Adenylate cyclase type 9 antagonizes cAMP accumulation and regulates endothelial signalling involved in atheroprotection.

AIMS: The adenylate cyclase type 9 (ADCY9) gene appears to determine atherosclerotic outcomes in patients treated with dalcetrapib. In mice, we recently demonstrated that Adcy9 inactivation potentiates endothelial function and inhibits atherogenesis. The objective of this study was to characterize the contribution of ADCY9 to the regulation of endothelial signalling pathways involved in atherosclerosis. METHODS AND RESULTS: We show that ADCY9 is expressed in the endothelium of mouse aorta and femoral arteries. We demonstrate that ADCY9 inactivation in cultured endothelial cells paradoxically increases cAMP accumulation in response to the adenylate cyclase activators forskolin and vasoactive intestinal peptide (VIP). Reciprocally, ADCY9 overexpression decreases cAMP production. Using mouse femoral artery arteriography, we show that Adcy9 inactivation potentiates VIP-induced endothelial-dependent vasodilation. Moreover, Adcy9 inactivation reduces mouse atheroma endothelial permeability in different vascular beds. ADCY9 overexpression reduces forskolin-induced phosphorylation of Ser157-vasodilator-stimulated phosphoprotein (VASP) and worsens thrombin-induced fall of RAP1 activity, both leading to increased endothelial permeability. ADCY9 inactivation in thrombin-stimulated human coronary artery endothelial cells results in cAMP accumulation, increases p-Ser157-VASP, and inhibits endothelial permeability. MLC2 phosphorylation and actin stress fibre increases in response to thrombin were reduced by ADCY9 inactivation, suggesting actin cytoskeleton regulation. Finally, using the Miles assay, we demonstrate that Adcy9 regulates thrombin-induced endothelial permeability in vivo in normal and atherosclerotic animals. CONCLUSION: Adcy9 is expressed in endothelial cells and regulates local cAMP and endothelial functions including permeability relevant to atherogenesis.

Bulb growth potential is independent of leaf longevity for the spring ephemeral Erythronium americanum Ker-Gawl.

Growth in most spring ephemerals is decreased under warmer temperatures. Although photosynthetic activities are improved at warmer temperatures, leaves senesce earlier, which prevents the bulb from reaching a larger size. A longer leaf life duration during a warm spring, therefore, may improve bulb mass. We tested this hypothesis by modulating leaf life span of Erythronium americanum through the application of Promalin® (PRO; cytokinins and gibberellins) that prolonged or silver thiosulfate (STS) that reduced leaf duration. Gas exchange and chlorophyll fluorescence were measured along with leaf and bulb carbohydrate concentrations. Plants were also pulse labelled with 13CO2 to monitor sugar transport to the bulb. Lower photosynthetic rates and shorter leaf life span of STS plants reduced the amount of carbon that they assimilated during the season, resulting in a smaller bulb compared with control plants. PRO plants maintained their photosynthetic rates for a longer period than control plants, yet final bulb biomass did not differ between them. We conclude that seasonal growth for E. americanum is not limited by leaf life duration under warm growing conditions, but rather by limited sink growth capacity. Under global warming, spring geophytes might be at risk of being reduced in size and, eventually, reproducing less frequently.

ADCY9 (Adenylate Cyclase Type 9) Inactivation Protects From Atherosclerosis Only in the Absence of CETP (Cholesteryl Ester Transfer Protein).

