Inhibition of N1-Src kinase by a specific SH3 peptide ligand reveals a role for N1-Src in neurite elongation by L1-CAM

In the mammalian brain the ubiquitous tyrosine kinase, C-Src, undergoes splicing to insert short sequences in the SH3 domain to yield N1- and N2-Src. We and others have previously shown that the N-Srcs have altered substrate specificity and kinase activity compared to C-Src. However, the exact functions of the N-Srcs are unknown and it is likely that N-Src signalling events have been misattributed to C-Src because they cannot be distinguished by conventional Src inhibitors that target the kinase domain. By screening a peptide phage display library, we discovered a novel ligand (PDN1) that targets the unique SH3 domain of N1-Src and inhibits N1-Src in cells. In cultured neurons, PDN1 fused to a fluorescent protein inhibited neurite outgrowth, an effect that was mimicked by shRNA targeting the N1-Src microexon. PDN1 also inhibited L1-CAM-dependent neurite elongation in cerebellar granule neurons, a pathway previously shown to be disrupted in Src(−/−) mice. PDN1 therefore represents a novel tool for distinguishing the functions of N1-Src and C-Src in neurons and is a starting point for the development of a small molecule inhibitor of N1-Src

Pituitary and systemic autoimmunity in a case of intrasellar germinoma

Germinomas arising in the sella turcica are difficult to differentiate from autoimmune hypophysitis because of similar clinical and pathological features. This differentiation, nevertheless, is critical for patient care due to different treatments of the two diseases. We report the case of an 11-year-old girl who presented with diabetes insipidus and growth retardation, and was found to have an intra- and supra-sellar mass. Initial examination of the pituitary biopsy showed diffuse lymphocytic infiltration of the adenohypophysis and absent placental alkaline phosphatase expression, leading to a diagnosis of hypophysitis and glucocorticoid treatment. Because of the lack of clinical and radiological response, the pituitary specimen was re-examined, revealing this time the presence of scattered c-kit and Oct4 positive germinoma cells. The revised diagnosis prompted the initiation of radiotherapy, which induced disappearance of the pituitary mass. Immunological studies showed that the patient’s serum recognized antigens expressed by the patient’s own germinoma cells, as well as pituitary antigens like growth hormone and systemic antigens like the Sjögren syndrome antigen B and alpha-enolase. The study first reports the presence of pituitary and systemic antibodies in a patient with intrasellar germinoma, and reminds us that diffuse lymphocytic infiltration of the pituitary gland and pituitary antibodies does not always indicate a diagnosis of autoimmune hypophysitis

Phosphorylation by Dyrk1A of Clathrin Coated Vesicle-Associated Proteins: Identification of the Substrate Proteins and the Effects of Phosphorylation

Dyrk1A phosphorylated multiple proteins in the clathrin-coated vesicle (CCV) preparations obtained from rat brains. Mass spectrometric analysis identified MAP1A, MAP2, AP180, and α- and β-adaptins as the phosphorylated proteins in the CCVs. Each protein was subsequently confirmed by [32P]-labeling and immunological methods. The Dyrk1A-mediated phosphorylation released the majority of MAP1A and MAP2 and enhanced the release of AP180 and adaptin subunits from the CCVs. Furthermore, Dyrk1A displaced adaptor proteins physically from CCVs in a kinase-concentration dependent manner. The clathrin heavy chain release rate, in contrast, was not affected by Dyrk1A. Surprisingly, the Dyrk1A-mediated phosphorylation of α- and β-adaptins led to dissociation of the AP2 complex, and released only β-adaptin from the CCVs. AP180 was phosphorylated by Dyrk1A also in the membrane-free fractions, but α- and β-adaptins were not. Dyrk1A was detected in the isolated CCVs and was co-localized with clathrin in neurons from mouse brain sections and from primary cultured rat hippocampus. Previously, we proposed that Dyrk1A inhibits the onset of clathrin-mediated endocytosis in neurons by phosphorylating dynamin 1, amphiphysin 1, and synaptojanin 1. Current results suggest that besides the inhibition, Dyrk1A promotes the uncoating process of endocytosed CCVs

Impact of diets with different proportions of linseed and sunflower oils on the growth, liver histology, immunological and chemical blood parameters, and proximate composition of pikeperch Sander lucioperca (L.)

