A comparison of national approaches to setting ecological status boundaries in phytobenthos assessment for the European Water Framework Directive: results of an intercalibration exercise

The European Union (EU)’s Water Framework Directive (WFD) requires that all Member States participate in intercalibration exercises in order to ensure that ecological status concepts and assessment levels are consistent across the EU. This paper describes one such exercise, performed by the countries in the Central/Baltic Geographical Intercalibration Group stretching from Ireland in the west to Estonia in the east and from the southern parts of Scandinavia to the northern regions of Spain and Italy (but excluding alpine regions, which were intercalibrated separately). In this exercise, methods used to measure ecological status of rivers using benthic diatoms were compared. Ecological status is estimated as the ratio between the observed value of a biological element and the value expected in the absence of significant human impact. Approaches to defining the ‘reference sites’, from which these ‘expected’ values were derived, varied from country to country. Minimum criteria were established as part of the exercise but there was still considerable variation between national reference values, reflecting typological differences that could not be resolved during the exercise. A simple multimetric index was developed to compare boundary values using two widely used diatom metrics. Boundary values for high/good status and good/moderate status set by each participant were converted to their equivalent values of this intercalibration metric using linear regression. Variation of ±0.05 EQR units around the median value was considered to be acceptable and the exercise provided a means for those Member States who fell significantly above or below this line to review their approaches and, if necessary, adjust their boundaries. Handling editor: J. Padisak     

Investigation of 17 candidate genes for personality traits confirms effects of the HTR2A gene on novelty seeking

Genes involved in serotonergic and dopaminergic neurotransmission have been hypothesized to affect different aspects of personality, but findings from genetic association studies did not provide conclusive results so far. In previous studies, however, only one or a few polymorphisms within single genes were investigated neglecting the possibility that the genetic associations might be more complex comprising several genes or gene regions. To overcome this limitation, we performed an extended genetic association study analyzing 17 serotonergic ( SLC6A4, HTR1A, HTR1B, HTR2A, HTR2C, HTR3A, HTR6, MAOA, TPH1, TPH2 ) and dopaminergic genes ( SLC6A3, DRD2, DRD3, DRD4, COMT, MAOA, TH, DBH ), which have been previously reported to be implicated with personality traits.
One hundred and ninety-five single nucleotide polymorphisms (SNPs) within these genes were genotyped with the Illumina BeadChip technology (HumanHap300, Human-1) in a sample of 366 mentally healthy Caucasians. Additionally, we tried to replicate our results in an independent sample of further 335 Caucasians. Personality traits in both samples were assessed with the German version of Cloninger's Tridimensional Personality Questionnaire.
From 30 SNPs showing associations at a nominal level of significance, two intronic SNPs, rs2770296 and rs927544, both located in the HTR2A gene, withstood correction for multiple testing. These SNPs were associated with the personality trait novelty seeking . The effect of rs927544 could be replicated for the novelty seeking subscale extravagance , and the same SNP was also associated with extravagance inthe combined samples.
Our results show that HTR2A polymorphisms modulate facets of novelty seeking behaviour in healthy adults suggesting that serotonergic neurotransmission is involved in this phenotype.     

Hypoxia. Hypoxia in the pathogenesis of systemic sclerosis

Autoimmunity, microangiopathy and tissue fibrosis are hallmarks of systemic sclerosis (SSc). Vascular alterations and reduced capillary density decrease blood flow and impair tissue oxygenation in SSc. Oxygen supply is further reduced by accumulation of extracellular matrix (ECM), which increases diffusion distances from blood vessels to cells. Therefore, severe hypoxia is a characteristic feature of SSc and might contribute directly to the progression of the disease. Hypoxia stimulates the production of ECM proteins by SSc fibroblasts in a transforming growth factor-β-dependent manner. The induction of ECM proteins by hypoxia is mediated via hypoxia-inducible factor-1α-dependent and -independent pathways. Hypoxia may also aggravate vascular disease in SSc by perturbing vascular endothelial growth factor (VEGF) receptor signalling. Hypoxia is a potent inducer of VEGF and may cause chronic VEGF over-expression in SSc. Uncontrolled over-expression of VEGF has been shown to have deleterious effects on angiogenesis because it leads to the formation of chaotic vessels with decreased blood flow. Altogether, hypoxia might play a central role in pathogenesis of SSc by augmenting vascular disease and tissue fibrosis.     