BACKGROUND: Pharmacogenomic studies have shown that ADCY9 genotype determines the effects of the CETP (cholesteryl ester transfer protein) inhibitor dalcetrapib on cardiovascular events and atherosclerosis imaging. The underlying mechanisms responsible for the interactions between ADCY9 and CETP activity have not yet been determined. METHODS: Adcy9-inactivated ( Adcy9Gt/Gt) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETPtg Adcy9Gt/Gt and CETPtg Adcy9WT), were submitted to an atherogenic protocol (injection of an AAV8 [adeno-associated virus serotype 8] expressing a PCSK9 [proprotein convertase subtilisin/kexin type 9] gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, vasorelaxation, telemetry, and adipose tissue magnetic resonance imaging were evaluated. RESULTS: Adcy9Gt/Gt mice had a 65% reduction in aortic atherosclerosis compared to WT ( P<0.01). CD68 (cluster of differentiation 68)-positive macrophage accumulation and proliferation in plaques were reduced in Adcy9Gt/Gt mice compared to WT animals ( P<0.05 for both). Femoral artery endothelial-dependent vasorelaxation was improved in Adcy9Gt/Gt mice (versus WT, P<0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase, and endothelial-dependent hyperpolarization pathways were all responsible for the improvement of vasodilatation in Adcy9Gt/Gt ( P<0.01 for all). Aortic endothelium from Adcy9Gt/Gt mice allowed significantly less adhesion of splenocytes compared to WT ( P<0.05). Adcy9Gt/Gt mice gained more weight than WT with the atherogenic diet; this was associated with an increase in whole body adipose tissue volume ( P<0.01 for both). Feed efficiency was increased in Adcy9Gt/Gt compared to WT mice ( P<0.01), which was accompanied by prolonged cardiac RR interval ( P<0.05) and improved nocturnal heart rate variability ( P=0.0572). Adcy9 inactivation-induced effects on atherosclerosis, endothelial function, weight gain, adipose tissue volume, and feed efficiency were lost in CETPtg Adcy9Gt/Gt mice ( P>0.05 versus CETPtg Adcy9WT). CONCLUSIONS: Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, and improved endothelial function and autonomic tone.

Light acclimation strategies change from summer green to spring ephemeral as wild-leek plants age.

PREMISE OF THE STUDY: Spring-ephemeral forest-herbs emerge early to take advantage of the high-light conditions preceding canopy closure; they complete their life cycle in a few weeks, then senesce as the tree canopy closes. Summer greens acclimate their leaves to shade and thus manage to maintain a net carbon gain throughout summer. Differences in phenology among life stages within a species have been reported in tree saplings, whose leaf activity may extend beyond the period of shade conditions caused by mature trees. Similar phenological acclimation has seldom been studied in forest herbs. METHODS: We compared wild-leek bulb growth and leaf phenology among plants from seedling to maturity and from under 4 to 60% natural light availability. We also compared leaf chlorophyll content and chl a/b ratio among seedlings and adult plants in a natural population as an indicator of photosynthetic capacity and acclimation to light environment. KEY RESULTS: Overall, younger plants senesced later than mature ones. Increasing light availability delayed senescence in mature plants, while hastening seedling senescence. In natural populations, only seedlings acclimated to the natural reduction in light availability through time. CONCLUSIONS: Wild-leek seedlings exhibit a summer-green phenology, whereas mature plants behave as true spring ephemerals. Growth appears to be more source-limited in seedlings than in mature plants. This modulation of phenological strategy, if confirmed in other species, would require a review of the current classification of species as either spring ephemerals, summer greens, wintergreens, or evergreens.

Invader disruption of belowground plant mutualisms reduces carbon acquisition and alters allocation patterns in a native forest herb.

Invasive plants impose novel selection pressures on naïve mutualistic interactions between native plants and their partners. As most plants critically rely on root fungal symbionts (RFSs) for soil resources, invaders that disrupt plant-RFS mutualisms can significantly depress native plant fitness. Here, we investigate the consequences of RFS mutualism disruption on native plant fitness in a glasshouse experiment with a forest invader that produces known anti-fungal allelochemicals. Over 5 months, we regularly applied either green leaves of the allelopathic invader Alliaria petiolata, a nonsystemic fungicide to simulate A. petiolata's effects, or green leaves of nonallelopathic Hesperis matronalis (control) to pots containing the native Maianthemum racemosum and its RFSs. We repeatedly measured M. racemosum physiology and harvested plants periodically to assess carbon allocation. Alliaria petiolata and fungicide treatment effects were indistinguishable: we observed inhibition of the RFS soil hyphal network and significant reductions in M. racemosum physiology (photosynthesis, transpiration and conductance) and allocation (carbon storage, root biomass and asexual reproduction) in both treatments relative to the control. Our findings suggest a general mechanistic hypothesis for local extinction of native species in ecosystems challenged by allelopathic invaders: RFS mutualism disruption drives carbon stress, subsequent declines in native plant vigor, and, if chronic, declines in RFS-dependent species abundance.

