Targeting of nonkaryophilic cell-permeable peptides into the nuclei of intact cells by covalently attached nuclear localization signals

Dermaseptins are a family of antimicrobial peptides that lyse target bacterial cells by destabilization of their membranes. Here we present a novel application of a peptide derived from the dermaseptin S4, S4(13). At nontoxic concentrations, fluorescently labeled S4(13) was able to penetrate intact cultured HeLa cells but essentially failed to enter their nuclei despite its low molecular weight. Covalent attachment of nuclear localization signal (NLS) motifs of the SV40-T-antigen and of the HIV-1 Rev protein (ARM) conferred karyophilic properties upon the S4(13). The resulting peptides, which were designated as PV-S4(13) and RR-S4(13) penetrated into intact HeLa cells and were able to accumulate within the cells' nuclei. In studies with digitonin-permeabilized cells, nuclear uptake of the PV-S4(13) and the RR-S4(13) peptides showed the same features that characterize active nuclear import. Nuclear import was observed at 37 degrees C, was ATP-dependent, and was inhibited by the free peptides bearing the SV40 NLS and the Rev and Tat ARMs. Microinjected S4(13) remained in the cytoplasm while microinjected RR-S4(13) was translocated into the cells' nuclei. The new type of cell-permeable "karyophilic" peptides described here may be of potential application as a lead compound for therapeutic purposes, as a tool to study nucleocytoplasmic shuttling in intact cells, and for the delivery of peptides to the nucleus.     

Amelioration of dextran sulfate colitis by butyrate: role of heat shock protein 70 and NF-kappaB

Butyrate enemas have been demonstrated to ameliorate inflammation in ulcerative colitis. The mechanism of this protective effect of butyrate is not known, and this study examines the effect of butyrate on epithelial function, inducible heat shock protein 70 (HSP70) expression, and NF-kappaB activation in experimental colitis. Colitis was induced in rats by oral dextran sulfate sodium (DSS) and by butyrate or saline enemas. Mucosal barrier function was assessed by electrical resistance and [14C]mannitol permeability. HSP70 production was determined by [35S]methionine labeling, Western blot analysis, and immunohistochemistry. Activation of heat shock factors (HSFs) and NF-kappaB was evaluated by EMSA. Butyrate showed a significant protection against the decrease in cell viability, increase in mucosal permeability, and polymorphonuclear neutrophil infiltration seen in DSS colitis. Butyrate inhibited HSP70 expression in DSS colitis and also inhibited the activation of HSF and NF-kappaB. Thus butyrate enema was found to be cytoprotective in DSS colitis, an effect partly mediated by suppressing activation of HSP70 and NF-kappaB.     

Structure and Binding Interface of the Cytosolic Tails of αXβ2 Integrin

Background

Integrins are signal transducer proteins involved in a number of vital physiological processes including cell adhesion, proliferation and migration. Integrin molecules are hetero-dimers composed of two distinct subunits, α and β. In humans, 18 α and 8 β subunits are combined into 24 different integrin molecules. Each of the subunit comprises a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT). The CTs of integrins are vital for bidirectional signal transduction and in maintaining the resting state of the receptors. A large number of intracellular proteins have been found to interact with the CTs of integrins linking integrins to the cytoskeleton.

Methodology/Principal Findings

In this work, we have investigated structure and interactions of CTs of the leukocyte specific integrin αXβ2. We determined the atomic resolution structure of a myristoylated CT of αX in perdeuterated dodecylphosphocholine (DPC) by NMR spectroscopy. Our results reveal that the 35-residue long CT of αX adopts an α-helical conformation for residues F4-N17 at the N-terminal region. The remaining residues located at the C-terminal segment of αX delineate a long loop of irregular conformations. A segment of the loop maintains packing interactions with the helical structure by an extended non-polar surface of the αX CT. Interactions between αX and β2 CTs are demonstrated by ¹⁵N-¹H HSQC NMR experiments. We find that residues constituting the polar face of the helical conformation of αX are involved in interactions with the N-terminal residues of β2 CT. A docked structure of the CT complex indicates that a network of polar and/or salt-bridge interactions may sustain the heteromeric interactions.

