Akt as a Victim,Villain and Potential Hero in Parkinson’s Disease Pathophysiology and Treatment

There are two major purposes of this essay. The first is to summarize existing evidence that irrespective of the initiating causes, neuron death and degeneration in Parkinson’s disease (PD) are due to the common feature of failure of signaling by Akt, a kinase involved in neuron survival and maintenance of synaptic contacts. The second is to consider possible means by which such a failure of Akt signaling might be benignly prevented or reversed in neurons affected by PD, so as to treat PD symptoms, block disease progression, and potentially, promote recovery.     

Bacteria associated with Artemia spp. along the salinity gradient of the solar salterns at Eilat (Israel)

The crustacean genus Artemia naturally inhabits various saline and hypersaline environments and is the most frequently laboratory-hatched animal for live feed in mari- and aquaculture. Because of its high economic importance, Artemia-bacteria interactions were so far studied mostly in laboratory strains. In this study, we focused our attention on the Artemia-associated microbiota in its natural environment in the solar salterns of Eilat, Israel. We applied a culture-independent method (clone libraries) to investigate the bacterial community structure associated with Artemia in five evaporation ponds with salinities from slightly above seawater (5%) to the point of saturation (32%), in two different developmental stages: in nauplii and in the intestine of adult animals. Bacteria found in naupliar and adult stages were classified within the Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria and Cyanobacteria. The halophilic proteobacterial genera Halomonas spp. and Salinivibrio spp. dominated the Artemia microbiota in both stages in all ponds. We also analysed a clone library of entire adult animals, revealing a novel bacterial phylogenetic lineage. This is the first molecular study of bacteria associated with two developmental stages of Artemia along a salinity gradient.     

Mechanism underlying oxidative stress-mediated lipotoxicity: exposure of J774.2 macrophages to triacylglycerols facilitates mitochondrial reactive oxygen species production and cellular necrosis

The aim of this study was to elucidate death pathways in macrophages resulting from exposure to triacylglycerols (TG), mechanisms which may be relevant to the development of atherosclerosis. A commercial TG emulsion (lipid emulsion, LE; 0.1-1.5 mg lipids/ml) was added to J774.2 cells in culture. Within the first 24 h after TG treatment, cellular reactive oxygen species (ROS) levels were strongly elevated and basal caspase-3 activity was attenuated. In contrast, after 48 h, ROS production was arrested. TG-mediated ROS production was demonstrated to be via mitochondrial complex 1 of the electron-transfer chain since the inhibitor of complex 1 rotenone significantly attenuated the cellular ROS levels in TG-treated cells. The TG effect culminated in cell death, with no caspase-3 activation. We therefore evaluated the effect of TG on apoptotic cells showing high caspase activity. TG induced elevated ROS levels and suppressed caspase-3 in apoptotic cells pretreated for 24 h with cycloheximide. Dual staining with propidium iodide and Annexin V followed by flow cytometric analysis showed that TG facilitated cell death with clear necrotic characteristics. To elucidate whether the necrotic cell death process is indeed oxidant dependent, antioxidant protection was studied. Treatment with N-acetylcysteine (NAC) (0.5 mM), ascorbic acid (0.5 mM), and resveratrol (0.2 mM) protected against the TG lipotoxic effect, while, surprisingly, lipophilic antioxidants did not. The combination of NAC, ascorbic acid, and resveratrol, each at much lower concentrations, had a synergistic protective effect. In conclusion, we show here for the first time that exposure to TG can directly regulate lipotoxicity in macrophages by inducing mitochondria-mediated prolonged oxidative stress; this, in turn, can inactivate the apoptotic caspase system, resulting in necrotic cell death which can be prevented by specific antioxidants.     

