Prolonged increase of corticosterone secretion by chronic social stress does not necessarily impair immune functions.

The influence of a chronic social stress upon immunity was investigated in Wistar rats, submitted for four weeks to two different behavioral situations, balanced in a factorial design: housing with three females and membership rotation. The combination of these two factor led to adrenal enlargement (43.3%), thymus involution (39.5%) and increased basal corticosterone levels, all indices of activation of the hypothalamic-hypophysis-adrenal axis. However, neither natural killer cell activity, splenocyte reactivity to mitogen nor the rate of spontaneous development of antibodies against Mycoplasma pulmonis, a common pathogen of the respiratory tract, were changed in the endocrine activated animals. Analysis of the data on kinetics of stress at 1, 7 and 28 days after the initial mixing of the animals gave the same results. These data question the immunosuppressant activity usually conferred to corticosteroids, at least when adrenal hyperactivity is induced by chronic environmental stressors.     

Genetic structure and life history are key factors in species distribution models of spiny lobsters

AimWe incorporated genetic structure and life history phase in species distribution models (SDMs) constructed for a widespread spiny lobster, to reveal local adaptations specific to individual subspecies and predict future range shifts under the RCP 8.5 climate change scenario.LocationIndo‐West Pacific.MethodsMaxEnt was used to construct present‐day SDMs for the spiny lobster Panulirus homarus and individually for the three genetically distinct subspecies of which it comprises. SDMs incorporated both sea surface and benthic (seafloor) climate layers to recreate discrete influences of these habitats during the drifting larval and benthic juvenile and adult life history phases. Principle component analysis (PCA) was used to infer environmental variables to which individual subspecies were adapted. SDM projections of present‐day habitat suitability were compared with predictions for the year 2,100, under the RCP 8.5 climate change scenario.ResultsIn the PCA, salinity best explained P. h. megasculptus habitat suitability, compared with current velocity in P. h. rubellus and sea surface temperature in P. h. homarus. Drifting and benthic life history phases were adapted to different combinations of sea surface and benthic environmental variables considered. Highly suitable habitats for benthic phases were spatially enveloped within more extensive sea surface habitats suitable for drifting larvae. SDMs predicted that present‐day highly suitable habitats for P. homarus will decrease by the year 2,100.Main conclusionsIncorporating genetic structure in SDMs showed that individual spiny lobster subspecies had unique adaptations, which could not be resolved in species‐level models. The use of sea surface and benthic climate layers revealed the relative importance of environmental variables during drifting and benthic life history phases. SDMs that included genetic structure and life history were more informative in predictive models of climate change effects.     

Towards an understanding of the role of intrinsic protein disorder on plant adaptation to environmental challenges

Intrinsic protein disorder is an interesting structural feature where fully functional proteins lack a three-dimensional structure in solution. In this work, we estimated the relative content of intrinsic protein disorder in 96 plant proteomes including monocots and eudicots. In this analysis, we found variation in the relative abundance of intrinsic protein disorder among these major clades; the relative level of disorder is higher in monocots than eudicots. In turn, there is an inverse relationship between the degree of intrinsic protein disorder and protein length, with smaller proteins being more disordered. The relative abundance of amino acids depends on intrinsic disorder and also varies among clades. Within the nucleus, intrinsically disordered proteins are more abundant than ordered proteins. Intrinsically disordered proteins are specialized in regulatory functions, nucleic acid binding, RNA processing, and in response to environmental stimuli. The implications of this on plants’ responses to their environment are discussed.     

Hippocampal Neuroligin-2 Overexpression Leads to Reduced Aggression and Inhibited Novelty Reactivity in Rats

Disturbances of the excitation/inhibition (E/I) balance in the brain were recently suggested as potential factors underlying disorders like autism and schizophrenia resulting in associated behavioral alterations including changes in social and emotional behavior as well as abnormal aggression. Neuronal cell adhesion molecules (nCAMs) and mutations in these genes were found to be strongly implicated in the pathophysiology of these disorders. Neuroligin2 (nlgn2) is a postsynaptic cell adhesion molecule, which is predominantly expressed at inhibitory synapses and required for synapse specification and stabilization. Changes in the expression of nlgn2 were shown to result in alterations of social behavior as well as altered inhibitory synaptic transmission, hence modifying the E/I balance. In our study, we focused on the role of nlgn2 in the dorsal hippocampus in the regulation of emotional and social behaviors. To this purpose, we injected an AAV construct overexpressing nlgn2 in the hippocampus of rats and investigated the effects on behavior and on markers for the E/I ratio. We could show an increase in GAD65, a GABA-synthesizing protein in neuronal terminals, and furthermore, reduced exploration of novel stimuli and less offensive behavior. Our data suggest nlgn2 in the hippocampus to be strongly implicated in maintaining the E/I balance in the brain and thereby modulating social and emotional behavior.     