The aim of the study was to determine the impact of applying different proportions of linseed (LO) and sunflower (SFO) oils in pikeperch diets on growth, histological changes in the liver, immunological and blood chemical parameters. The fish were fed isoenergetic and isoprotein feeds containing SFO (group 100SFO) or LO (group 100LO) in quantities of 67 g kg/feed, and a mixture of oils: 47 g SFO and 20 g LO kg/feed (group 70SFO/30LO) and 20 g SFO and 47 g LO kg/feed (group 30SFO/70LO). Dietary ratios of polyunsaturated fatty acids from the n-3 and n-6 series (n3/n6 index) were 0.36–2.15. Pikeperch were reared for 56 days in three replicates for each dietary treatment. Various dietary oils and ratios of n3/n6 did not impact fish growth, feed conversion ratio, viscerosomatic and hepatosomatic index, and size of the hepatocytes. Feeding the fish high quantities of LO and SO oils (groups 100LO and 100SFO) reduced the immunological response of the phagocytes and lymphocytes in the fish. Moreover, this resulted in significant differences among groups in the quantity of linolenic and linoleic acid in whole fish bodies, viscera, fillets, and livers. Various quantities of vegetable oils in the fish diets did not impact the quantity of arachidonic, eicosapentaenoic and docosahexaenoic acid in the fillets and livers. The immunological index and low quantities of linoleic acid in the fillets obtained in group 30SFO/70LO indicate that the n3/n6 dietary ratio of 1.35 was the most advantageous for feeding juvenile pikeperch feeds with vegetable oils

Curcumin and resveratrol inhibit nuclear factor-kappaB-mediated cytokine expression in adipocytes

<p>Abstract</p> <p>Background</p> <p>Adipocytes express inflammatory mediators that contribute to the low-level, chronic inflammation found in obese subjects and have been linked to the onset of cardiovascular disorders and insulin resistance associated with type 2 diabetes mellitus. A reduction in inflammatory gene expression in adipocytes would be expected to reverse this low-level, inflammatory state and improve cardiovascular function and insulin sensitivity. The natural products, curcumin and resveratrol, are established anti-inflammatory compounds that mediate their effects by inhibiting activation of NF-κB signaling. In the present study, we examined if these natural products can inhibit NF-κB activation in adipocytes and in doing so reduce cytokine expression.</p> <p>Methods</p> <p>Cytokine (TNF-α, IL-1β, IL-6) and COX-2 gene expression in 3T3-L1-derived adipocytes was measured by quantitative real-time PCR (qRT-PCR) with or without TNFα-stimulation. Cytokine protein and prostaglandin E₂(PGE₂) expression were measured by ELISA. Effects of curcumin and resveratrol were evaluated by treating TNFα-stimulated adipocytes with each compound and 1) assessing the activation state of the NF-κB signaling pathway and 2) measuring inflammatory gene expression by qRT-PCR and ELISA.</p> <p>Results</p> <p>Both preadipocytes and differentiated adipocytes express the genes for TNF-α, IL-6, and COX-2, key mediators of the inflammatory response. Preadipocytes were also found to express IL-1β; however, IL-1β expression was absent in differentiated adipocytes. TNF-α treatment activated NF-κB signaling in differentiated adipocytes by inducing IκB degradation and NF-κB translocation to the nucleus, and as a result increased IL-6 (6-fold) and COX-2 (2.5-fold) mRNA levels. TNF-α also activated IL-1β gene expression in differentiated adipocytes, but had no effect on endogenous TNF-α mRNA levels. No detectable TNFα or IL-1β was secreted by adipocytes. Curcumin and resveratrol treatment inhibited NF-κB activation and resulted in a reduction of TNF-α, IL-1β, IL-6, and COX-2 gene expression (IC₅₀= 2 μM) and a reduction of secreted IL-6 and PGE₂(IC₅₀~ 20 μM).</p> <p>Conclusion</p> <p>Curcumin and resveratrol are able to inhibit TNFα-activated NF-κB signaling in adipocytes and as a result significantly reduce cytokine expression. These data suggest that curcumin and resveratrol may provide a novel and safe approach to reduce or inhibit the chronic inflammatory properties of adipose tissue.</p

