Early apoptosis-related changes triggered by HSV-1 in individual neuronlike cells

Early events of apoptosis following HSV-1 infection were investigated at the single-cell level using intensified fluorescence digital-imaging microscopy. The results provide evidence that infection of differentiated ND7 neuronlike cells by HSV-1 triggers detectable alterations indicative of physiological changes associated with the early stages of apoptosis. Less than 1 h after infection with HSV-1 (KOS strain) or K26GFP (GFP being fused to HSV-1 capsid protein VP26) we observed (i) moderate decrease in mitochondrial membrane potential (about 20%), (ii) exposure of phosphatidyl serine, (iii) morphological change in the mitochondria that became spherical instead of filamentous, and (iv) activation of caspase-8. Within 3 h changes reverted to normal, which indicated that apoptosis was counteracted very early following HSV-1 infection. Similar results were obtained with KOS-TK27GFP, lacking TK and UL24 proteins, suggesting that TK and UL24 play no role in apoptosis. In Vero cells mitochondrial changes characteristic of the apoptotic process were not observed following HSV-1 infection. The UV-inactivated K26GFP had the capacity to induce apoptosis in neuronlike cells. This real-time multiparametric analysis, in combination with relevant viral mutants, could be a useful approach for dissecting the roles of various viral genes in modulating apoptotic pathways during infection.     

Molecular characterization influencing metal resistance in the Cupriavidus/Ralstonia genomes

Our environment is stressed with a load of heavy and toxic metals. Microbes, abundant in our environment, are found to adapt well to this metal-stressed condition. A comparative study among five Cupriavidus/Ralstonia genomes can offer a better perception of their evolutionary mechanisms to adapt to these conditions. We have studied codon usage among 1051 genes common to all these organisms and identified 15 optimal codons frequently used in highly expressed genes present within 1051 genes. We found the core genes of Cupriavidus metallidurans CH34 have a different optimal codon choice for arginine, glycine and alanine in comparison with the other four bacteria. We also found that the synonymous codon usage bias within these 1051 core genes is highly correlated with their gene expression. This supports that translational selection drives synonymous codon usage in the core genes of these genomes. Synonymous codon usage is highly conserved in the core genes of these five genomes. The only exception among them is C. metallidurans CH34. This genomewide shift in synonymous codon choice in C. metallidurans CH34 may have taken place due to the insertion of new genes in its genomes facilitating them to survive in heavy metal containing environment and the co-evolution of the other genes in its genome to achieve a balance in gene expression. Structural studies indicated the presence of a longer N-terminal region containing a copper-binding domain in the cupC proteins of C. metallidurans CH3 that helps it to attain higher binding efficacy with copper in comparison with its orthologs.     

Studies of ice melting using molecular dynamics

In this paper, the melting of ice 1h is studied using molecular dynamics (MD). Common potential functions employed in the MD simulations include SPC/E, TIP4P, TIP5P, TIP4/ice and TIP5P/E. We first conducted melting of ice bulks and then studied the melting speed of the ice/water interface during ice melting. It is found that various potentials result in similar ice-melting phenomena. The result is compared with the analytical solution for phase change problem. We also studied size effects and temperature gradient effects on ice melting.     

Membrane potential responses controlling chemodispersal of Paramecium caudatum from quinine

Membrane potential responses of a ciliate protozoan Paramecium caudatum to the external application of quinine were investigated in relation to its motile activities. Wild-type specimens swimming in the reference solution did not enter into a quinine-containing (0.5 mM) test solution due to avoiding responses exhibited at the border between the two solutions, and therefore stayed in the reference solution (chemodispersal). Squirting of a quinine-containing test solution over a wild-type specimen evoked a train of action potentials superimposed on a depolarizing chemoreceptor potential. Squirting of a quinine-containing test solution over a CNR-mutant specimen defective in voltage-gated Ca²⁺ channel evoked only chemoreceptor potentials, which consisted of an initial transient depolarization, a following transient hyperpolarization and a sustained depolarization. A current-evoked action potential became larger in its amplitude and longer in its duration with the external application of quinine. Under the voltage-clamp condition, the fast inward current did not change whereas the delayed outward current decreased with the external application of quinine. It is concluded that quinine is a potent repellent for Paramecium because it produces a depolarizing chemoreceptor potential which evokes action potentials and prolongs the duration of the action potential.     

Dysfunction of rat liver mitochondria by selenite: induction of mitochondrial permeability transition through thiol-oxidation

Selenium is an essential trace element in mammals and is thought to play a chemopreventive role in human cancer, possibly by inducing tumor cell apoptosis. Mitochondria play a pivotal role in the induction of apoptosis in many cell types. The effects of selenite on mitochondrial function were therefore investigated. Selenite induced the oxidation and cross-linking of protein thiol groups, mitochondrial permeability transition (MPT), a decrease in the mitochondrial membrane potential, and the release of cytochrome c in mitochondria isolated from rat liver. Induction of the MPT by selenite was prevented by cyclosporin A, EGTA, or N-ethylmaleimide. These results thus indicate that selenite induces the MPT as a result of direct modification of protein thiol groups, resulting in the release of cytochrome c and a loss of mitochondrial membrane potential.     

