BTK-2, a new inhibitor of the Kv1.1 potassium channel purified from Indian scorpion Buthus tamulus

A novel inhibitor of voltage-gated potassium channel was isolated and purified to homogeneity from the venom of the red scorpion Buthus tamulus. The primary sequence of this toxin, named BTK-2, as determined by peptide sequencing shows that it has 32 amino acid residues with six conserved cysteines. The molecular weight of the toxin was found to be 3452 Da. It was found to block the human potassium channel hKv1.1 (IC(50)=4.6 microM). BTK-2 shows 40-70% sequence similarity to the family of the short-chain toxins that specifically block potassium channels. Multiple sequence alignment helps to categorize the toxin in the ninth subfamily of the K+ channel blockers. The modeled structure of BTK-2 shows an alpha/beta scaffold similar to those of the other short scorpion toxins. Comparative analysis of the structure with those of the other toxins helps to identify the possible structure-function relationship that leads to the difference in the specificity of BTK-2 from that of the other scorpion toxins. The toxin can also be used to study the assembly of the hKv1.1 channel.     

Slit protein-mediated inhibition of CXCR4-induced chemotactic and chemoinvasive signaling pathways in breast cancer cells

Slit, which mediates its function by binding to the Roundabout (Robo) receptor, has been shown to regulate neuronal and CXCR4-mediated leukocyte migration. Slit-2 was shown to be frequently inactivated in lung and breast cancers because of hypermethylation of its promoter region. Furthermore, the CXCR4/CXCL12 axis has been reported recently to be actively involved in breast cancer metastasis to target organs such as lymph nodes, lung, and bone. In this study, we sought to characterize the effect of Slit (=Slit-2) on the CXCL12/CXCR4-mediated metastatic properties of breast cancer cells. We demonstrate here that breast cancer cells and tissues derived from breast cancer patients express Robo 1 and 2 receptors. We also show that Slit treatment inhibits CXCL12/CXCR4-induced breast cancer cell chemotaxis, chemoinvasion, and adhesion, the fundamental components that promote metastasis. Slit had no significant effect on the CXCL12-induced internalization process of CXCR4. In addition, characterization of signaling events revealed that Slit inhibits CXCL12-induced tyrosine phosphorylation of focal adhesion components such as RAFTK/Pyk2 at residues 580 and 881, focal adhesion kinase at residue 576, and paxillin. We found that Slit also inhibits CXCL12-induced phosphatidylinositol 3-kinase, p44/42 MAP kinase, and metalloproteinase 2 and 9 activities. However, it showed no effect on JNK and p38 MAP kinase activities. To our knowledge, this is the first report to analyze in detail the effect of Slit on breast cancer cell motility as well as its effect on the critical components of the cancer cell chemotactic machinery. Studies of the Slit-Robo complex may foster new anti-chemotactic approaches to block cancer cell metastasis.     

Evolutionary origin of the medaka Y chromosome

Genetic sex determination in an XX-XY chromosome system can be realized through a locus on the Y chromosome that makes the undifferentiated gonad develop into a testis. Although this mechanism is widespread, only in two cases so far have the corresponding master male sex-determining genes been identified. One is Sry, which initiates testes determination in most mammals. The other is dmrt1bY (syn. dmy), from the fish medaka, Oryzias latipes. The mammalian Y is roughly estimated to be over 200 million years old. The medaka Y may be considerably younger. A comparative analysis of the genus Oryzias revealed that one sister species of the medaka has dmrt1bY on a homologous Y chromosome, whereas in another closely related species only a non-sex-linked pseudogene is present. In all other species, dmrt1bY was not detected. The divergence time for the different species was determined with mitochondrial DNA sequences. The timing was confirmed by independent calculations based on dmrt1 sequences. We show that the medaka sex-determining gene originated approximately 10 million years ago. This makes dmrt1bY and the corresponding Y chromosome the youngest male sex-determining system, at least in vertebrates, known so far.     

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.     