Responses of two understory herbs, Maianthemum canadense and Eurybia macrophylla, to experimental forest warming: early emergence is the key to enhanced reproductive output.

PREMISE OF THE STUDY: Understory herbs might be the most sensitive plant form to global warming in deciduous forests, yet they have been little studied in the context of climate change. METHODS: A field experiment set up in Minnesota, United States simulated global warming in a forest setting and provided the opportunity to study the responses of Maianthemum canadense and Eurybia macrophylla in their natural environment in interaction with other components of the ecosystem. Effects of +1.7° and +3.4°C treatments on growth, reproduction, phenology, and gas exchange were evaluated along with treatment effects on light, water, and nutrient availability, potential drivers of herb responses. KEY RESULTS: Overall, growth and gas exchanges of these two species were modestly affected by warming. They emerged up to 16 (E. macrophylla) to 17 d (M. canadense) earlier in the heated plots than in control plots, supporting early-season carbon gain under high light conditions before canopy closure. This additional carbon gain in spring likely supported reproduction. Eurybia macrophylla only flowered in the heated plots, and both species had some aspect of reproduction that was highest in the +1.7°C treatment. The reduced reproductive effort in the +3.4°C plots was likely due to reduced soil water availability, counteracting positive effects of warming. CONCLUSIONS: Global warming might improve fitness of herbaceous species in deciduous forests, mainly by advancing their spring emergence. However, other impacts of global warming such as drier soils in the summer might partly reduce the carbon gain associated with early emergence.

Phosphorylation of DEP-1/PTPRJ on threonine 1318 regulates Src activation and endothelial cell permeability induced by vascular endothelial growth factor.

The protein tyrosine phosphatase DEP-1/PTPRJ positively regulates Src family kinases and critical biological functions in endothelial and hematopoietic cells. Phosphorylation of DEP-1 on Y1311/Y1320 mediates the association and activation of Src, and promotes Src-dependent angiogenic responses including endothelial cell permeability. We have identified T1318 as a phosphorylated residue proximal to Y1320. The aim of this study was to determine if T1318 phosphorylation exerts a regulatory role over the function of DEP-1. We show that phosphorylation of DEP-1 on Y1320 was reduced when T1318 was mutated. This led to the decreased association of DEP-1 T1318A with Src, and defective Src activation in both HEK 293T and VEGF-stimulated endothelial cells. Consistent with these findings, VEGF-induced tyrosine phosphorylation of VE-cadherin, its association to ß-arrestin1/2, and cell permeability were impaired in cells expressing DEP-1 T1318A. Conversely, expression of the phosphomimetic mutant DEP-1 T1318E constitutively enhanced the phosphorylation of Y1320 and VE-cadherin over that induced by WT DEP-1, and resulted in increased VEGF-dependent permeability. DEP-1 T1318 is part of a CK2 consensus phosphorylation site and was identified as a CK2 substrate. Modulation of CK2 expression or activity in endothelial cells regulated T1318 phosphorylation, and correlated with the status of Y1320 phosphorylation, Src activation, and cell permeability. CK2-dependent phosphorylation of DEP-1 T1318 promotes Y1320 phosphorylation and Src activation upon VEGF stimulation. Phosphorylation of T1318 is thus part of a regulatory mechanism that channels the activity of DEP-1 towards Src to allow its optimal activation and the promotion of endothelial cell permeability.

SYN2 is an autism predisposing gene: loss-of-function mutations alter synaptic vesicle cycling and axon outgrowth.

An increasing number of genes predisposing to autism spectrum disorders (ASDs) has been identified, many of which are implicated in synaptic function. This 'synaptic autism pathway' notably includes disruption of SYN1 that is associated with epilepsy, autism and abnormal behavior in both human and mice models. Synapsins constitute a multigene family of neuron-specific phosphoproteins (SYN1-3) present in the majority of synapses where they are implicated in the regulation of neurotransmitter release and synaptogenesis. Synapsins I and II, the major Syn isoforms in the adult brain, display partially overlapping functions and defects in both isoforms are associated with epilepsy and autistic-like behavior in mice. In this study, we show that nonsense (A94fs199X) and missense (Y236S and G464R) mutations in SYN2 are associated with ASD in humans. The phenotype is apparent in males. Female carriers of SYN2 mutations are unaffected, suggesting that SYN2 is another example of autosomal sex-limited expression in ASD. When expressed in SYN2  knockout neurons, wild-type human Syn II fully rescues the SYN2 knockout phenotype, whereas the nonsense mutant is not expressed and the missense mutants are virtually unable to modify the SYN2 knockout phenotype. These results identify for the first time SYN2  as a novel predisposing gene for ASD and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies ASD.

