Astrocytic αVβ3 integrin inhibits neurite outgrowth and promotes retraction of neuronal processes by clustering Thy-1

Thy-1 is a membrane glycoprotein suggested to stabilize or inhibit growth of neuronal processes. However, its precise function has remained obscure, because its endogenous ligand is unknown. We previously showed that Thy-1 binds directly to α(V)β(3) integrin in trans eliciting responses in astrocytes. Nonetheless, whether α(V)β(3) integrin might also serve as a Thy-1-ligand triggering a neuronal response has not been explored. Thus, utilizing primary neurons and a neuron-derived cell line CAD, Thy-1-mediated effects of α(V)β(3) integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous α(V)β(3) integrin restricted neurite outgrowth. Likewise, α(V)β(3)-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(-) CAD cells. In differentiating primary neurons exposed to α(V)β(3)-Fc, fewer and shorter dendrites were detected. This effect was abolished by cleavage of Thy-1 from the neuronal surface using phosphoinositide-specific phospholipase C (PI-PLC). Moreover, α(V)β(3)-Fc also induced retraction of already extended Thy-1(+)-axon-like neurites in differentiated CAD cells as well as of axonal terminals in differentiated primary neurons. Axonal retraction occurred when redistribution and clustering of Thy-1 molecules in the plasma membrane was induced by α(V)β(3) integrin. Binding of α(V)β(3)-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, α(V)β(3)-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that α(V)β(3) integrin is a ligand for Thy-1 that upon binding not only restricts the growth of neurites, but also induces retraction of already existing processes by inducing Thy-1 clustering. We propose that these events participate in bi-directional astrocyte-neuron communication relevant to axonal repair after neuronal damage.     

Potato snakin-1 gene silencing affects cell division, primary metabolism, and cell wall composition

Snakin-1 (SN1) is an antimicrobial cysteine-rich peptide isolated from potato (Solanum tuberosum) that was classified as a member of the Snakin/Gibberellic Acid Stimulated in Arabidopsis protein family. In this work, a transgenic approach was used to study the role of SN1 in planta. Even when overexpressing SN1, potato lines did not show remarkable morphological differences from the wild type; SN1 silencing resulted in reduced height, which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of the adaxial epidermis of mature leaves revealed that silenced lines had 70% to 90% increases in mean cell size with respect to wild-type leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-green fluorescent protein fusion protein was localized in plasma membrane, and bimolecular fluorescence complementation assays revealed that SN1 self-interacted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry, Fourier transform infrared spectroscopy, and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in seven cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism, and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense.     

In utero glucocorticoid exposure reduces fetal skeletal muscle mass in rats independent of effects on maternal nutrition

Maternal stress and undernutrition can occur together and expose the fetus to high glucocorticoid (GLC) levels during this vulnerable period. To determine the consequences of GLC exposure on fetal skeletal muscle independently of maternal food intake, groups of timed-pregnant Sprague-Dawley rats (n = 7/group) were studied: ad libitum food intake (control, CON); ad libitum food intake with 1 mg dexamethasone/l drinking water from embryonic day (ED)13 to ED21 (DEX); pair-fed (PF) to DEX from ED13 to ED21. On ED22, dams were injected with [(3)H]phenylalanine for measurements of fetal leg muscle and diaphragm fractional protein synthesis rates (FSR). Fetal muscles were analyzed for protein and RNA contents, [(3)H]phenylalanine incorporation, and MuRF1 and atrogin-1 (MAFbx) mRNA expression. Fetal liver tyrosine aminotransferase (TAT) expression was quantified to assess fetal exposure to GLCs. DEX treatment reduced maternal food intake by 13% (P < 0.001) and significantly reduced placental mass relative to CON and PF dams. Liver TAT expression was elevated only in DEX fetuses (P < 0.01). DEX muscle protein masses were 56% and 70% than those of CON (P < 0.01) and PF (P < 0.05) fetuses, respectively; PF muscles were 80% of CON (P < 0.01). Muscle FSR decreased by 35% in DEX fetuses (P < 0.001) but were not different between PF and CON. Only atrogin-1 expression was increased in DEX fetus muscles. We conclude that high maternal GLC levels and inadequate maternal food intake impair fetal skeletal muscle growth, most likely through different mechanisms. When combined, the effects of decreased maternal intake and maternal GLC intake on fetal muscle growth are additive.     