Conclusions/Significance

The current study provides important insights into the conservation of interactions and structures among different CTs of integrins.     

Truncated tyrosine kinase B brain-derived neurotrophic factor receptor directs cortical neural stem cells to a glial cell fate by a novel signaling mechanism

During development of the mammalian cerebral cortex neural stem cells (NSC) first generate neurons and subsequently produce glial cells. The mechanism(s) responsible for this developmental shift from neurogenesis to gliogenesis is unknown. Brain-derived neurotrophic factor (BDNF) is believed to play important roles in the development of the mammalian cerebral cortex; it enhances neurogenesis and promotes the differentiation and survival of newly generated neurons. Here, we provide evidence that a truncated form of the BDNF receptor tyrosine kinase B (trkB-t) plays a pivotal role in directing embryonic mouse cortical NSC to a glial cell fate. Expression of trkB-t promotes differentiation of NSC toward astrocytes while inhibiting neurogenesis both in cell culture and in vivo. The mechanism by which trkB-t induces astrocyte genesis is not simply the result of inhibition of full-length receptor with intrinsic tyrosine kinase activity signaling. Instead, binding of BDNF to trkB-t activates a signaling pathway (involving a G-protein and protein kinase C) that induced NSC to become glial progenitors and astrocytes. Thus, the increased expression of trkB-t in the embryonic cerebral cortex that occurs coincident with astrocyte production plays a pivotal role in the developmental transition from neurogenesis to gliogenesis. Our findings suggest a mechanism by which a single factor (BDNF) regulates the production of the two major cell types in the mammalian cerebral cortex.     

Effect of inland water salinity on growth performance and nutritional physiology in growing milkfish, Chanos chanos (Forsskal): field and laboratory studies

To investigate the effect of inland groundwater salinity on growth performance, feed conversion efficiency, nutrient retention and intestinal enzyme activity in milkfish, two experiments were conducted. In the first experiment (Expt I), a 100‐day monoculture of Chanos chanos [mean body weight (BW): 2.2 g] at different salinities (0, 10, 15, 20 and 25‰) was carried out in ponds fertilized with cowdung (about 10 000 kg ha^?1 year^?1) and poultry droppings (about 3000 kg ha^?1 year^?1). The fish were fed a compounded supplementary diet (containing 40% protein) at 5% BW day^?1. Studies have revealed that growth increased with each increase in the salinity level; the highest values in weight gain and energy assimilated were observed in ponds maintained at 25‰ salinity [weight: 322.2 g and specific growth rate (SGR): 8.3]. Highest values of condition factor (0.7) and exponential value (n) of the length–weight relationship (LWR; n = 3.25) were also observed in ponds maintained at 25‰ salinity. Dissolved oxygen (DO), biological oxygen demand (BOD), pH and nutrient release remained at the optimal level during the culture period. High values of chlorophyll a, net primary productivity (NPP), phytoplankton and zooplankton population coincided with the highest values of alkalinity and turbidity in ponds maintained at 25‰ salinity. Multivariate analysis revealed a significant positive correlation of chlorides (r = 0.91), conductivity (r = 0.89) and hardness (r = 0.96) with fish growth. Productivity indicating parameters viz. NPP (r = 0.45), nitrate (r = 0.94) and o‐PO₄ (r = 0.52) also showed a significant positive correlation with fish weight gain. In the second experiment (Expt II), milkfish (mean BW: 3.7 g) fry were exposed to different levels of salinity (0.0, 10, 15, 20, 25 and 30‰), and maintained for 90 days in the laboratory. Significantly (P < 0.05) high growth (percentage increase in BW: 183.1 and SGR: 1.2), feed conversion efficiency (64.5%) and intestinal enzyme activity (protease 5.1, amylase 4.1 and cellulolytic 3.2) were observed in the group maintained at 25 ppt salinity in comparison with other groups similarly maintained at low or high salinity levels. Carcass composition, muscle and liver glycogen levels were also significantly (P < 0.05) affected by salinity changes. The significance of these findings is discussed in this paper.     

