-adapted E. coli. However, the bovine STEC/ETEC strain formed a remote cluster with two STECs of bovine origin. All three STEC/ETEC strains harbored several other virulence genes, apart from stx and est, and lacked ETEC colonization factors. Two STEC/ETEC strains produced both toxins and one strain Stx only.

Conclusions

This study shows that pathogroup-associated virulence genes of different E. coli can co-exist in strains originating from different phylogenetic lineages. The possibility of virulence genes to be associated with several E. coli pathogroups should be taken into account in strain typing and in epidemiological surveillance. Development of novel hybrid E. coli strains may cause a new public health risk, which challenges the traditional diagnostics of E. coli infections.     

Context dependency and metacommunity structuring in boreal headwater streams

We studied the relative importance of spatial and environmental factors as determinants of algal, bryophyte, and macroinvertebrate metacommunities in two boreal drainage basins differing in spatial extent. We used eigenfunction spatial analysis to model the spatial relationships among sites and distance‐based redundancy analysis to partition the variability in biotic communities between the spatial filters generated through spatial eigenfunction analysis and the environmental factors measured in the field. In the smaller study area, each metacommunity was structured mostly by environmental factors. This was evidenced by the fact that either the pure environmental effect was significant or environmental factors were strongly spatially structured. In the larger study area, only pure environmental effects were significant. These findings suggest that the environmental control prevails in boreal headwater streams. However, our findings also suggest that the specific details of the community‐environment and community–space relationships are dependent on the focal organism group and drainage basin.     

Reference Values for Clinical Laboratory Parameters in Young Adults in Maputo,Mozambique

Background

Clinical laboratory reference values from North American and European populations are currently used in most Africans countries due to the absence of locally derived reference ranges, despite previous studies reporting significant differences between populations. Our aim was to define reference ranges for both genders in 18 to 24 year-old Mozambicans in preparation for clinical vaccine trials.

Methods

A cross-sectional study including 257 volunteers (102 males and 155 females) between 18 and 24 years was performedat a youth clinic in Maputo, Mozambique. All volunteers were clinically healthy and human immunodeficiency virus, Hepatitis B virus and syphilis negative.Median and 95% reference ranges were calculated for immunological, hematological and chemistry parameters. Ranges were compared with those reported based on populations in other African countries and the US. The impact of applying US NIH Division of AIDS (DAIDS) toxicity tables was assessed.

Results

The immunology ranges were comparable to those reported for the US and western Kenya.There were significant gender differences in CD4⁺ T cell values 713 cells/µL in males versus 824 cells/µL in females (p<0.0001). Hematologic values differed from the US values but were similar to reports of populations in western Kenya and Uganda. The lower and upper limits of the ranges for hemoglobin, hematocrit, red blood cells, white blood cells and lymphocytes were somewhat lower than those from these African countries. The chemistry values were comparable to US values, with few exceptions. The upper limits for ALT, AST, bilirubin, cholesterol and triglycerides were higher than those from the US. DAIDStables for adverse events predicted 297 adverse events and 159 (62%) of the volunteers would have been excluded.

Conclusion

This study is the first to determine normal laboratory parameters in Mozambique. Our results underscore the necessity of establishing region-specific clinical reference ranges for proper patient management and safe conduct of clinical trials.