Vasoregression Linked to Neuronal Damage in the Rat with Defect of Polycystin-2

Background

Neuronal damage is correlated with vascular dysfunction in the diseased retina, but the underlying mechanisms remain controversial because of the lack of suitable models in which vasoregression related to neuronal damage initiates in the mature retinal vasculature. The aim of this study was to assess the temporal link between neuronal damage and vascular patency in a transgenic rat (TGR) with overexpression of a mutant cilia gene polycystin-2.

Methods

Vasoregression, neuroglial changes and expression of neurotrophic factors were assessed in TGR and control rats in a time course. Determination of neuronal changes was performed by quantitative morphometry of paraffin-embedded vertical sections. Vascular cell composition and patency were assessed by quantitative retinal morphometry of digest preparations. Glial activation was assessed by western blot and immunofluorescence. Expression of neurotrophic factors was detected by quantitative PCR.

Findings

At one month, number and thickness of the outer nuclear cell layers (ONL) in TGR rats were reduced by 31% (p<0.001) and 17% (p<0.05), respectively, compared to age-matched control rats. Furthermore, the reduction progressed from 1 to 7 months in TGR rats. Apoptosis was selectively detected in the photoreceptor in the ONL, starting after one month. Nevertheless, TGR and control rats showed normal responses in electroretinogram at one month. From the second month onwards, TGR retinas had significantly increased acellular capillaries (p<0.001), and a reduction of endothelial cells (p<0.01) and pericytes (p<0.01). Upregulation of GFAP was first detected in TGR retinas after 1 month in glial cells, in parallel with an increase of FGF2 (fourfold) and CNTF (60 %), followed by upregulation of NGF (40 %) at 3 months.

Interpretation

Our data suggest that TGR is an appropriate animal model for vasoregression related to neuronal damage. Similarities to experimental diabetic retinopathy render this model suitable to understand general mechanisms of maturity-onset vasoregression.     

Plasma zinc concentrations of mothers and the risk of oral clefts in their children in Utah

BACKGROUND: The role of maternal zinc nutrition in human oral clefts (OCs) is unclear. We measured plasma zinc concentrations (PZn) of case and control mothers to evaluate the associations between PZn and risk of OCs with and without other malformations. METHODS: Case mothers were ascertained by the Utah Birth Defects Network and control mothers were selected from Utah birth certificates by matching for child gender and delivery month and year. Maternal blood was collected >1 year after the last pregnancy. PZn was available for 410 case mothers who were divided into four subgroups: isolated cleft lip with or without cleft palate (CL/P‐I, n = 231), isolated cleft palate (CP‐I, n = 74), CL/P with other malformations (CLP‐M, n = 42), and CP with other malformations (CP‐M, n = 63). PZn was available for 447 control mothers. The mean age of children at blood sampling was 3.7 years for all cases combined and 4.3 years for controls. RESULTS: Mean PZns of all groups were similar, and low PZn (<11.0 μmol/L) was found in 59% of cases and 62% of controls. Risk of OCs did not vary significantly across PZn quartiles for the four subgroups individually and all OC groups combined. CONCLUSIONS: We previously reported that poor maternal zinc status was a risk factor for OCs in the Philippines, where OC prevalence is high and maternal PZn is low. In Utah, however, no such association was found, suggesting that poor maternal zinc status may become a risk factor only when zinc status is highly compromised. Birth Defects Research (Part A), 2009. © 2008 Wiley‐Liss, Inc.     

Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells

Background

Human multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity.

Results

After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G₁ phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation.

Conclusion

Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.     