Nuclear import and export signals in control of Nrf2

Nrf2 binds to the antioxidant response element and regulates expression and antioxidant induction of a battery of chemopreventive genes. In this study, we have identified nuclear import and export signals of Nrf2 and show that the nuclear import and export of Nrf2 is regulated by antioxidants. We demonstrate that Nrf2 contains a bipartite nuclear localization signal (NLS) and a leucine-rich nuclear export signal, which regulate Nrf2 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nrf2 accumulates in the nucleus within 15 min of antioxidant treatment and is exported out of nucleus by 8 h after treatment. Nrf2 mutant lacking the NLS failed to enter the nucleus and displayed diminished expression and induction of the downstream NAD(P)H:quinone oxidoreductase 1 gene. The Nrf2 NLS sequence, when fused to green fluorescence protein, resulted in the nuclear accumulation of green fluorescence protein, indicating that this signal sequence was sufficient to direct nuclear localization of Nrf2. A nuclear export signal (NES) was characterized in the C terminus of Nrf2, the deletion of which caused Nrf2 to accumulate predominantly in the nucleus. The Nrf2 NES was sensitive to leptomycin B and could function as an independent export signal when fused to a heterologous protein. Further studies demonstrate that NES-mediated nuclear export of Nrf2 is required for degradation of Nrf2 in the cytosol. These results led to the conclusion that Nrf2 localization between cytosol and nucleus is controlled by both nuclear import and export of Nrf2, and the overall distribution of Nrf2 is probably the result from a balance between these two processes. Antioxidants change this balance in favor of nuclear accumulation of Nrf2, leading to activation of chemopreventive proteins. Once this is achieved, Nrf2 exits the nucleus for binding to INrf2 and degradation.     

Evaluation of cyclooxygenase 2 derived endogenous prostacyclin in mouse preimplantation embryo development in vitro

Cyclooxygenase (COX) plays an important role in prostaglandin (PG) synthesis and has two isoforms, COX1 and COX2. PGI synthase (PGIS) catalyzes the isomeization of PGH(2) to prostacyclin (PGI(2)). It is reported that COX2 derived PGI2(2) plays a critical role in blastocyst implantation and decidualization and PGI2 mediates its function via PPARdelta receptor. It is also known that cyclooxygenase derived prostaglandins play an important role in mouse blastocyst hatching in vitro. In this study we hypothesized that COX2 derived PGI2 plays an important role in preimplantation embryonic development by increasing the cell number. To examine this hypothesis, 8-cell stage mouse embryos were cultured in the presence of selective inhibitors of COX1 (SC560), COX2 (NS398) and PGIS (U51605) respectively. COX2 and PGIS inhibitor significantly reduced the blastocyst development and presence of PGI2 analogue along with these inhibitors restored the blastocyst development by increasing the total number of embryonic cells. Our immunohistochemical analysis showed that COX1 is expressed at 2-cell, 8-cell, compaction and blastocyst stage whereas COX2 expression starts from eight cell stage embryos. PGIS and PPARdelta expression starts at 2-cell stage of development. Our results suggest that PGI(2) may affect blastomeres number via the so called hypothesis of PPARdelta nuclear receptor in autocrine manner.     

Influence of Nutrient Availability and Quorum Sensing on the Formation of Metabolically Inactive Microcolonies Within Structurally Heterogeneous Bacterial Biofilms: An Individual-Based 3D Cellular Automata Model

The resistance of bacterial biofilms to antibiotic treatment has been attributed to the emergence of structurally heterogeneous microenvironments containing metabolically inactive cell populations. In this study, we use a three-dimensional individual-based cellular automata model to investigate the influence of nutrient availability and quorum sensing on microbial heterogeneity in growing biofilms. Mature biofilms exhibited at least three structurally distinct strata: a high-volume, homogeneous region sandwiched between two compact sections of high heterogeneity. Cell death occurred preferentially in layers in close proximity to the substratum, resulting in increased heterogeneity in this section of the biofilm; the thickness and heterogeneity of this lowermost layer increased with time, ultimately leading to sloughing. The model predicted the formation of metabolically dormant cellular microniches embedded within faster-growing cell clusters. Biofilms utilizing quorum sensing were more heterogeneous compared to their non-quorum sensing counterparts, and resisted sloughing, featuring a cell-devoid layer of EPS atop the substratum upon which the remainder of the biofilm developed. Overall, our study provides a computational framework to analyze metabolic diversity and heterogeneity of biofilm-associated microorganisms and may pave the way toward gaining further insights into the biophysical mechanisms of antibiotic resistance.     

