LEC-2, a highly variable lectin in the lichen Peltigera membranacea

Lectins are a diverse group of carbohydrate binding proteins often involved in cellular interactions. A lectin gene, lec-2, was identified in the mycobiont of the lichen Peltigera membranacea. Sequencing of lec-2 open reading frames from 21 individual samples showed an unexpectedly high level of polymorphism in the deduced protein (LEC-2), which was sorted into nine haplotypes based on amino acid sequence. Calculations showed that the rates of nonsynonymous versus synonymous nucleotide substitutions deviated significantly from the null hypothesis of neutrality, indicating strong positive selection. Molecular modeling revealed that most amino acid replacements were around the putative carbohydrate-binding pocket, indicating changes in ligand binding. Lectins have been thought to be involved in the recognition of photobiont partners in lichen symbioses, and the hypothesis that positive selection of LEC-2 is driven by variation in the Nostoc photobiont partner was tested by comparing mycobiont LEC-2 haplotypes and photobiont genotypes, as represented by the rbcLX region. It was not possible to pair up the two types of marker sequences without conflicts, suggesting that positive selection of LEC-2 was not due to variation in photobiont partners.     

Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis

Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.     

Naringin Treatment Improves Functional Recovery by Increasing BDNF and VEGF Expression, Inhibiting Neuronal Apoptosis After Spinal Cord Injury

The aim of this study was to determine the therapeutic efficacy of starting naringin treatment 1 day after spinal cord injury (SCI) in rat and to investigate the underlying mechanism. SCI was induced using the modified weight-drop method in Sprague-Dawley rats. The SCI animals were randomly divided into three groups: vehicle-treated group; 20 mg/kg naringin-treated group; 40 mg/kg naringin-treated group, and additionally with sham group (laminectomy only). Locomotors functional recovery was assessed during the 6 weeks post operation period by performing open-field locomotors tests and inclined-plane tests. At the end of the study, the segments of spinal cord encompassing the injury site were removed for histopathological analysis. Immunohistochemistry was performed to observe the expression of the brain-derived neurotrophic factor (BDNF). The expression of vascular endothelial growth factor (VEGF), B-cell CLL/lymphoma-2 (Bcl-2), BCL-2-associated X protein (Bax) and caspase-3 were detected by Western blot analysis. The apoptotic neural cells were assessed using the TUNEL method. The results showed that the naringin-treated animals had significantly better locomotor function recovery, less myelin loss, and higher expression of BDNF and VEGF. In addition, naringin treatment significantly increased in Bcl-2:Bax ratio, reduced the enzyme activity of caspase-3 and decreased the number of apoptotic cells after SCI. These findings suggest that naringin treatment starting 1 day after SCI can significantly improve locomotor recovery, and this neuroprotective effect may be related to the upregulation of BDNF and VEGF and the inhibition of neural apoptosis. Therefore, naringin may be useful as a promising therapeutic agent for SCI.     

Cell-type-based analysis of microRNA profiles in the mouse brain

MicroRNAs (miRNA) are implicated in brain development and function but the underlying mechanisms have been difficult to study in part due to the cellular heterogeneity in neural circuits. To systematically analyze miRNA expression in neurons, we have established a miRNA tagging and affinity-purification (miRAP) method that is targeted to cell types through the Cre-loxP binary system in mice. Our studies of the neocortex and cerebellum reveal the expression of a large fraction of known miRNAs with distinct profiles in glutamatergic and GABAergic neurons and subtypes of GABAergic neurons. We further detected putative novel miRNAs, tissue or cell type-specific strand selection of miRNAs, and miRNA editing. Our method thus will facilitate a systematic analysis of miRNA expression and regulation in specific neuron types in the context of neuronal development, physiology, plasticity, pathology, and disease models, and is generally applicable to other cell types and tissues.     

Eps8 protein facilitates phagocytosis by increasing TLR4-MyD88 protein interaction in lipopolysaccharide-stimulated macrophages

Toll-like receptors (TLRs) are crucial in macrophage phagocytosis, which is pivotal in host innate immune response. However, the detailed mechanism is not fully defined. Here, we demonstrated that the induction of Src and Eps8 in LPS-treated macrophages was TLR4- and MyD88-dependent, and their attenuation reduced LPS-promoted phagocytosis. Confocal microscopy indicated the colocalization of Eps8 and TLR4 in the cytosol and at the phagosome. Consistently, both Eps8 and TLR4 were present in the same immunocomplex regardless of LPS stimulation. Inhibition of this complex formation by eps8 siRNA or overexpression of pleckstrin homology domain-truncated Eps8 (i.e. 261-p97(Eps8)) decreased LPS-induced TLR4-MyD88 interaction and the following activation of Src, focal adhesion kinase, and p38 MAPK. Importantly, attenuation of Eps8 impaired the bacterium-killing ability of macrophages. Thus, Eps8 is a key regulator of the LPS-stimulated TLR4-MyD88 interaction and contributes to macrophage phagocytosis.     

A stochastic model for DNA translocation through an electropore

A 1D Fokker-Planck simulation of DNA translocation through an electropore under finite pulses is presented. This study is motivated by applications relevant to DNA electrotransfer into biological cells via electroporation. The results review important insights. The translocation may occur on two disparate time scales, the electrophoretic time (~ms), and the diffusive time (~s), depending on the pulse length. Furthermore, a power-law correlation is observed, F-PST~(V(m)t(p))(a)/N(b), where F-PST is the final probability of successful translocation, V(m) is the transmembrane potential, t(p) is the pulse length, and N is the DNA length in segments. The values for a and b are close to 1 and 1.5, respectively. The simulated results are compared with previous data to interpret the trends. In particular, the diffusive time scale is used to explain the frequency dependence observed in electroporation experiments with uni- and bi-polar pulse trains. The predictions from the current model can be harnessed to help design experiments for the further understanding and quantification of DNA electrotransfer.     

iASPPsv antagonizes apoptosis induced by chemotherapeutic agents in MCF-7 cells and mouse thymocytes

iASPP was an inhibitory member of ASPP family and could specifically inhibit the apoptotic function of p53. iASPPsv was identified by our lab as the short isoform of iASPP, which encoded a 407aa protein and highly matched the carboxyl terminus of iASPP. In this study, iASPPsv was stably transfected into the breast cancer cell line MCF-7 by means of lentivirus to explore the effects of iASPPsv on biological functions of MCF-7. Thymocytes from iASPP/iASPPsv transgenic mice were also used to explore the effects of iASPP/iASPPsv on cell biological function. The results demonstrated that iASPPsv antagonized the growth inhibition induced by etoposide (VP-16) in MCF-7 cells. iASPPsv also down-regulated proapoptotic genes (Bax, Puma and Noxa) expression to inhibit apoptosis caused by VP-16. Moreover, iASPP and iASPPsv could both help the thymocytes of transgenic mice to resist the growth inhibition and apoptosis caused by dexamethasone (Dex) or VP-16. At the same time, DNA double strand break damage accumulated in either iASPPsv MCF-7 cells or iASPP/iASPPsv thymocytes. These findings showed that iAPSS/iASPPsv reduced the growth inhibition and apoptosis induced by Dex or VP-16, with DNA damage accumulating which might promote the pathogenesis and/or progression of cancer.