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells.Biological membranes are conceptually simple structures that may be generated in vitro according to simple physicochemical principles. In vivo, however, membranes are highly complex and host a plethora of proteins that mediate the transfer of molecules and communication across the membrane. Proteins may be trapped in membrane by their transmembrane domains, anchored by lipid tails, or attach to membrane-integral proteins. A further level of complexity is seen when membrane proteins are not equally distributed but occupy only a limited fraction of the available surface (i.e. when they are polarly localized or when they form small membrane subdomains in the micrometer range). The question of how membrane proteins are retained locally and prevented from diffusing freely is of high importance to cell biology. Polarly localized proteins may be retained in their respective domains by membrane fences; in such a situation, polarly localized proteins are mobile in their domains but cannot diffuse through tightly packed scaffold proteins forming a molecular fence within the membrane. Membrane fences delimiting polar domains have been described in different organisms. For example, diffusion between membrane compartments is prevented in budding yeast (Saccharomyces cerevisiae) at the level of the bud neck (Barral et al., 2000; Takizawa et al., 2000); in ciliated vertebrate cells, between ciliary and periciliary membranes (Hu et al., 2010); in epithelial cells, between apical and basolateral membranes (van Meer and Simons, 1986); in neurons, between axon and soma (Kobayashi et al., 1992; Winckler et al., 1999; Nakada et al., 2003); and in spermatozoa, at the level of the annulus (Myles et al., 1984; Nehme et al., 1993). The existence of membrane scaffolds that prevent free protein diffusion has also been described in bacteria (Baldi and Barral, 2012; Schlimpert et al., 2012). In plants, we have shown the existence of a strict membrane fence in the root endodermis, where a median domain splits the cell in two lateral halves occupied by different sets of proteins (Alassimone et al., 2010). The situation in the plant endodermis is analogous to the separation of animal epithelia into apical and basolateral domains; indeed, a parallel between epithelia and endodermal cells has been drawn, despite the different origin of multicellularity in plants and animals (Grebe, 2011).The protein complexes responsible for the formation of membrane fences have been identified. Septins are a family of proteins able to oligomerize and form filaments (Saarikangas and Barral, 2011); their role in the formation of membrane fences has been demonstrated in several organisms and cellular situations, including the yeast bud neck (Barral et al., 2000; Takizawa et al., 2000), animal cilia (Hu et al., 2010), and mammalian spermatozoa (Ihara et al., 2005; Kissel et al., 2005; Kwitny et al., 2010). At the axonal initial segment of neurons, AnkyrinG is necessary to establish and maintain a membrane scaffold where different membrane proteins are immobilized and stabilized (Hedstrom et al., 2008; Sobotzik et al., 2009). In Caulobacter crescentus, the stalk protein Stp forms a complex that prevents diffusion between the cell body and stalk and between stalk compartments. Claudins and occludin are the main components of epithelial tight junctions (Furuse et al., 1993, 1998). Occludins are four-membrane-span proteins and belong to the MARVEL protein family (Sánchez-Pulido et al., 2002), as do Tricellulin and MARVELD3, which are also tight junction-associated proteins (Furuse et al., 1993; Ikenouchi et al., 2005; Steed et al., 2009).In Arabidopsis (Arabidopsis thaliana), our group identified a family of proteins that form a membrane fence in the endodermis (Roppolo et al., 2011). These CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASP1 to CASP5) are four-transmembrane proteins that form a median domain referred to as the Casparian strip membrane domain (CSD). CASPs are initially targeted to the whole plasma membrane, then they are quickly removed from lateral plasma membranes and remain localized exclusively at the CSD; there, they show an extremely low turnover, although they are eventually removed (Roppolo et al., 2011). The membrane proteins NOD26-LIKE INTRINSIC PROTEIN5;1 and BORON TRANSPORTER1 are restricted from diffusing through the CSD and remain polarly localized in the outer and inner lateral membranes, respectively; a fluorescent lipophilic molecule, when integrated in the outer endodermal membrane, was blocked at the level of the CSD and could not diffuse into the inner membrane (Roppolo et al., 2011). Besides making a plasma membrane diffusion barrier, CASPs have an important role in directing the modification of the cell wall juxtaposing their membrane domain: by interacting with secreted peroxidases, they mediate the deposition of lignin and the building up of the Casparian strips (Roppolo et al., 2011; Naseer et al., 2012; Lee et al., 2013). The two CASP activities, making membrane scaffolds and directing a modification of the cell wall, can be uncoupled: indeed, (1) formation of the CASP domain is independent from the deposition of lignin, and (2) interaction between CASPs and peroxidases can take place outside the CSD when CASPs are ectopically expressed (Lee et al., 2013).As CASPs are currently the only known proteins forming membrane fences in plants and because of their essential role in directing a local cell wall modification, we were interested in characterizing the repertoire of a large number of CASP-like (CASPL) proteins in the plant kingdom. Our aim was to provide the molecular basis for the discovery of additional membrane domains in plants and for the identification of proteins involved in local cell wall modifications. We extended our phylogenetic analysis outside of the plant kingdom and found conservation between CASPLs and the MARVEL protein family. Conserved residues are located in transmembrane domains, and we provide evidence suggesting that these domains are involved in CASP localization. We explored the potential use of the CASPL module in plants by investigating CASPL expression patterns and their ability to form membrane domains in the endodermis. Moreover, we related the appearance of the Casparian strips in the plant kingdom to the emergence of a CASP-specific signature that was not found in the genomes of plants lacking Casparian strips.     

