Gα12 Inhibits α2β1 Integrin–mediated Madin-Darby Canine Kidney Cell Attachment and Migration on Collagen-I and Blocks Tubulogenesis

Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that Gα12 inhibits interaction of MDCK cells with collagen-I, the major ligand for α2β1 integrin. Activating Gα12 (QL point mutation or stimulating endogenous Gα12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with Gα12-regulated attachment to collagen-I, Gα12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in Gα12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. Gα12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of α2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated Gα12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, Gα12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for Gα12–integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.     

The influence of historical landscape change on genetic variation and population structure of a terrestrial salamander (Plethodon cinereus)

Forest loss and fragmentation is expected to shape the genetic structure of amphibian populations and reduce genetic variation. Another factor widely understood to have impacted these same parameters in North America is the range expansion that occurred following glacial retreat at the end of the Pleistocene. The Eastern Red-Backed Salamander (Plethodon cinereus) has been subjected to both processes. In this context, we investigated the historical events that are likely to have shaped genetic variation in this species using a panel of six microsatellite markers screened on individuals sampled across ten localities in northeastern Indiana, USA. We found low genetic diversity across forest patches and minimal differentiation. We expected population structure associated with forest fragmentation to result from genetic drift in isolation but instead found that a balance between gene flow and drift was ~50 times more likely. Ratios of allele number and range (M), and coalescent modeling of population demography suggested the occurrence of marked historic decline in effective population size across the region. Taken together, the data point to a loss of genetic variation which preceded deforestation over the past 200 years. This result indicates an important role for ancient demographic processes in shaping current genetic variation that may make it difficult to detect the effect of recent habitat fragmentation.     

The open state gating mechanism of gramicidin a requires relative opposed monomer rotation and simultaneous lateral displacement

The gating mechanism of the open state of the gramicidin A (gA) channel is studied by using a new Monte Carlo Normal Mode Following (MC-NMF) technique, one applicable even without a target structure. The results demonstrate that the lowest-frequency normal mode (NM) at approximately 6.5 cm(-1) is the crucial mode that initiates dissociation. Perturbing the gA dimer in either direction along this NM leads to opposed, nearly rigid-body rotations of the gA monomers around the central pore axis. Tracking this NM by using the eigenvector-following technique reveals the channel's gating mechanism: dissociation via relative opposed monomer rotation and simultaneous lateral displacement. System evolution along the lowest-frequency eigenvector shows that the large-amplitude motions required for gating (dissociation) are not simple relative rigid-body motions of the monomers. Gating involves coupling intermonomer hydrogen bond breaking, backbone realignment, and relative monomer tilt with complex side chain reorganization at the intermonomer junction.     

Depletion of a single nucleoporin, Nup107, induces apoptosis in eukaryotic cells

Nuclear pores are large protein complexes that cross the nuclear envelope, which is the double membrane surrounding the eukaryotic cell nucleus. There are about on average 2,000 nuclear pore complexes (NPCs) in the nuclear envelope of a vertebrate cell, but it varies depending on cell type and the stage in the life cycle. The proteins that make up the NPC are known as nucleoporins. In mammalian cells, Nup107 is the homolog of yeast Nup84p nucleoporin. Nup107 contains a leucine zipper motif in its carboxyl-terminal region and numerous kinase consensus sites, but does not contain FG repeats. Previously it was reported that NUP88 and NUP107 are over expressed in many types of cancers including colon, breast, prostrate, etc. In this study, we were interested in investigating the role of NUP107 in grade 4 Astrocytoma, i.e., Glioblastoma multiforme cultured cell line. We transfected human Astrocytoma cells with Nup107-specific siRNA duplexes. The level of mRNA for Nup107 was monitored by RT-PCR, 24, 48, and 72 h after the initial transfection. Nup107 mRNA was significantly diminished by 24 h after transfection and we took that as our incubation time. Next we studied the effect of this inhibition on cell viability. We did a Trypan Blue cell viability assay and it showed increased cell death in NUP107 transfected cells than untreated control. We further tried to analyze the nature of the cell death whether apoptotic or necrotic by doing apoptosis assays like ssDNA ELISA assay, Caspase-3, and Caspase-8 assays. All the assays showed that siRNA transfected cells are undergoing increased apoptosis than control.     

Comparison of methods for measuring oxygen consumption in tumor cells in vitro

The oxygen consumption rate of tumor cells affects tumor oxygenation and response to therapies. Highly sensitive methods for determining cellular oxygen consumption are, therefore, needed to identify treatments that can modulate this parameter. We compared the performances of three different methods for measuring cellular oxygen consumption: electron paramagnetic resonance (EPR) oximetry, the Clark electrode, and the MitoXpress fluorescent assay. To compare the assays, we used K562 cells in the presence of rotenone and hydrocortisone, compounds that are known to inhibit the mitochondrial electron transport chain to different extents. The EPR method was the only one that could identify both rotenone and hydrocortisone as inhibitors of tumor cell oxygen consumption. The Clark electrode and the fluorescence assay demonstrated a significant decrease in cellular oxygen consumption after administration of the most potent inhibitor (rotenone) but failed to show any significant effect of hydrocortisone. EPR oximetry is, therefore, the most sensitive method for identifying inhibitors of oxygen consumption on cell assays, whereas the Clark electrode offers the unique opportunity to add external compounds during experiments and still shows great sensitivity in studying enzyme and chemical reactions that consume oxygen (non-cell assays). Finally, the MitoXpress fluorescent assay has the advantage of a high-sample throughput and low bulk requirements but at the cost of a lower sensitivity.     