Regulation of energy liberation during steady sarcomere shortening

Energy liberation rate (E) during steady muscle shortening is a monotonic increasing or biphasic function of the shortening velocity (V). The study examines three plausible hypotheses for explaining the biphasic E-V relationship (EVR): 1) the cross-bridge (XB) turnover rate from non-force-generating (weak) to force-generating (strong) conformation decreases as V increases; 2) XB kinetics is determined by the number of strong XBs (XB-XB cooperativity); and 3) the affinity of troponin for calcium is modulated by the number of strong XBs (XB-Ca cooperativity). The relative role of the various energy-regulating mechanisms is not well defined. The hypotheses were tested by coupling calcium kinetics with XB cycling. All three hypotheses yield identical steady-state characteristics: 1) hyperbolic force-velocity relationship; 2) quasi-linear stiffness-force relationship; and 3) biphasic EVR, where E declines at high V due to decrease in the number of cycling XBs or in the weak-to-strong transition rate. The hypotheses differ in the ability to describe the existence of both monotonic and biphasic EVRs and in the effect of intracellular free calcium concentration ([Ca2+]i) on the EVR peak. Monotonic and biphasic EVRs with a shift in EVR peak to higher velocity at higher [Ca2+]i are obtained only by XB-Ca cooperativity. XB-XB cooperativity provides only biphasic EVRs. A direct effect of V on XB kinetics predicts that EVR peak is obtained at the same velocity independently of [Ca2+]i. The study predicts that measuring the dependence of the EVR on [Ca2+]i allows us to test the hypotheses and to identify the dominant energy-regulating mechanism. The established XB-XB and XB-Ca mechanisms provide alternative explanations to the various reported EVRs.     

Fine-Resolution Mapping of TF Binding and Chromatin Interactions

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ATP synthesis catalyzed by the ATPase complex from Rhodospirillum rubrum reconstituted into phospholipid vesicles together with bacteriorhodopsin

The coupling factor ATPase complex extracted by Triton X-100 from the photosynthetic bacterium Rhodospirillum rubrum could be incorporated into phospholipid vesicles after removal of the Triton. Vesicles reconstituted with this F₀ · F₁-type ATPase together with bacteriorhodopsin were found to catalyze, in the light, net ATP synthesis which was inhibited by the energy transfer inhibitors oligomycin and N,N-dicyclohexylcarbodiimide as well as by uncouplers. In vesicles reconstituted with the crude ATPase up to 50% of the observed rate of phosphorylation was independent on light and bacteriorhodopsin and insensitive to the above-listed inhibitors. This dark activity was, however, completely blocked by the adenylate kinase inhibitor, p¹,p⁵-di(adenosine-5′)pentaphosphate, which did not affect at all the net light-dependent phosphorylation nor the ATP-³²P_i exchange reaction. Vesicles reconstituted with the purified ATPase catalyzed only the light- and bacteriorhodopsin-dependent diadenosine pentaphosphate-insensitive phosphorylation. The rate of this photophosphorylation was found to be proportional to the amount of ATPase and bacteriorhodopsin, and linear for at least 20 min of illumination. These results indicate that the purified ATPase contains the complete assembly of subunits required to transduce electrochemical gradient energy into chemical energy.     

Environmental implications of skeletal micro-density and porosity variation in two scleractinian corals

The correlations between skeletal parameters (bulk density, micro-density and porosity), coral age and sea surface temperature were assessed along a latitudinal gradient in the zooxanthellate coral Balanophyllia europaea and in the azooxanthellate coral Leptopsammia pruvoti. In both coral species, the variation of bulk density was more influenced by the variation of porosity than of micro-density. With increasing polyp age, B. europaea formed denser and less porous skeletons while L. pruvoti showed the opposite trend, becoming less dense and more porous. B. europaea skeletons were generally less porous (more dense) than those of L. pruvoti, probably as a consequence of the different habitats colonized by the two species. Increasing temperature had a negative impact on the zooxanthellate species, leading to an increase of porosity. In contrast, micro-density increased with temperature in the azooxanthellate species. It is hypothesized that the increase in porosity with increasing temperatures observed in B. europaea could depend on an attenuation of calcification due to an inhibition of the photosynthetic process at elevated temperatures, while the azooxanthellate species appears more resistant to variations of temperature, highlighting possible differences in the sensitivity/tolerance of these two coral species to temperature changes in face of global climate change.     

Cbp1, a fungal virulence factor under positive selection,forms an effector complex that drives macrophage lysis

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We observe a high rate of fixation of non-synonymous substitutions in the Cbp1 coding sequences, indicating that Cbp1 is under positive selection. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel “binocular” fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a fungal virulence factor under positive selection that localizes to the cytosol to trigger host cell lysis.