Up-regulation of Cavβ3 subunit in primary sensory neurons increases voltage-activated Ca2+ channel activity and nociceptive input in neuropathic pain

High voltage-activated calcium channels (HVACCs) are essential for synaptic and nociceptive transmission. Although blocking HVACCs can effectively reduce pain, this treatment strategy is associated with intolerable adverse effects. Neuronal HVACCs are typically composed of α(1), β (Cavβ), and α(2)δ subunits. The Cavβ subunit plays a crucial role in the membrane expression and gating properties of the pore-forming α(1) subunit. However, little is known about how nerve injury affects the expression and function of Cavβ subunits in primary sensory neurons. In this study, we found that Cavβ(3) and Cavβ(4) are the most prominent subtypes expressed in the rat dorsal root ganglion (DRG) and dorsal spinal cord. Spinal nerve ligation (SNL) in rats significantly increased mRNA and protein levels of the Cavβ(3), but not Cavβ(4), subunit in the DRG. SNL also significantly increased HVACC currents in small DRG neurons and monosynaptic excitatory postsynaptic currents of spinal dorsal horn neurons evoked from the dorsal root. Intrathecal injection of Cavβ(3)-specific siRNA significantly reduced HVACC currents in small DRG neurons and the amplitude of monosynaptic excitatory postsynaptic currents of dorsal horn neurons in SNL rats. Furthermore, intrathecal treatment with Cavβ(3)-specific siRNA normalized mechanical hyperalgesia and tactile allodynia caused by SNL but had no significant effect on the normal nociceptive threshold. Our findings provide novel evidence that increased expression of the Cavβ(3) subunit augments HVACC activity in primary sensory neurons and nociceptive input to dorsal horn neurons in neuropathic pain. Targeting the Cavβ(3) subunit at the spinal level represents an effective strategy for treating neuropathic pain.     

Secretome analysis of thermophilic mould Myceliophthora thermophila cultivated on rice straw and hydrolysis of lignocellulosic biomass for bioethanol production

Abstract

Myceliophthora thermophila encodes for large number of carbohydrate-active enzymes (CAZymes) involved in lignocellulosic biomass degradation. The mould was grown on rice straw in solid state fermentation at pH 5.0 and 45?°C that produced high levels of cellulolytic and xylanolytic enzymes i.e. 2218.12, 515.23, 478.23, 13.34?U/g DMR for xylanase, CMCase, FPase and β-glucosidase, respectively. The secretome analysis of M. thermophila BJAMDU5 by mass spectroscopy, described 124 different proteins with majority of CAZymes consisting of glycosyl hydrolases (GH), lytic polysaccharide mono-oxygenases (LPMO), carbohydrate esterases (CE) and polysaccharide lyases (PL). Furthermore, the enzyme cocktail of the mould was evaluated for hydrolysis of steam treated rice straw that produced 184.59?mg/g substrate reducing sugars after 24?h, which was used for production of bioethanol by using fast fermenting yeast Saccharomyces cerevisiae resulting in high production of bioethanol.     

Lentiviral reporter constructs for fluorescence tracking of the temporospatial pattern of Smad3 signaling

Canonical TGF-beta is involved in cell differentiation, tissue maintenance, and wound healing, but also plays a central role in the pathogenesis of diseases such as cancer Here we describe a lentivirus-based reporter vector system expressing green fluorescent protein (GFP) or red fluorescent protein (RFP) under the control of a Smad3-responsive element (CAGA)12 that allows observation of the temporospatial pattern of endogeneous Smad3-mediated signaling on a cellular level. Use of this method will be valuable to identify cells with active Smad3 signaling and investigate the role of endogenous Smad3 signaling in complex systems such as co-cultures in vitro, or in tumors during tumor cell invasion and metastasis in vivo.