The effects of garlic-derived sulfur compounds on cell proliferation, caspase 3 activity, thiol levels and anaerobic sulfur metabolism in human hepatoblastoma HepG2 cells

The aim of the present studies was to determine whether the mechanism of biological action of garlic-derived sulfur compounds in human hepatoma (HepG2) cells can be dependent on the presence of labile sulfane sulfur in their molecules. We investigated the effect of allyl sulfides from garlic: monosulfide, disulfide and trisulfide on cell proliferation and viability, caspase 3 activity and hydrogen peroxide (H(2)O(2)) production in HepG2 cells. In parallel, we also examined the influence of the previously mentioned compounds on the levels of thiols, glutathione, cysteine and cysteinyl-glycine, and on the level of sulfane sulfur and the activity of its metabolic enzymes: rhodanese, 3-mercaptopyruvate sulfurtransferase and cystathionase. Among the compounds under study, diallyl trisulfide (DATS), a sulfane sulfur-containing compound, showed the highest biological activity in HepG2 cells. This compound increased the H(2)O(2) formation, lowered the thiol level and produced the strongest inhibition of cell proliferation and the greatest induction of caspase 3 activity in HepG2 cells. DATS did not affect the activity of sulfurtransferases and lowered sulfane sulfur level in HepG2 cells. It appears that sulfane sulfur containing DATS can be bioreduced in cancer cells to hydroperthiol that leads to H(2)O(2) generation, thereby influencing transmission of signals regulating cell proliferation and apoptosis.     

The impact of the glial spatial buffering on the K+ Nernst potential

Astrocytes play a critical role in CNS metabolism, regulation of volume and ion homeostasis of the interstitial space. Of special relevance is their clearance of K⁺ that is released by active neurons into the extracellular space. Mathematical analysis of a modified Nernst equation for the electrochemical equilibrium of neuronal plasma membranes, suggests that K⁺ uptake by glial cells is not only relevant during neuronal activity but also has a non-neglectable impact on the basic electrical membrane properties, specifically the resting membrane potential, of neurons and might be clinically valuable as a factor in the genetics and epigenetics of the epilepsy and tuberous sclerosis complex.     

Zipping and Unzipping of Adenylate Kinase: Atomistic Insights into the Ensemble of Open ↔ Closed Transitions

Adenylate kinase (AdK), a phosphotransferase enzyme, plays an important role in cellular energy homeostasis. It undergoes a large conformational change between an open and a closed state, even in the absence of substrate. We investigate the apo-AdK transition at the atomic level both with free-energy calculations and with our new dynamic importance sampling (DIMS) molecular dynamics method. DIMS is shown to sample biologically relevant conformations as verified by comparing an ensemble of hundreds of DIMS transitions to AdK crystal structure intermediates. The simulations reveal in atomic detail how hinge regions partially and intermittently unfold during the transition. Conserved salt bridges are seen to have important structural and dynamic roles; in particular, four ionic bonds that open in a sequential, zipper-like fashion and, thus, dominate the free-energy landscape of the transition are identified. Transitions between the closed and open conformations only have to overcome moderate free-energy barriers. Unexpectedly, the closed state and the open state encompass broad free-energy basins that contain conformations differing in domain hinge motions by up to 40°. The significance of these extended states is discussed in relation to recent experimental Förster resonance energy transfer measurements. Taken together, these results demonstrate how a small number of cooperative key interactions can shape the overall dynamics of an enzyme and suggest an “all-or-nothing” mechanism for the opening and closing of AdK. Our efficient DIMS molecular dynamics computer simulation approach can provide a detailed picture of a functionally important macromolecular transition and thus help to interpret and suggest experiments to probe the conformational landscape of dynamic proteins such as AdK.     

Pushing, pulling and trapping--modes of motor protein supported protein translocation

Protein translocation across the cellular membranes is an ubiquitous and crucial activity of cells. This process is mediated by translocases that consist of a protein conducting channel and an associated motor protein. Motor proteins interact with protein substrates and utilize the free energy of ATP binding and hydrolysis for protein unfolding, translocation and unbinding. Since motor proteins are found either at the cis- or trans-side of the membrane, different mechanisms for translocation have been proposed. In the Power stroke model, cis-acting motors are thought to push, while trans-motors pull on the substrate protein during translocation. In the Brownian ratchet model, translocation occurs by diffusion of the unfolded polypeptide through the translocation pore while directionality is achieved by trapping and refolding. Recent insights in the structure and function of the molecular motors suggest that different mechanisms can be employed simultaneously.     

Panicle Water Potential, a Physiological Trait to Identify Drought Tolerance in Rice

Two upland rice varieties （IRAT109, IAPAR9） and one lowland rice variety （Zhenshan 97B） were planted in summer and treated with both normal （full water） and drought stress in the reproductive stage. Panicle water potential （PWP） and leaf water potential （LWP） were measured every 1.0-1.5 h over 24 h on sunny days. Both PWP and LWP of upland varieties started to decrease later, maintained a higher level and recovered more quickly than that of the lowland variety. The results show that PWP can be used as an indicator of plant water status based on the parallel daily changes, and the high correlation between PWP and LWP. Similar correlations were also observed between PWP, LWP and eight traits related to plant growth and grain yield formation. PWP seemed to be more effective for distinguishing the upland rice varieties with different drought-tolerant ability. Differences in PWP and LWP between upland and lowland rice varieties were also observed at noon even under normal water conditions, implying the incorporation of the drought-tolerant mechanism to improve the photosynthesis and yield of traditional paddy rice.