The plant ionome revisited by the nutrient balance concept.

Tissue analysis is commonly used in ecology and agronomy to portray plant nutrient signatures. Nutrient concentration data, or ionomes, belongs to the compositional data class, i.e., multivariate data that are proportions of some whole, hence carrying important numerical properties. Statistics computed across raw or ordinary log-transformed nutrient data are intrinsically biased, hence possibly leading to wrong inferences. Our objective was to present a sound and robust approach based on a novel nutrient balance concept to classify plant ionomes. We analyzed leaf N, P, K, Ca, and Mg of two wild and six domesticated fruit species from Canada, Brazil, and New Zealand sampled during reproductive stages. Nutrient concentrations were (1) analyzed without transformation, (2) ordinary log-transformed as commonly but incorrectly applied in practice, (3) additive log-ratio (alr) transformed as surrogate to stoichiometric rules, and (4) converted to isometric log-ratios (ilr) arranged as sound nutrient balance variables. Raw concentration and ordinary log transformation both led to biased multivariate analysis due to redundancy between interacting nutrients. The alr- and ilr-transformed data provided unbiased discriminant analyses of plant ionomes, where wild and domesticated species formed distinct groups and the ionomes of species and cultivars were differentiated without numerical bias. The ilr nutrient balance concept is preferable to alr, because the ilr technique projects the most important interactions between nutrients into a convenient Euclidean space. This novel numerical approach allows rectifying historical biases and supervising phenotypic plasticity in plant nutrition studies.

Tyrosine phosphorylation of DEP-1/CD148 as a mechanism controlling Src kinase activation, endothelial cell permeability, invasion, and capillary formation.

DEP-1/CD148 is a receptor-like protein tyrosine phosphatase with antiproliferative and tumor-suppressive functions. Interestingly, it also positively regulates Src family kinases in hematopoietic and endothelial cells, where we showed it promotes VE-cadherin-associated Src activation and endothelial cell survival upon VEGF stimulation. However, the molecular mechanism involved and its biologic functions in endothelial cells remain ill-defined. We demonstrate here that DEP-1 is phosphorylated in a Src- and Fyn-dependent manner on Y1311 and Y1320, which bind the Src SH2 domain. This allows DEP-1-catalyzed dephosphorylation of Src inhibitory Y529 and favors the VEGF-induced phosphorylation of Src substrates VE-cadherin and Cortactin. Accordingly, RNA interference (RNAi)-mediated knockdown of DEP-1 or expression of DEP-1 Y1311F/Y1320F impairs Src-dependent biologic responses mediated by VEGF including permeability, invasion, and branching capillary formation. In addition, our work further reveals that above a threshold expression level, DEP-1 can also dephosphorylate Src Y418 and attenuate downstream signaling and biologic responses, consistent with the quiescent behavior of confluent endothelial cells that express the highest levels of endogenous DEP-1. Collectively, our findings identify the VEGF-dependent phosphorylation of DEP-1 as a novel mechanism controlling Src activation, and show this is essential for the proper regulation of permeability and the promotion of the angiogenic response.

Novel α1 and γ2 GABAA receptor subunit mutations in families with idiopathic generalized epilepsy.

