Early Activation of STAT3 Regulates Reactive Astrogliosis Induced by Diverse Forms of Neurotoxicity

Astrogliosis, a cellular response characterized by astrocytic hypertrophy and accumulation of GFAP, is a hallmark of all types of central nervous system (CNS) injuries. Potential signaling mechanisms driving the conversion of astrocytes into “reactive” phenotypes differ with respect to the injury models employed and can be complicated by factors such as disruption of the blood-brain barrier (BBB). As denervation tools, neurotoxicants have the advantage of selective targeting of brain regions and cell types, often with sparing of the BBB. Previously, we found that neuroinflammation and activation of the JAK2-STAT3 pathway in astrocytes precedes up regulation of GFAP in the MPTP mouse model of dopaminergic neurotoxicity. Here we show that multiple mechanistically distinct mouse models of neurotoxicity (MPTP, AMP, METH, MDA, MDMA, KA, TMT) engender the same neuroinflammatory and STAT3 activation responses in specific regions of the brain targeted by each neurotoxicant. The STAT3 effects seen for TMT in the mouse could be generalized to the rat, demonstrating cross-species validity for STAT3 activation. Pharmacological antagonists of the neurotoxic effects blocked neuroinflammatory responses, pSTAT3^tyr705 and GFAP induction, indicating that damage to neuronal targets instigated astrogliosis. Selective deletion of STAT3 from astrocytes in STAT3 conditional knockout mice markedly attenuated MPTP-induced astrogliosis. Monitoring STAT3 translocation in GFAP-positive cells indicated that effects of MPTP, METH and KA on pSTAT3^tyr705 were localized to astrocytes. These findings strongly implicate the STAT3 pathway in astrocytes as a broadly triggered signaling pathway for astrogliosis. We also observed, however, that the acute neuroinflammatory response to the known inflammogen, LPS, can activate STAT3 in CNS tissue without inducing classical signs of astrogliosis. Thus, acute phase neuroinflammatory responses and neurotoxicity-induced astrogliosis both signal through STAT3 but appear to do so through different modules, perhaps localized to different cell types.     

The Detection of Inherent Homologous Recombination Between Repeat Sequences in H. pylori 26695 by the PCR-Based Method

Helicobacter pylori infects more than half of the world’s population, making it the most widespread infection of bacteria. It has high genetic diversity and has been considered as one of the most variable bacterial species. In the present study, a PCR-based method was used to detect the presence and the relative frequency of homologous recombination between repeat sequences (>500 bp) in H. pylori 26695. All the recombinant structures have been confirmed by sequencing. The inversion generated between inverted repeats showed distinct features from the recombination for duplication or deletion between direct repeats. Meanwhile, we gave the mathematic reasoning of a general formula for the calculation of relative recombination frequency and indicated the conditions for its application. This formula could be extensively applied to detect the frequency of homologous recombination, site-specific recombination, and other types of predictable recombination. Our results should be helpful for better understanding the genome evolution and adaptation of bacteria.     

Thrombin potentiates d‐aspartate efflux from cultured astrocytes under conditions of K+ homeostasis disruption

Thrombin levels increase in brain during ischemia and hemorrhagic episodes, and may contribute to excitotoxic neural damage. This study examined the effect of thrombin on glutamate efflux from rat cortical cultured astrocytes using ³H‐d ‐aspartate as radiotracer. The glutamate efflux was initiated by addition of 100 mM K⁺ plus 1 mM ouabain (K/O) to replicate extracellular and intracellular ionic changes that occur during cerebral ischemia. Upon exposure to K/O, astrocytes swelled slowly and progressively with no evidence of volume regulation. The K/O‐induced swelling was inhibited by 65% with bumetanide and 25% with BaCl₂, suggesting contribution of Na⁺/K⁺/Cl^? co‐transporter and Kir channels. K/O‐elicited ³H‐d ‐aspartate that consisted of two phases. The first transient component of the release corresponded to 13.5% of total ³H‐d ‐aspartate loaded. It was markedly reduced (61%) by the glutamate transporter blocker DL‐threo‐b‐Benzyloxyaspartic acid and weakly inhibited (21%) by the volume‐sensitive anion channel blocker 4‐[(2‐Butyl‐6,7dichloro‐2‐cyclopentyl‐2,3‐dihidro‐1oxo‐1H‐inden‐5‐yl)oxy] butanoic acid (DCPIB). During the second sustained phase of release, cells lost 45% of loaded of ³H‐d ‐aspartate via a mechanism that was insensitive to DL‐threo‐b‐Benzyloxyaspartic acid but nearly completely suppressed by DCPIB. Thrombin (5 U/mL) had only marginal effects on the first phase but strongly potentiated (more than two‐fold) ³H‐d ‐aspartate efflux in the second phase. The effect of thrombin effect was proportional to cell swelling and completely suppressed by DCPIB. Overall our data showed that under K/O swelling conditions, thrombin potently enhance glutamate release via volume‐sensitive anion channel. Similar mechanisms may contribute to brain damage in neural pathologies which are associated with cell swelling, glutamate efflux and increased thrombin levels.     