Fluctuation of oxidative stress indicators in Salix nigra seeds during priming

Salix nigra seeds subjected to increased humidification show a decrease in normal germination (NG) during early imbibition followed by a recovery in that parameter at increasing imbibition times. Since photo-oxidized seeds contain high levels of reactive oxygen species (ROS), it is possible to infer that the atypical decrease in NG is a consequence of a higher ROS mobilization at early imbibition and the subsequent recovery from an increase in antioxidant activity. In this study, several oxidative stress indicators were evaluated in photo-oxidized seeds subjected to priming. ROS production was studied using electronic spin resonance spectroscopy, spontaneous chemiluminescence (SCL), spectrophotometry (with XTT), and histochemical (with DAB and NBT) and cytochemical (with CeCl(3)) techniques. Four indicators of molecular damage were monitored: lipid peroxidation, pigment destruction, protein oxidation, and membrane integrity. Antioxidant activity was evaluated by changes in the enzymes SOD, CAT, APX, and POX. The results revealed that the decrease in NG at the beginning of priming occurs by an oxidative burst, as determined by increases in both SCL and superoxide anion radical (O2(·-)) Such oxidative burst generates lipid peroxidation, protein oxidation, and a decrease in both pigment content and enzyme activities. With increasing hydration, damages are progressively reversed and NG restored, which coincides with the increased activity of antioxidant defences. It is proposed that these novel observations regarding the occurrence of an oxidative burst are related to the high basal ROS levels and the high membrane content retained in the mature embryo tissues.     

Physicochemical,Thermal and Rheological Properties of Native and Oxidized Starch from Corn Landraces and Hybrids

Food Biophysics - Native starch was isolated from three corn landraces and a hybrid variety. The starches were oxidized with several concentrations of NaOCl (0.8, 2 and 5% w/w active chlorine). The...     

Study of the Affinity between the Protein Kinase PKA and Peptide Substrates Derived from Kemptide Using Molecular Dynamics Simulations and MM/GBSA

We have carried out a protocol in computational biochemistry including molecular dynamics (MD) simulations and MM/GBSA free energy calculations on the complex between the protein kinase A (PKA) and the specific peptide substrate Kemptide (LRRASLG). We made the same calculations on other PKA complexes that contain Kemptide derivatives (with mutations of the arginines, and with deletions of N and C-terminal amino acids). We predicted shifts in the free energy changes from the free PKA to PKA-substrate complex (ΔΔG_E→ES) when Kemptide structure is modified (we consider that the calculated shifts correlate with the experimental shifts of the free energy changes from the free PKA to the transition states (ΔΔG_E→TS) determined by the catalytic efficiency (k_cat/K_M) changes). Our results demonstrate that it is possible to predict the kinetic properties of protein kinases using simple computational biochemistry methods. As an additional benefit, these methods give detailed molecular information that permit the analysis of the atomic forces that contribute to the affinity between protein kinases and their substrates.     

Molecular Phylogeny of Weakfish Species of the Stellifer Group (Sciaenidae,Perciformes) of the Western South Atlantic Based on Mitochondrial and Nuclear Data

The phylogenetic relationships within the Stellifer group of weakfishes (Stellifer, Odontoscion, Ophioscion, and Bairdiella) were evaluated using 2723 base pairs comprising sequences of nuclear (rhodopsin, TMO-4C4, RAG-1) and mitochondrial (16S rRNA and COI) markers obtained from specimens of nine species. Our results indicate a close relationship between Bairdiella and Odontoscion, and also that the genus Stellifer is not monophyletic, but rather that it consists of two distinct lineages, one clade containing S. microps/S. naso/S. brasiliensis and the other, S. rastrifer/S. stellifer/Stellifer sp. B, which is closer to Ophioscion than the former clade. The O. punctatissimus populations from the northern and southern Brazilian coast were also highly divergent in both nuclear (0.8% for rhodopsin and 0.9% for RAG-1) and mitochondrial sequences (2.2% for 16S rRNA and 7.3% for COI), which we conclude is consistent with the presence of two distinct species. The morphological similarities of the members of the Stellifer group is reinforced by the molecular data from both the present study and previous analyses, which have questioned the taxonomic status of the Stellifer group. If, on the one hand, the group is in fact composed of four genera (Stellifer, Ophioscion, Odontoscion, and Bairdiella), one of the two Stellifer clades should be reclassified as a new genus. However, if the close relationship and the reduced genetic divergence found within the group is confirmed in a more extensive study, including representatives of additional taxa, this, together with the morphological evidence, would support downgrading the whole group to a single genus. Obviously, these contradictory findings reinforce the need for a more systematic taxonomic revision of the Stellifer group as a whole.