Genetic Evidence That Chain Length and Branch Point Distributions Are Linked Determinants of Starch Granule Formation in Arabidopsis

The major component of starch is the branched glucan amylopectin. Structural features of amylopectin, such as the branching pattern and the chain length distribution, are thought to be key factors that enable it to form semicrystalline starch granules. We varied both structural parameters by creating Arabidopsis (Arabidopsis thaliana) mutants lacking combinations of starch synthases (SSs) SS1, SS2, and SS3 (to vary chain lengths) and the debranching enzyme ISOAMYLASE1-ISOAMYLASE2 (ISA; to alter branching pattern). The isa mutant accumulates primarily phytoglycogen in leaf mesophyll cells, with only small amounts of starch in other cell types (epidermis and bundle sheath cells). This balance can be significantly shifted by mutating different SSs. Mutation of SS1 promoted starch synthesis, restoring granules in mesophyll cell plastids. Mutation of SS2 decreased starch synthesis, abolishing granules in epidermal and bundle sheath cells. Thus, the types of SSs present affect the crystallinity and thus the solubility of the glucans made, compensating for or compounding the effects of an aberrant branching pattern. Interestingly, ss2 mutant plants contained small amounts of phytoglycogen in addition to aberrant starch. Likewise, ss2ss3 plants contained phytoglycogen, but were almost devoid of glucan despite retaining other SS isoforms. Surprisingly, glucan production was restored in the ss2ss3isa triple mutants, indicating that SS activity in ss2ss3 per se is not limiting but that the isoamylase suppresses glucan accumulation. We conclude that loss of only SSs can cause phytoglycogen production. This is readily degraded by isoamylase and other enzymes so it does not accumulate and was previously unnoticed.Starch, the major storage carbohydrate in plants, is composed of two α-1,4- and α-1,6-linked glucan polymers: moderately branched amylopectin and predominantly linear amylose. Amylopectin, which constitutes approximately 80% of most starches, is synthesized by three enzyme activities. Starch synthases (SSs) transfer the glucosyl moiety of ADP-Glc to a glucan chain, forming a new α-1,4 glucosidic linkage, extending the linear chains. Branching enzymes (BEs) cleave some α-1,4 linkages and reattach chains of six Glc units or more via α-1,6 linkages, creating branch points. Debranching enzymes (DBEs) hydrolyze some of these branches, tailoring the structure of the polymer. However, the way in which the individual enzymes work together to create crystallization-competent amylopectin remains unclear.The coordinated actions of SSs, BEs, and DBEs are thought to produce a glucan with a tree-like architecture in which the branch points are nonrandomly positioned. According to models of amylopectin, clusters of unbranched chain segments are formed. Within these clusters, adjacent chains form double helices, which align in parallel giving rise to crystalline lamellae. These alternate with amorphous lamellae containing the branch points and chain segments that span the clusters (Zeeman et al., 2010). In the context of this amylopectin model, glucan chains can be categorized according to their length and connection to other chains. The A chains are external chains that do not carry other branches. The B chains carry one or more branches (either an A chain or another B chain) and have both external and internal segments. The B chains can span one or more clusters (e.g. a B₁ chain spans one cluster). The C chain is the single chain that has a reducing end (Manners, 1989). The A chains tend to be the shortest, having an average chain length (ACL) of 12 to 16, depending on the species (Hizukuri, 1986). Together with the B₁ chains, the A chains are thought to make up the crystalline clusters. Longer chains such as B₂ chains (ACL 20–24) or B₃ chains (ACL 42–48) are presumed to connect clusters (Hizukuri, 1986). Amylose is a distinct polymer synthesized within the amylopectin matrix by granule-bound SS (Tatge et al., 1999). Mutants lacking granule-bound SS also lack amylose but still make starch granules, showing that amylose synthesis is not required for this (Zeeman et al., 2010).The structural properties of amylopectin contrast with those of glycogen, the Glc polymer synthesized in organisms such as fungi, animals, and most bacteria. Glycogen also consists of α-1,4-linked Glc chains with α-1,6-linked branches, but differs in three major ways from amylopectin. First, its external branches are