Epilepsy is a heterogeneous neurological disease affecting approximately 50 million people worldwide. Genetic factors play an important role in both the onset and severity of the condition, with mutations in several ion-channel genes being implicated, including those encoding the GABA(A) receptor. Here, we evaluated the frequency of additional mutations in the GABA(A) receptor by direct sequencing of the complete open reading frame of the GABRA1 and GABRG2 genes from a cohort of French Canadian families with idiopathic generalized epilepsy (IGE). Using this approach, we have identified three novel mutations that were absent in over 400 control chromosomes. In GABRA1, two mutations were found, with the first being a 25-bp insertion that was associated with intron retention (i.e. K353delins18X) and the second corresponding to a single point mutation that replaced the aspartate 219 residue with an asparagine (i.e. D219N). Electrophysiological analysis revealed that K353delins18X and D219N altered GABA(A) receptor function by reducing the total surface expression of mature protein and/or by curtailing neurotransmitter effectiveness. Both defects would be expected to have a detrimental effect on inhibitory control of neuronal circuits. In contrast, the single point mutation identified in the GABRG2 gene, namely P83S, was indistinguishable from the wildtype subunit in terms of surface expression and functionality. This finding was all the more intriguing as the mutation exhibited a high degree of penetrance in three generations of one French Canadian family. Further experimentation will be required to understand how this mutation contributes to the occurrence of IGE in these individuals.

SYN1 loss-of-function mutations in autism and partial epilepsy cause impaired synaptic function.

Several genes predisposing to autism spectrum disorders (ASDs) with or without epilepsy have been identified, many of which are implicated in synaptic function. Here we report a Q555X mutation in synapsin 1 (SYN1), an X-linked gene encoding for a neuron-specific phosphoprotein implicated in the regulation of neurotransmitter release and synaptogenesis. This nonsense mutation was found in all affected individuals from a large French-Canadian family segregating epilepsy and ASDs. Additional mutations in SYN1 (A51G, A550T and T567A) were found in 1.0 and 3.5% of French-Canadian individuals with autism and epilepsy, respectively. The majority of these SYN1 mutations were clustered in the proline-rich D-domain which is substrate of multiple protein kinases. When expressed in synapsin I (SynI) knockout (KO) neurons, all the D-domain mutants failed in rescuing the impairment in the size and trafficking of synaptic vesicle pools, whereas the wild-type human SynI fully reverted the KO phenotype. Moreover, the nonsense Q555X mutation had a dramatic impact on phosphorylation by MAPK/Erk and neurite outgrowth, whereas the missense A550T and T567A mutants displayed impaired targeting to nerve terminals. These results demonstrate that SYN1 is a novel predisposing gene to ASDs, in addition to epilepsy, and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies the pathogenesis of both diseases.

Source-sink imbalance increases with growth temperature in the spring geophyte Erythronium americanum.

Spring geophytes produce larger storage organs and present delayed leaf senescence under lower growth temperature. Bulb and leaf carbon metabolism were investigated in Erythronium americanum to identify some of the mechanisms that permit this improved growth at low temperature. Plants were grown under three day/night temperature regimes: 18/14 °C, 12/8 °C, and 8/6 °C. Starch accumulated more slowly in the bulb at lower temperatures probably due to the combination of lower net photosynthetic rate and activation of a 'futile cycle' of sucrose synthesis and degradation. Furthermore, bulb cell maturation was delayed at lower temperatures, potentially due to the delayed activation of sucrose synthase leading to a greater sink capacity. Faster starch accumulation and the smaller sink capacity that developed at higher temperatures led to early starch saturation of the bulb. Thereafter, soluble sugars started to accumulate in both leaf and bulb, most probably inducing decreases in fructose-1,6-bisphosphatase activity, triose-phosphate utilization in the leaf, and the induction of leaf senescence. Longer leaf life span and larger bulbs at lower temperature appear to be due to an improved equilibrium between carbon fixation capacity and sink strength, thereby allowing the plant to sustain growth for a longer period of time before feedback inhibition induces leaf senescence.

Impact of growth form and carbohydrate reserves on tolerance to simulated deer herbivory and subsequent recovery in Liliaceae.

