Oxidative stress-induced DNA damage and cell cycle regulation in B65 dopaminergic cell line

Reactive oxygen species and oxidative stress are associated with neuronal cell death in many neurodegenerative conditions. However, the exact molecular mechanisms triggered by oxidative stress in neurodegeneration are still unclear. This study used the B65 rat neuroblastoma cell line as a model to study the molecular events that occur after H₂O₂ treatment. Treatment of B65 cells with H₂O₂ rapidly up-regulated the DNA damage pathway involved in double-strand breakage. Subsequently, proteins involved in p53 regulation, such as sirtuin 1 and STAT1, were modified. In addition, H₂O₂ treatment altered the pattern of cell cycle protein expression. Specifically, a decrease was found in the expression of cyclin D1, cdk4 and surprisingly the levels of cyclin A and the retinoblastoma protein phosphorylated at ser780 were increased. Furthermore, this study shows that pre-treatment of B65 cells with 50 µM trolox confers almost total protection against apoptotic cell death and restores the cell cycle. Likewise, the increase in retinoblastoma phosphorylation was attenuated by KU-55993, a selective ATM inhibitor, and also by trolox. These observations indicate that DNA damage and oxidative stress are responsible for cell cycle regulation. In summary, this study describes the molecular mechanisms involved in cell cycle alterations induced by oxidative stress in B65 cells. These findings highlight the relevance of ATM in the regulation of cell cycle after oxidative stress.     

Water permeation dynamics of AqpZ: a tale of two states

Molecular dynamics simulations of aquaporin Z homotetramer which is a membrane protein facilitating rapid water movement through the plasma membrane of Escherichia coli were performed. Initial configurations were taken from the open and closed states of crystal structures separately. The resulting water osmotic permeability (pf) and diffusive permeability (pd) displayed distinct features. Consistent with previous studies, the side chain conformation of arginine189 was found to mediate the water permeability. A potential of mean force (PMF) as a function of the distance between NH1 of R189 and carbonyl oxygen of A117 was constructed based on the umbrella sampling technique. There are multiple local minima and transition states on the PMF. The assignment of the open or closed state was supported by the permeability pf, calculated within trajectories in umbrella sampling simulations. Our study disclosed a detailed mechanism of the gated water transport.     

Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH- and cysteine protease-independent FcγR pathway

Public health measures successfully contained outbreaks of the severe acute respiratory syndrome coronavirus (SARS-CoV) infection. However, the precursor of the SARS-CoV remains in its natural bat reservoir, and reemergence of a human-adapted SARS-like coronavirus remains a plausible public health concern. Vaccination is a major strategy for containing resurgence of SARS in humans, and a number of vaccine candidates have been tested in experimental animal models. We previously reported that antibody elicited by a SARS-CoV vaccine candidate based on recombinant full-length Spike-protein trimers potentiated infection of human B cell lines despite eliciting in vivo a neutralizing and protective immune response in rodents. These observations prompted us to investigate the mechanisms underlying antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro. We demonstrate here that anti-Spike immune serum, while inhibiting viral entry in a permissive cell line, potentiated infection of immune cells by SARS-CoV Spike-pseudotyped lentiviral particles, as well as replication-competent SARS coronavirus. Antibody-mediated infection was dependent on Fcγ receptor II but did not use the endosomal/lysosomal pathway utilized by angiotensin I converting enzyme 2 (ACE2), the accepted receptor for SARS-CoV. This suggests that ADE of SARS-CoV utilizes a novel cell entry mechanism into immune cells. Different SARS vaccine candidates elicit sera that differ in their capacity to induce ADE in immune cells despite their comparable potency to neutralize infection in ACE2-bearing cells. Our results suggest a novel mechanism by which SARS-CoV can enter target cells and illustrate the potential pitfalls associated with immunization against it. These findings should prompt further investigations into SARS pathogenesis.     

In vivo cytochrome and alternative pathway respiration in leaves of Arabidopsis thaliana plants with altered alternative oxidase under different light conditions

The in vivo activity of the alternative pathway (ν(alt)) has been studied using the oxygen isotope fractionation method in leaves of Arabidopsis thaliana modified for the expression of the AtAOX1a gene by anti-sense (AS-12) or overexpression (XX-2). Under non-stressful conditions, ν(alt) was similar in all plant lines regardless of its different alternative pathway capacities (V(alt)). Total leaf respiration (V(t)) and V(alt) were directly related to growth light conditions while electron partitioning between the cytochrome pathway (CP) and alternative pathway (AP) was unchanged by light levels. Interestingly, the AP functioned at full capacity in anti-sense plants under both growth light conditions. The role of the AP in response to a high light stress induced by short-term high light treatment (HLT) was also studied. In wild type and XX-2, both CP and AP rates increased proportionally after HLT while in AS-12, where the AP was unable to increase its rate, the CP accommodated all the increase in respiration. The results obtained under high light stress suggest that flexibility in the response of the mitochondrial electron transport chain is involved in sustaining photosynthetic rates in response to this stress while the saturated AP in AS-12 plants may contribute to the observed increase in photoinhibition.     