Pathogenic <i>SPTBN1</i> variants cause an autosomal dominant neurodevelopmental syndrome

SPTBN1 encodes βII-spectrin, the ubiquitously expressed β-spectrin that forms micrometer-scale networks associated with plasma membranes. Mice deficient in neuronal βII-spectrin have defects in cortical organization, developmental delay and behavioral deficiencies. These phenotypes, while less severe, are observed in haploinsufficient animals, suggesting that individuals carrying heterozygous SPTBN1 variants may also show measurable compromise of neural development and function. Here we identify heterozygous SPTBN1 variants in 29 individuals with developmental, language and motor delays; mild to severe intellectual disability; autistic features; seizures; behavioral and movement abnormalities; hypotonia; and variable dysmorphic facial features. We show that these SPTBN1 variants lead to effects that affect βII-spectrin stability, disrupt binding to key molecular partners, and disturb cytoskeleton organization and dynamics. Our studies define SPTBN1 variants as the genetic basis of a neurodevelopmental syndrome, expand the set of spectrinopathies affecting the brain and underscore the critical role of βII-spectrin in the central nervous system

Acute increase of alpha-synuclein inhibits synaptic vesicle recycling evoked during intense stimulation

This work was supported by grants from the NIH/National Institute of Neurological Disorder and Stroke RO1 NS078165 (to J.R.M.), the Morton Cure Paralysis Fund (to J.R.M.), and the Branfman Family Foundation (to J.M.G.) and by a Dorothea Bennett graduate fellowship (to D.J.B.)

Diverse and Active Roles for Adipocytes During Mammary Gland Growth and Function

The mammary gland is unique in its requirement to develop in close association with a depot of adipose tissue that is commonly referred to as the mammary fat pad. As discussed throughout this issue, the mammary fat pad represents a complex stromal microenvironment that includes a variety of cell types. In this article we focus on adipocytes as local regulators of epithelial cell growth and their function during lactation. Several important considerations arise from such a discussion. There is a clear and close interrelationship between different stromal tissue types within the mammary fat pad and its adipocytes. Furthermore, these relationships are both stage- and species-dependent, although many questions remain unanswered regarding their roles in these different states. Several lines of evidence also suggest that adipocytes within the mammary fat pad may function differently from those in other fat depots. Finally, past and future technologies present a variety of opportunities to model these complexities in order to more precisely delineate the many potential functions of adipocytes within the mammary glands. A thorough understanding of the role for this cell type in the mammary glands could present numerous opportunities to modify both breast cancer risk and lactation performance

Synaptic vesicle recycling is unaffected in the Ts65Dn mouse model of Down syndrome

Down syndrome (DS) is the most common genetic cause of intellectual disability, and arises from trisomy of human chromosome 21. Accumulating evidence from studies of both DS patient tissue and mouse models has suggested that synaptic dysfunction is a key factor in the disorder. The presence of several genes within the DS trisomy that are either directly or indirectly linked to synaptic vesicle (SV) endocytosis suggested that presynaptic dysfunction could underlie some of these synaptic defects. Therefore we determined whether SV recycling was altered in neurons from the Ts65Dn mouse, the best characterised model of DS to date. We found that SV exocytosis, the size of the SV recycling pool, clathrin-mediated endocytosis, activity-dependent bulk endocytosis and SV generation from bulk endosomes were all unaffected by the presence of the Ts65Dn trisomy. These results were obtained using battery of complementary assays employing genetically-encoded fluorescent reporters of SV cargo trafficking, and fluorescent and morphological assays of fluid-phase uptake in primary neuronal culture. The absence of presynaptic dysfunction in central nerve terminals of the Ts65Dn mouse suggests that future research should focus on the established alterations in excitatory / inhibitory balance as a potential route for future pharmacotherapy