PREMISE OF THE STUDY: Over-browsing of the understory vegetation by white-tailed deer has been a cause of decline in many plant populations. Liliaceae are particularly sensitive, yet individual species differ in their tolerance to deer herbivory. In this paper, we examine whether differences in clonal habit, carbon allocation patterns, and phenology influence the capacity of a species to tolerate and recover from repeated herbivory. • METHODS: Flowering ramets of Clintonia borealis, Maianthemum canadense, and Trillium erectum were subjected to total defoliation for one or two springs. • KEY RESULTS: Survival was highest in the nonclonal species, T. erectum, most probably due to its very large carbohydrate reserves. Nutrient reserves were less affected than carbohydrate reserves by defoliation, confirming the importance of carbohydrate reserves for survival. However, faster recovery following episodes of defoliation was observed not in the species that sprouted the earliest, T. erectum, but in the clonal species, M. canadense, which had the smallest carbohydrate reserves but also a lower shoot to root ratio than the other clonal species, C. borealis. All plants that were defoliated for 2 years only partially recovered in terms of leaf area, plant biomass, and carbohydrate and nutrient reserves, confirming the overall sensitivity of these species to simulated deer herbivory. • CONCLUSIONS: High carbohydrate reserves and consequently low shoot to root ratios appear to increase tolerance to herbivory, whereas clonal species recover faster than nonclonal species. The role played by carbohydrates reserves suggests that these species could benefit from slightly higher light conditions in areas subjected to high deer pressure.

The alternative respiratory pathway allows sink to cope with changes in carbon availability in the sink-limited plant Erythronium americanum.

Mechanisms that allow plants to cope with a recurrent surplus of carbon in conditions of imbalance between source and sink activity has not received much attention. The response of sink growth and metabolism to the modulation of source activity was investigated using elevated CO(2) and elevated O(3) growth conditions in Erythronium americanum. Sink activity was monitored via slice and mitochondrial respiratory rates, sucrose hydrolysis activity, carbohydrates, and biomass accumulation throughout the growth season, while source activity was monitored via gas exchanges, rubisco and phosphoenolpyruvate carboxylase activities, carbohydrates, and respiratory rates. Elevated CO(2) increased the net photosynthetic rate by increasing substrate availability for rubisco. Elevated O(3) decreased the net photosynthetic rate mainly through a reduction in rubisco activity. Despite this modulation of the source activity, neither plant growth nor starch accumulation were affected by the treatments. Sucrose synthase activity was higher in the sink under elevated CO(2) and lower under elevated O(3), thereby modulating the pool of glycolytic intermediates. The alternative respiratory pathway was similarly modulated in the sink, as seen with both the activity and capacity of the pathway, as well as with the alternative oxidase abundance. In this sink-limited species, the alternative respiratory pathway appears to balance carbon availability with sink capacity, thereby avoiding early feedback-inhibition of photosynthesis in conditions of excess carbon availability.

Low temperature maximizes growth of Crocus vernus (L.) Hill via changes in carbon partitioning and corm development.

In Crocus vernus, a spring bulbous species, prolonged growth at low temperatures results in the development of larger perennial organs and delayed foliar senescence. Because corm growth is known to stop before the first visual sign of leaf senescence, it is clear that factors other than leaf duration alone determine final corm size. The aim of this study was to determine whether reduced growth at higher temperatures was due to decreased carbon import to the corm or to changes in the partitioning of this carbon once it had reached the corm. Plants were grown under two temperature regimes and the amount of carbon fixed, transported, and converted into a storable form in the corm, as well as the partitioning into soluble carbohydrates, starch, and the cell wall, were monitored during the growth cycle. The reduced growth at higher temperature could not be explained by a restriction in carbon supply or by a reduced ability to convert the carbon into starch. However, under the higher temperature regime, the plant allocated more carbon to cell wall material, and the amount of glucose within the corm declined earlier in the season. Hexose to sucrose ratios might control the duration of corm growth in C. vernus by influencing the timing of the cell division, elongation, and maturation phases. It is suggested that it is this shift in carbon partitioning, not limited carbon supply or leaf duration, which is responsible for the smaller final biomass of the corm at higher temperatures.

Novel de novo SHANK3 mutation in autistic patients.

A number of studies have confirmed that genetic factors play an important role in autism spectrum disorder (ASD). More recently de novo mutations in the SHANK3 gene, a synaptic scaffolding protein, have been associated with the ASD phenotype. As part of our gene discovery strategy, we sequenced the SHANK3 gene in a cohort of 427 ASD subjects and 190 controls. Here, we report the identification of two putative causative mutations: one being a de novo deletion at an intronic donor splice site and one missense transmitted from an epileptic father. We were able to confirm the deleterious effect of the splice site deletion by RT-PCR using mRNA extracted from cultured lymphoblastoid cells. The missense mutation, a leucine to proline at amino acid position 68, is perfectly conserved across all species examined, and would be predicted to disrupt an alpha-helical domain. These results further support the role of SHANK3 gene disruption in the etiology of ASD.