considerably shorter (6–8 Glc units compared with 12–16 in amylopectin). Second, the branch frequency (10%) is twice as high as in amylopectin. Third, its branch points are assumed to be distributed homogeneously, whereas branching in amylopectin is thought to be nonhomogeneous. These differences prevent the formation and parallel alignment of double helices in glycogen, rendering it soluble. Glycogen synthesis requires only a single glycogen synthase enzyme and a single glycogen BE, whereas several SS and BE isoforms are involved in amylopectin synthesis. In Arabidopsis (Arabidopsis thaliana), there are four SSs (SS1–SS4) and two BEs (BE2 and BE3; Li et al., 2003; Streb and Zeeman, 2012). In addition, Arabidopsis has three DBEs. ISOAMYLASE1-ISOAMYLASE2 (hereafter referred to simply as ISA), a heteromultimeric enzyme composed of the two subunits ISA1 and ISA2, is implicated in amylopectin synthesis (Delatte et al., 2005). The other two DBEs, ISA3 and LIMIT DEXTRINASE (LDA), are implicated in starch degradation (Delatte et al., 2006).Loss of specific SS isoforms has different effects on the starch amount, amylopectin chain length distribution (CLD), and starch granule morphology, suggesting distinct functions for each isoform. For example, amylopectin from SS1-deficient mutants of Arabidopsis (Delvallé et al., 2005; Szydlowski et al., 2011) and rice (Oryza sativa; Fujita et al., 2006) has fewer chains with a degree of polymerization (DP; i.e. chain length) between 8 and 12 and more chains with a DP between 17 and 20 compared with the wild-type starches. This is consistent with in vitro data for the maize (Zea mays; Commuri and Keeling, 2001) and rice SSI enzymes (Fujita et al., 2006), which preferentially elongate short chains of DP 6 or 7 up to a length of DP 10. This indicates that SSI functions to elongate the short chains created by BEs by a few Glc units (Commuri and Keeling, 2001; Delvallé et al., 2005). Comparable studies in SS2-deficient mutants reveal amylopectin with more chains with DP 6 to 11, but depletion in chains with DP 13 to 20 compared with the corresponding wild-type amylopectins. Thus, SS2 is suggested to elongate shorter chains (e.g. those made by SS1) to a length of between DP 13 and 20 (Edwards et al., 1999; Yamamori et al., 2000; Umemoto et al., 2002; Morell et al., 2003; Zhang et al., 2004, 2008). SS3 was proposed to be important for the generation of long, cluster-spanning chains (Jeon et al., 2010; Tetlow and Emes, 2011), as well as contributing to A chain and B₁ chain elongation (Edwards et al., 1999; Zhang et al., 2005, 2008). By contrast, SS4 appears to have a specialized role in initiating or coordinating granule formation (Roldán et al., 2007; Crumpton-Taylor et al., 2012, 2013). Arabidopsis ss4 mutants have just one round starch granule per chloroplast rather than five or more lenticular granules observed in the wild type.Partial loss of BE activity in maize (Stinard et al., 1993), rice (Mizuno et al., 1993), and potato (Solanum tuberosum; Schwall et al., 2000) leads to starches with high apparent amylose, most likely caused by the accumulation of less frequently branched amylopectin. A total lack of branching activity in Arabidopsis be2be3 mutants, however, abolishes starch production. Instead, maltose accumulates, suggesting that linear glucans are produced, but degraded by α- and β-amylases (Dumez et al., 2006).Loss of DBE of the ISA1 class causes a dramatic phenotype, with production of a soluble glucan (phytoglycogen) in place of starch. This has been observed in starch-synthesizing tissues of several species, including Chlamydomonas reinhardtii cells (Mouille et al., 1996), Arabidopsis leaves (Delatte et al., 2005; Wattebled et al., 2005), and the endosperms of maize (Zea Mays; James et al., 1995), rice (Oryza sativa; Nakamura et al., 1997), and barley (Hordeum vulgare) seeds (Burton et al., 2002). Phytoglycogen has structural similarities to glycogen in that both are water soluble and have a higher branch frequency than amylopectin. Accordingly, it was proposed that the trimming of glucans produced by SS and BE isoforms by ISA1 removes branches that interfere with the formation of secondary and tertiary structures (i.e. organized arrays of double helices), thereby facilitating amylopectin biosynthesis and crystallization (Ball et al., 1996). Compared with ISA1, the other two DBEs (LDA and ISA3) have different substrate specificities, both preferring substrates with short outer chains, such as β-limit dextrins, suggesting that their role is primarily in starch