Effects of Adenovirus Type 5 E1A Isoforms on Viral Replication in Arrested Human Cells

Human adenovirus has evolved to infect and replicate in terminally differentiated human epithelial cells, predominantly those within the airway, the gut, or the eye. To overcome the block to viral DNA replication present in these cells, the virus expresses the Early 1A proteins (E1A). These immediate early proteins drive cells into S-phase and induce expression of all other viral early genes. During infection, several E1A isoforms are expressed with proteins of 289, 243, 217, 171, and 55 residues being present for human adenovirus type 5. Here we examine the contribution that the two largest E1A isoforms make to the viral life cycle in growth-arrested normal human fibroblasts. Viruses that express E1A289R were found to replicate better than those that do not express this isoform. Importantly, induction of several viral genes was delayed in a virus expressing E1A243R, with several viral structural proteins undetectable by western blot. We also highlight the changes in E1A isoforms detected during the course of viral infection. Furthermore, we show that viral DNA replication occurs more efficiently, leading to higher number of viral genomes in cells infected with viruses that express E1A289R. Finally, induction of S-phase specific genes differs between viruses expressing different E1A isoforms, with those having E1A289R leading to, generally, earlier activation of these genes. Overall, we provide an overview of adenovirus replication using modern molecular biology approaches and further insights into the contribution that E1A isoforms make to the life cycle of human adenovirus in arrested human fibroblasts.     

A novel GAA-repeat-expansion-based mouse model of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a GAA repeat expansion mutation within intron 1 of the FXN gene, resulting in reduced levels of frataxin protein. We have previously reported the generation of human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing 90–190 GAA repeats, but the presence of multiple GAA repeats within these mice is considered suboptimal. We now describe the cellular, molecular and behavioural characterisation of a newly developed YAC transgenic FRDA mouse model, designated YG8sR, which we have shown by DNA sequencing to contain a single pure GAA repeat expansion. The founder YG8sR mouse contained 120 GAA repeats but, due to intergenerational expansion, we have now established a colony of YG8sR mice that contain ~200 GAA repeats. We show that YG8sR mice have a single copy of the FXN transgene, which is integrated at a single site as confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We have identified significant behavioural deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8sR FRDA mice compared with control Y47R and wild-type (WT) mice. We have also detected increased somatic GAA repeat instability in the brain and cerebellum of YG8sR mice, together with significantly reduced expression of FXN, FAST-1 and frataxin, and reduced aconitase activity, compared with Y47R mice. Furthermore, we have confirmed the presence of pathological vacuoles within neurons of the dorsal root ganglia (DRG) of YG8sR mice. These novel GAA-repeat-expansion-based YAC transgenic FRDA mice, which exhibit progressive FRDA-like pathology, represent an excellent model for the investigation of FRDA disease mechanisms and therapy.KEY WORDS: GAA repeat, Friedreich’s ataxia, FRDA, YG8sR, Mouse model     

The role of NCAM in auditory fear conditioning and its modulation by stress: a focus on the amygdala

Chronic stress in rodents was shown to induce structural shrinkage and functional alterations in the hippocampus that were linked to spatial memory impairments. Effects of chronic stress on the amygdala have been linked to a facilitation of fear conditioning. Although the underlying molecular mechanisms are still poorly understood, increasing evidence highlights the neural cell adhesion molecule (NCAM) as an important molecular mediator of stress‐induced structural and functional alterations. In this study, we investigated whether altered NCAM expression levels in the amygdala might be related to stress‐induced enhancement of auditory fear conditioning and anxiety‐like behavior. In adult C57BL/6J wild‐type mice, chronic unpredictable stress resulted in an isoform‐specific increase of NCAM expression (NCAM‐140 and NCAM‐180) in the amygdala, as well as enhanced auditory fear conditioning and anxiety‐like behavior. Strikingly, forebrain‐specific conditional NCAM‐deficient mice (NCAM‐floxed mice that express the cre‐recombinase under the control of the promoter of the α‐subunit of the calcium‐calmodulin‐dependent protein kinase II), whose amygdala NCAM expression levels are reduced, displayed impaired auditory fear conditioning which was not altered following chronic stress exposure. Likewise, chronic stress in these conditional NCAM‐deficient mice did not modify NCAM expression levels in the amygdala or hippocampus, while they showed enhanced anxiety‐like behavior, questioning the involvement of NCAM in this type of behavior. Together, our results strongly support the involvement of NCAM in the amygdala in the consolidation of auditory fear conditioning and highlight increased NCAM expression in the amygdala among the mechanisms whereby stress facilitates fear conditioning processes.     