Disruption of AP1S1, causing a novel neurocutaneous syndrome, perturbs development of the skin and spinal cord.

Adaptor protein (AP) complexes regulate clathrin-coated vesicle assembly, protein cargo sorting, and vesicular trafficking between organelles in eukaryotic cells. Because disruption of the various subunits of the AP complexes is embryonic lethal in the majority of cases, characterization of their function in vivo is still lacking. Here, we describe the first mutation in the human AP1S1 gene, encoding the small subunit sigma1A of the AP-1 complex. This founder splice mutation, which leads to a premature stop codon, was found in four families with a unique syndrome characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratodermia (MEDNIK). To validate the pathogenic effect of the mutation, we knocked down Ap1s1 expression in zebrafish using selective antisens morpholino oligonucleotides (AMO). The knockdown phenotype consisted of perturbation in skin formation, reduced pigmentation, and severe motility deficits due to impaired neural network development. Both neural and skin defects were rescued by co-injection of AMO with wild-type (WT) human AP1S1 mRNA, but not by co-injecting the truncated form of AP1S1, consistent with a loss-of-function effect of this mutation. Together, these results confirm AP1S1 as the gene responsible for MEDNIK syndrome and demonstrate a critical role of AP1S1 in development of the skin and spinal cord.

Carbon dioxide enrichment does not reduce leaf longevity or alter accumulation of carbon reserves in the woodland spring ephemeral Erythronium americanum.

BACKGROUND AND AIMS: Woodland spring ephemerals exhibit a relatively short epigeous growth period prior to canopy closure. However, it has been suggested that leaf senescence is induced by a reduction in the carbohydrate sink demand, rather than by changes in light availability. To ascertain whether a potentially higher net carbon (C) assimilation rate could shorten leaf lifespan due to an accelerated rate of storage, Erythronium americanum plants were grown under ambient (400 ppm) and elevated (1100 ppm) CO2 concentrations. METHODS: During this growth-chamber experiment, plant biomass, bulb starch concentration and cell size, leaf phenology, gas exchange rates and nutrient concentrations were monitored. KEY RESULTS: Plants grown at 1100 ppm CO2 had greater net C assimilation rates than those grown at 400 ppm CO2. However, plant size, final bulb mass, bulb filling rate and timing of leaf senescence did not differ. CONCLUSIONS: Erythronium americanum fixed more C under elevated than under ambient CO2 conditions, but produced plants of similar size. The similar bulb growth rates under both CO2 concentrations suggest that the bulb filling rate is dependant on bulb cell elongation rate, rather than on C availability. Elevated CO2 stimulated leaf and bulb respiratory rates; this might reduce feed-back inhibition of photosynthesis and avoid inducing premature leaf senescence.

Carbon partitioning in a split-root system of arbuscular mycorrhizal plants is fungal and plant species dependent.

• Root carbon (C) partitioning in two host plant species colonized by one of three arbuscular mycorrhizal (AM) fungal species was investigated. • Split-root systems of barley (Hordeum vulgare) and sugar maple (Acer saccharum) were inoculated on one side with one of three AM fungi. Leaves were labelled with 14 CO2 3 wk after inoculation. Plants were harvested 24 h later and the root systems from the mycorrhizal (M) and nonmycorrhizal (NM) sides were analysed separately for 14 C. • Partitioning of 14 C between M and NM sides varied depending on the fungal and host plant species used. Gigaspora rosea showed a strong C-sink capacity with both plant species, Glomus intraradices showed a strong C-sink capacity with barley, and Glomus mosseae did not affect 14 C partitioning. The C-sink strength of the M barley roots inoculated with G. rosea or G. intraradices was linearly correlated with the degree of colonization. • The use of three AM fungal and two plant species allowed us to conclude that C-sink strength of AM fungi depends on both partners involved in the symbiosis.