degradation. Consistently, mutating these genes in Arabidopsis causes a starch-excess phenotype rather than phytoglycogen accumulation (Delatte et al., 2006).Although it is now widely accepted that a degree of debranching occurs to control branch number and positioning in amylopectin, the importance of this for crystalline starch production is still uncertain. Several studies have shown that some cell types in isa1-deficient mutants still produce some starch (e.g. epidermal and bundle sheath cells in Arabidopsis mutants; Delatte et al., 2005), indicating that other factors can also affect the partitioning between phytoglycogen and starch.No starch granules are made in the Arabidopsis isa1isa2isa3lda quadruple mutant, which lacks all three DBEs (Streb et al., 2008). Although suggestive of redundancy between the DBEs, the loss of each enzyme has distinct effects on amylopectin or phytoglycogen structure, consistent with their different substrate specificities. Furthermore, the loss of starch granules in isa1isa2isa3lda was shown to be at least partly due to the actions of α-amylase; typical α-amylolytic products (short malto-oligosaccharides) accumulated alongside phytoglycogen. Mutation of the gene encoding the chloroplastic α-AMYLASE3 (AMY3) eliminated these short malto-oligosaccharides and restored starch granule biosynthesis in all cell types examined. This unexpected result showed that crystalline glucans can be produced in the absence of DBE activity, despite an altered branching pattern. Streb et al. (2008) proposed that AMY3 shortens external chains of the glucans made by SSs and BEs so that they cannot form double helices with their neighbors. This idea is consistent with models for amylopectin, in which a suitable CLD is a critical factor in the formation of the secondary and higher-order crystalline structures (Gidley and Bulpin, 1987; Pfannemüller, 1987). Thus, factors that affect the CLD, such as a failure to sufficiently elongate new branches or concomitant chain degradation by amylases, should also affect crystallinity. Indeed, early studies of maize mutants (that were subsequently shown to be affected in DBE and SS activities) reported that loss of SS in a DBE mutant background altered the ratio of starch to phytoglycogen compared with the DBE mutants alone (Cameron and Cole, 1954; Creech, 1965).The aim of this work was to use genetics to systematically vary both branch point position and chain lengths and determine the impact on glucan amount, structure, and starch granule formation in Arabidopsis. We analyzed mutants lacking combinations of SSs (to vary chain lengths) in the absence of the debranching enzyme ISA1-ISA2 (to change branch point distribution/frequency). This revealed that the length of external chains is a key factor in the production of a crystallization-competent glucan. Remarkably, our results also provide evidence for phytoglycogen production due to mutations just in SSs. Our results indicate that this phenomenon is largely masked by the presence of ISA1-ISA2, which degrades the aberrant glucan instead of trimming it to amylopectin.     

Conditional ablation of integrin alpha-6 in mouse epidermis leads to skin fragility and inflammation

Hemidesmosomes (HDs) are essential anchorage junctions which mediate the firm attachment of epithelia to the underlying basement membranes, of which one main component is the integrin α6β4. These specific junctions are also able to trigger signalling pathways, via the recruitment and interactions of signalling molecules with HD components such as the cytoplasmic tail of the β4 integrin or the plakin plectin. HDs must also assemble and disassemble depending on the tissue context for example during tissue remodelling. Alterations of HD components or their loss result in skin blistering disorders known as epidermolysis bullosa. Since mice lacking integrin α6 die at birth with severe skin blistering, we have produced a mouse line in which epidermal deletion of integrin α6 can be controlled by tamoxifen injection. We observed that the deletion was mosaic, but that hairless skin such as ears, tails and paws were affected and showed chronic inflammation associated with hyperproliferation, and expression of laminin-111. Interestingly, two cytokines, amphiregulin and epiregulin, previously found increased in integrin α6 deficient cultured keratinocytes, were also increased here in the affected skin. In detached areas, we validate clearly that the absence of integrin α6 leads to a delocalisation of plectin, and the complete disappearance of HD structures.