Triterpenoidal alkaloids from Buxus hyrcana and their enzyme inhibitory,anti-fungal and anti-leishmanial activities

From the aerial parts of Buxus hyrcana, three triterpenoidal alkaloids, 17-oxo-3-benzoylbuxadine (1), buxhyrcamine (2), and 31-demethylcyclobuxoviridine (3), along with 16 known compounds, cyclobuxoviridine (4), N_b-dimethylcyclobuxoviricine (5), E-buxenone (6), Z-buxenone (7), moenjodaramine (8), homomoenjodarmine (9), buxamine A (10), buxamine B (11), 31-hydroxybuxamine B (12), N₂₀-formylbuxaminol E (13), papillozine C (14), buxmicrophylline F (15), buxrugulosamine (16), cyclobuxophylline O (17), spirofornabuxine (18) and arbora-1,9(11)-dien-3-one (19) were isolated. Their structures were elucidated by using NMR spectroscopic methods. All of the compounds exhibited moderate to weak acetylcholinesterase, butyrylcholinesterase and glutathione S-transferase inhibitory activities. Compounds 1–19 also exhibited modest anti-fungal activities against Candida albicans. Compounds 1, 2, 8, 9 and 18 also exhibited weak anti-leishmanial activity.     

Oxo-bridged isomers of aza-trishomocubane sigma (σ) receptor ligands: Synthesis,in vitro binding,and molecular modeling

Isomeric oxo-bridged analogs of aza-trishomocubane sigma (σ) receptor ligands were synthesized and shown to display a reduced affinity for the σ receptor. In the case of phenethyl derivative 4, there was a concomitant introduction of high-affinity for the α_2C adrenergic receptor, and moderate affinity for the dopamine transporter. Molecular modeling was undertaken to rationalize these results.     

Your place or mine? A phylogenetic comparative analysis of marital residence in Indo-European and Austronesian societies

Accurate reconstruction of prehistoric social organization is important if we are to put together satisfactory multidisciplinary scenarios about, for example, the dispersal of human groups. Such considerations apply in the case of Indo-European and Austronesian, two large-scale language families that are thought to represent Neolithic expansions. Ancestral kinship patterns have mostly been inferred through reconstruction of kin terminologies in ancestral proto-languages using the linguistic comparative method, and through geographical or distributional arguments based on the comparative patterns of kin terms and ethnographic kinship 'facts'. While these approaches are detailed and valuable, the processes through which conclusions have been drawn from the data fail to provide explicit criteria for systematic testing of alternative hypotheses. Here, we use language trees derived using phylogenetic tree-building techniques on Indo-European and Austronesian vocabulary data. With these trees, ethnographic data and Bayesian phylogenetic comparative methods, we statistically reconstruct past marital residence and infer rates of cultural change between different residence forms, showing Proto-Indo-European to be virilocal and Proto-Malayo-Polynesian uxorilocal. The instability of uxorilocality and the rare loss of virilocality once gained emerge as common features of both families.     

Demography of an endangered endemic rupicolous cactus

The snow cover extent is an important factor for the structure and composition of arctic and alpine tundra communities. Over the last few decades, snowmelt in many arctic and alpine regions has advanced, causing the growing season to start earlier and last longer. In a field experiment in subarctic tundra in Interior Alaska, I manipulated the timing of snowmelt and measured the response in mortality, phenology, growth, and reproduction of the eight dominant plant species. I then tested whether the phenological development of these species was controlled by snowmelt date or by temperature (in particular growing degree days, GDD). In order to expand our understanding of plant sensitivity to snowmelt timing, I explored whether the response patterns can be generalized with regard to the temporal niche of each species. Differences in the phenology between treatments were only found for the first stages of the phenological development (=phenophases). The earlier the temporal niche (i.e., the sooner after snowmelt a species develops) the more its phenology was sensitive to snowmelt. Later phenophases were mostly controlled by GDD, especially in late-developing species. In no species did an earlier snowmelt and a longer growing season directly enhance plant fitness or fecundity, in spite of the changes in the timing of plant development. In conclusion, the temporal niche of a species’ phenological development could be a predictor of its response to snowmelt timing. However, only the first phenophases were susceptible to changes in snowmelt, and no short-term effects on plant fitness were found.     

A technique using reusable components for hand-infesting cornstalks with European corn borer (Lepidoptera: Crambidae) larvae

Field trials were conducted in 2005 and 2006 to evaluate the use of reusable wire nuts and nonreusable gelatin capsules for hand-infesting cornstalks with European corn borer, Ostrinia nubilalis (Hübner), (Lepidoptera: Crambidae) larvae. The reusable technique, which consists of a modified WingGard plastic wire connector (i.e., wire nut) as a containment device for larvae, was compared over three plant growth stages to a gelatin capsule technique. In 2005 and 2006, the wire nut technique resulted in significantly higher number of wire nuts still intact (i.e., undamaged, with or without a larva) on the stalk at 72 h after infestation compared with the gelatin capsule technique. In addition, the wire nut technique resulted in significantly higher number of tunnels per stalk compared with the gelatin capsule technique at all three corn growth stages during both years. In 2005, the mean +/- SEM number of tunnels per stalk was 0.53 +/- 0.03 in the wire nut technique compared with 0.13 +/- 0.03 tunnels per stalk in the gelatin capsule technique. In 2006, the mean number of tunnels per stalk was 0.45 +/- 0.03 in the wire nut technique compared with 0.08 +/- 0.02 tunnels per stalk in the gelatin capsule technique. In addition, the relative net precision in the wire-nut technique was approximately 2 times higher compared with the gelatin capsule technique.