Epitope mapping of anti‐myelin oligodendrocyte glycoprotein (MOG) antibodies in a mouse model of multiple sclerosis: microwave‐assisted synthesis of the peptide antigens and ELISA screening

The role of pathologic auto‐antibodies against myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is a highly controversial matter. As the use of animal models may enable to unravel the molecular mechanisms of the human disorder, numerous studies on multiple sclerosis are carried out using experimental autoimmune encephalomyelitis (EAE). In particular, the most extensively used EAE model is obtained by immunizing C57BL/6 mice with the immunodominant peptide MOG(35–55). In this scenario, we analyzed the anti‐MOG antibody response in this model using the recombinant refolded extracellular domain of the protein, MOG(1–117). To assess the presence of a B‐cell intramolecular epitope spreading mechanism, we tested also five synthetic peptides mapping the 1–117 sequence of MOG, including MOG(35–55). For this purpose, we cloned, expressed in Escherichia coli and on‐column refolded MOG(1–117), and we applied an optimized microwave‐assisted solid‐phase synthetic strategy to obtain the designed peptide sequences. Subsequently, we set up a solid‐phase immunoenzymatic assay testing both naïve and EAE mice sera and using MOG protein and peptides as antigenic probes. The results obtained disclose an intense IgG antibody response against both the recombinant protein and the immunizing peptide, while no response was observed against the other synthetic fragments, thus excluding the presence of an intramolecular epitope spreading mechanism. Furthermore, as the properly refolded recombinant probe is able to bind antibodies with greater efficiency compared with MOG(35–55), we hypothesize the presence of both linear and conformational epitopes on MOG(35–55) sequence. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Regulation of light-dependent Gqalpha translocation and morphological changes in fly photoreceptors

Heterotrimeric G-proteins relay signals between membrane-bound receptors and downstream effectors. Little is known, however, about the regulation of Galpha subunit localization within the natural endogenous environment of a specialized signaling cell. Here we show, using live Drosophila flies, that light causes massive and reversible translocation of the visual Gqalpha to the cytosol, associated with marked architectural changes in the signaling compartment. Molecular genetic dissection together with detailed kinetic analysis enabled us to characterize the translocation cycle and to unravel how signaling molecules that interact with Gqalpha affect these processes. Epistatic analysis showed that Gqalpha is necessary but not sufficient to bring about the morphological changes in the signaling organelle. Furthermore, mutant analysis indicated that Gqbeta is essential for targeting of Gqalpha to the membrane and suggested that Gqbeta is also needed for efficient activation of Gqalpha by rhodopsin. Our results support the 'two-signal model' hypothesis for membrane targeting in a living organism and characterize the regulation of both the activity-dependent Gq localization and the cellular architectural changes in Drosophila photoreceptors.     

库布齐沙漠南缘风沙-植被相互作用及其景观效应

干旱区尤其沙漠边缘地区的风沙与植被相互作用对塑造地表景观具有重要意义。选择库布齐沙漠南缘的油蒿灌丛地为研究区,开展了植被调查、风沙流观测和表层沉积物粒度采样测试,分析了顺风向植被盖度、风沙流结构与沉积物特征的沿程变化,探讨了风沙-植被相互作用及其对地表景观格局的影响。结果表明,风沙流与植被相互作用方式的改变使植物生长状况与地表蚀积模式发生变化,进而导致顺风向景观表现出明显的空间异质性。自上风向裸地过渡到均匀分布的新生油蒿和油蒿灌丛再至斑块状分布的灌丛沙堆,植被盖度与覆沙厚度先增大后减小,空气动力学粗糙度沿程不断增加且在过渡时其增幅最大,输沙率与沉积物粒度呈先减小后增大趋势,并在植被盖度与覆沙厚度最大处出现最小值。在沙漠边缘剥蚀高原上,起初适量风沙堆积促进油蒿定植与生长,均匀分布的油蒿灌丛进一步促进沙物质堆积,但当堆积厚度超过油蒿耐沙埋深度时发生退化,灌丛出现斑块状分布且风沙流在丘间地处侵蚀。据此,可理解为剥蚀高原风沙区景观异质性是风沙与植被相互协同与抑制作用的结果。