Inhibition of the yeast-mycelial transition and the phorogenesis of Mucorales by diamino butanone

Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.Abbreviations ODC ornithine decarboxylase - DAB 1,4-diamino butanone     

Inhibition of the yeast-mycelial transition and the phorogenesis of Mucorales by diamino butanone

Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.     

Symmetric array of the urothelium surface controlled by the lipid lattice composition

Proteins on the luminal surface of the mammalian membrane urothelium form an hexagonal two-dimensional (2D) lattice of approximately 12nm particles embedded in a lipid matrix. The establishment and/or maintenance of this two-dimensional lattice have not yet been elucidated. To understand the lipid involvement in these effects, which may have a potential role on the permeability barrier, the lattice structural changes induced by different dietary fatty acids were analyzed. Image analysis of the uranyl formate stained urothelial membrane from mice fed with a commercial diet (control) or a formula containing 5% (w/w) corn oil (CO) or olein (O) and our previous data of fluorescence anisotropy indicated that the olein diet profoundly alters the two-dimensional particle network. Besides the increase from 15nm (control) to 17nm center-to-center particle, a statistically significant increase of the particle size was induced by the olein diet. The same parameters on urothelial membranes from animals fed with a corn diet differed on the long-range super-array from the control values. A specific structural change of the urothelial plaque particle organization as a response to changes of lipid composition is described for the first time. These results are a starting point for further research of the lipid implications on the permeability barrier.     

Geographical variation in the use of intertidal rocky shores by the lizard Microlophus atacamensis in relation to changes in terrestrial productivity along the Atacama Desert coast

1. The movement of materials and organisms between ecosystems is a common process in nature. 2. In the present study we investigate the hypothesis that the movement of consumers between ecosystems depends not only on the differences in productivity between ecosystems and prey availability, but also on these animals' biological characteristics. 3. To address this hypothesis we investigated the changes in abundance, habitat utilization and diet of the lizard Microlophus atacamensis along its geographical range on the coast of the Atacama Desert. Within this range, intertidal rocky shore communities do not show important variations in their species composition and abundance, but terrestrial communities show a steep gradient of productivity associated with the increase in rainfall from north to south. 4. Our results show that the use of intertidal habitats and the consumption of intertidal prey by M. atacamensis change within its geographical range: in the North, the species uses intertidal areas and behaves as an herbivore consuming mostly algae, whereas in the South it expends most of the time in terrestrial habitats as a carnivore mainly of arthropods. 5. Our study gives new evidence for cross-ecosystem connections created by consumer movement between habitats of contrasting but variable productivity levels.     

Myxoma Virus Oncolytic Efficiency Can Be Enhanced Through Chemical or Genetic Disruption of the Actin Cytoskeleton

Myxoma virus (MYXV) is one of many animal viruses that exhibit oncolytic properties in transformed human cells. Compared to orthopoxviruses like vaccinia (VACV), MYXV spreads inefficiently, which could compromise its use in treating tumors and their associated metastases. The VACV F11 protein promotes virus exit and rapid spread by inhibiting Rho signalling, which results in a disruption of cortical actin. We have previously shown that although MYXV lacks an F11 homolog, the F11L gene can be introduced into MYXV promoting the spread of this Leporipoxvirus in natural host cells. Here we show that the F11-encoding (F11L⁺) MYXV strain replicates to higher levels in a number of human cancer cells. We also show that F11L⁺ MYXV induces better tumor control and prolonged survival of mice bearing MDA-MB-231 cancer cells. Furthermore, we show that this virus also spreads more efficiently from the site of growth in one injected tumor, to a second untreated tumor.While we focused mostly on the use of a modified MYXV we were able to show that the effects of F11 on MYXV growth in cancer cells could be mimicked through the use of pharmacological inhibition or siRNA-mediated silencing of key regulators of cortical actin (RhoA, RhoC, mDia1, or LIMK2). These data suggest that it may be possible to increase the oncolytic efficacy of wild-type MYXV using chemical inhibitors of RhoA/C or their downstream targets. Furthermore, since all viruses must overcome barriers to exit posed by structures like cortical actin, these findings suggest that the oncolytic activity of other viruses may be enhanced through similar strategies.