BchJ and BchM interact in a 1 : 1 ratio with the magnesium chelatase BchH subunit of Rhodobacter capsulatus

Substrate channeling between the enzymatic steps in the (bacterio)chlorophyll biosynthetic pathway catalyzed by magnesium chelatase (BchI/ChlI, BchD/ChlD and BchH/ChlH subunits) and S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase (BchM/ChlM) has been suggested. This involves delivery of magnesium-protoporphyrin IX from the BchH/ChlH subunit of magnesium chelatase to BchM/ChlM. Stimulation of BchM/ChlM activity by BchH/ChlH has previously been shown, and physical interaction of the two proteins has been demonstrated. In plants and cyanobacteria, there is an added layer of complexity, as Gun4 serves as a porphyrin (protoporphyrin IX and magnesium-protoporphyrin IX) carrier, but this protein does not exist in anoxygenic photosynthetic bacteria. BchJ may play a similar role to Gun4 in Rhodobacter, as it has no currently assigned function in the established pathway. Purified recombinant Rhodobacter capsulatus BchJ and BchM were found to cause a shift in the equilibrium amount of Mg-protoporphyrin IX formed in a magnesium chelatase assay. Analysis of this shift revealed that it was always in a 1 : 1 ratio with either of these proteins and the BchH subunit of the magnesium chelatase. The establishment of the new equilibrium was faster with BchM than with BchJ in a coupled magnesium chelatase assay. BchJ bound magnesium-protoporphyrin IX or formed a ternary complex with BchH and magnesium-protoporphyrin IX. These results suggest that BchJ may play a role as a general magnesium porphyrin carrier, similar to one of the roles of GUN4 in oxygenic organisms.     

Strategic Distribution of Protective Proteins within Bran Layers of Wheat Protects the Nutrient-Rich Endosperm

Bran from bread wheat (Triticum aestivum ‘Babbler’) grain is composed of many outer layers of dead maternal tissues that overlie living aleurone cells. The dead cell layers function as a barrier resistant to degradation, whereas the aleurone layer is involved in mobilizing organic substrates in the endosperm during germination. We microdissected three defined bran fractions, outer layers (epidermis and hypodermis), intermediate fraction (cross cells, tube cells, testa, and nucellar tissue), and inner layer (aleurone cells), and used proteomics to identify their individual protein complements. All proteins of the outer layers were enzymes, whose function is to provide direct protection against pathogens or improve tissue strength. The more complex proteome of the intermediate layers suggests a greater diversity of function, including the inhibition of enzymes secreted by pathogens. The inner layer contains proteins involved in metabolism, as would be expected from live aleurone cells, but this layer also includes defense enzymes and inhibitors as well as 7S globulin (specific to this layer). Using immunofluorescence microscopy, oxalate oxidase was localized predominantly to the outer layers, xylanase inhibitor protein I to the xylan-rich nucellar layer of the intermediate fraction and pathogenesis-related protein 4 mainly to the aleurone. Activities of the water-extractable enzymes oxalate oxidase, peroxidase, and polyphenol oxidase were highest in the outer layers, whereas chitinase activity was found only in assays of whole grains. We conclude that the differential protein complements of each bran layer in wheat provide distinct lines of defense in protecting the embryo and nutrient-rich endosperm.Wheat grain (Triticum aestivum) is a major cereal crop and staple food in many parts of the world. The endosperm is the main nutritional component and is extracted in milling to produce base ingredients such as flour and semolina. Crop yield and quality may be compromised by both environmental and biological stresses. Wheat varieties are known to vary in their resistance to such stresses, probably due to individual differences in defense protein levels (Demeke and Morris, 2002; Bonnin et al., 2005; Yarullina et al., 2005). Cereal grain contains many defense proteins that have been categorized according to their mode of action and structural similarities. A major class of these is the pathogenesis-related (PR) proteins, which include PR-1, PR-2 (β -1,3-glucanases), PR-3 (chitinases), PR-4 (wheatwin1), and PR-5 (thaumatin-like proteins; Selitrennikoff, 2001; Desmond et al., 2006). Other known defense proteins are xylanase inhibitor proteins (XIPs) and α -amylase inhibitor proteins (Mundy et al., 1984; Payan et al., 2003). All of these defense proteins have both general and specific roles that contribute to plant survival, although little is known of their location within the various grain tissues, particularly the multiple layers that constitute bran.Proteomic analysis of wheat grain has previously been applied to identify proteins in the germ and endosperm (Skylas et al., 2000; Wong et al., 2004; Mak et al., 2006), but analysis of bran and bran tissue fractions has not been reported. Collection of sufficiently pure bran tissue fractions has limited progress, mainly due to the strong bonds between the various bran tissue layers and endosperm in dry grain. Thus, a method to obtain bran layers free from contaminants, such as adjacent tissue and endosperm, is required to provide a sample suitable for proteomic analysis. Soaking whole grain in water causes the endosperm to soften, allowing it to be easily removed and washed from the bran; the bran becomes malleable enough to dissect. While this approach might not identify the proteome of dry grain fractions, it is the best available representation of the three distinct tissue fractions in grains, namely the outer layer (epidermis and hypodermis), intermediate layer (cross cells, tube cells, testa, and nucellar tissue), and inner layer (aleurone cells; Antoine et al., 2003, 2004). Using this method, water-soluble proteins that diffuse from the grain can be collected and identified.In this study we aimed (1) to dissect bran into the three separate tissue fractions described above and to identify the protein complement of each fraction using proteomics, (2) to confirm the location of three major defense proteins identified (one from each microfraction) using immunolocalization, and (3) to identify water-soluble proteins and assay any defense-related proteins for enzymatic activity.