Rubisco activity in Mediterranean species is regulated by the chloroplastic CO2 concentration under water stress

Water stress decreases the availability of the gaseous substrate for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) by decreasing leaf conductance to CO(2). In spite of limiting photosynthetic carbon assimilation, especially in those environments where drought is the predominant factor affecting plant growth and yield, the effects of water deprivation on the mechanisms that control Rubisco activity are unclear. In the present study, 11 Mediterranean species, representing different growth forms, were subject to increasing levels of drought stress, the most severe one followed by rewatering. The results confirmed species-specific patterns in the decrease in the initial activity and activation state of Rubisco as drought stress and leaf dehydration intensified. Nevertheless, all species followed roughly the same trend when Rubisco activity was related to stomatal conductance (g(s)) and chloroplastic CO(2) concentration (C(c)), suggesting that deactivation of Rubisco sites could be induced by low C(c), as a result of water stress. The threshold level of C(c) that triggered Rubisco deactivation was dependent on leaf characteristics and was related to the maximum attained for each species under non-stressing conditions. Those species adapted to low C(c) were more capable of maintaining active Rubisco as drought stress intensified.     

The Mediterranean evergreen Quercus ilex and the semi-deciduous Cistus albidus differ in their leaf gas exchange regulation and acclimation to repeated drought and re-watering cycles

Plants may exhibit some degree of acclimation after experiencing drought, but physiological adjustments to consecutive cycles of drought and re-watering (recovery) have scarcely been studied. The Mediterranean evergreen holm oak (Q. ilex) and the semi-deciduous rockrose (C. albidus) showed some degree of acclimation after the first of three drought cycles (S1, S2, and S3). For instance, during S2 and S3 both species retained higher relative leaf water contents than during S1, despite reaching similar leaf water potentials. However, both species showed remarkable differences in their photosynthetic acclimation to repeated drought cycles. Both species decreased photosynthesis to a similar extent during the three cycles (20-40% of control values). However, after S1 and S2, photosynthesis recovered only to 80% of control values in holm oak, due to persistently low stomatal (g(s)) and mesophyll (g(m)) conductances to CO(2). Moreover, leaf intrinsic water use efficiency (WUE) was kept almost constant in this species during the entire experiment. By contrast, photosynthesis of rockrose recovered almost completely after each drought cycle (90-100% of control values), while the WUE was largely and permanently increased (by 50-150%, depending on the day) after S1. This was due to a regulation which consisted in keeping g(s) low (recovering to 50-60% of control values after re-watering) while maintaining a high g(m) (even exceeding control values during re-watering). While the mechanisms to achieve such particular regulation of water and CO(2) diffusion in leaves are unknown, it clearly represents a unique acclimation feature of this species after a drought cycle, which allows it a much better performance during successive drought events. Thus, differences in the photosynthetic acclimation to repeated drought cycles can have important consequences on the relative fitness of different Mediterranean species or growth forms within the frame of climate change scenarios.     

Molecular evolution and network-level analysis of the N-glycosylation metabolic pathway across primates

N-glycosylation is one of the most important forms of protein modification, serving key biological functions in multicellular organisms. N-glycans at the cell surface mediate the interaction between cells and the surrounding matrix and may act as pathogen receptors, making the genes responsible for their synthesis good candidates to show signatures of adaptation to different pathogen environments. Here, we study the forces that shaped the evolution of the genes involved in the synthesis of the N-glycans during the divergence of primates within the framework of their functional network. We have found that, despite their function of producing glycan repertoires capable of evading rapidly evolving pathogens, genes involved in the synthesis of the glycans are highly conserved, and no signals of positive selection have been detected within the time of divergence of primates. This suggests strong functional constraints as the main force driving their evolution. We studied the strength of the purifying selection acting on the genes in relation to the network structure considering the position of each gene along the pathway, its connectivity, and the rates of evolution in neighboring genes. We found a strong and highly significant negative correlation between the strength of purifying selection and the connectivity of each gene, indicating that genes encoding for highly connected enzymes evolve slower and thus are subject to stronger selective constraints. This result confirms that network topology does shape the evolution of the genes and that the connectivity within metabolic pathways and networks plays a major role in constraining evolutionary rates.