Molecular Dynamics Simulations of Rhodopsin Point Mutants at the Cytoplasmic Side of Helices 3 and 6

Abstract

The present work reports on a structural analysis carried out through different computer simulations of a set of rhodopsin mutants with differential functional features in regard to the wild type. Most of these mutants, whose experimental features had previously been reported [Ramon et al. J Biol Chem 282, 14272–14282 (2007)], were designed to perturb a network of electrostatic interactions located at the cytoplasmic sides of transmembrane helices 3 and 6. Geometric and energetic features derived from the detailed analysis of a series of molecular dynamics simulations of the different rhodopsin mutants, involving positions 134(3.49), 247(6.30), and 251(6.34), suggest that the protein structure is sensitive to these mutations through the local changes induced that extend further to the secondary structure of neighboring helices and, ultimately, to the packing of the helical bundle. Overall, the results obtained highlight the complexity of the analyzed network of electrostatic interactions where the effect of each mutation on protein structure can produce rather specific features.     

Reduction of palladium and production of nano-catalyst by Geobacter sulfurreducens

The present study is the first report on the ability of Geobacter sulfurreducens PCA to reduce Pd(II) and produce Pd(0) nano-catalyst, using acetate as electron donor at neutral pH (7.0?±?0.1) and 30 °C. The microbial production of Pd(0) nanoparticles (NPs) was greatly enhanced by the presence of the redox mediator, anthraquinone-2,6-disulfonate (AQDS) when compared with controls lacking AQDS and cell-free controls. A cell dry weight (CDW) concentration of 800 mg/L provided a larger surface area for Pd(0) NPs deposition than a CDW concentration of 400 mg/L. Sample analysis by transmission electron microscopy revealed the formation of extracellular Pd(0) NPs ranging from 5 to 15 nm and X-ray diffraction confirmed the Pd(0) nature of the nano-catalyst produced. The present findings open the possibility for a new alternative to synthesize Pd(0) nano-catalyst and the potential application for microbial metal recovery from metal-containing waste streams.     

Allelic Diversity and Its Implications for the Rate of Adaptation

Genetic variation is usually estimated empirically from statistics based on population gene frequencies, but alternative statistics based on allelic diversity (number of allelic types) can provide complementary information. There is a lack of knowledge, however, on the evolutionary implications attached to allelic-diversity measures, particularly in structured populations. In this article we simulated multiple scenarios of single and structured populations in which a quantitative trait subject to stabilizing selection is adapted to different fitness optima. By forcing a global change in the optima we evaluated which diversity variables are more strongly correlated with both short- and long-term adaptation to the new optima. We found that quantitative genetic variance components for the trait and gene-frequency-diversity measures are generally more strongly correlated with short-term response to selection, whereas allelic-diversity measures are more correlated with long-term and total response to selection. Thus, allelic-diversity variables are better predictors of long-term adaptation than gene-frequency variables. This observation is also extended to unlinked neutral markers as a result of the information they convey on the demographic population history. Diffusion approximations for the allelic-diversity measures in a finite island model under the infinite-allele neutral mutation model are also provided.     

Identification of the infectious source of an unusual outbreak of histoplasmosis,in a hotel in Acapulco,state of Guerrero,Mexico

Three isolates of Histoplasma capsulatum were identified from mice lung, liver, and spleen inoculated with soil samples of the X hotel’s ornamental potted plants that had been fertilized with organic material known as compost. The presence of H. capsulatum in the original compost was detected using the dot-enzyme-linked immunosorbent assay. Nested-PCR, using a specific protein Hcp100 coding gene sequence, confirmed the fungal identification associated with an unusual histoplasmosis outbreak in Acapulco. Although, diversity between the H. capsulatum isolate from the hotel and some clinical isolates from Guerrero (positive controls) was observed using random amplification of polymorphic DNA based-PCR, sequence analyses of H-anti and ole fragment genes revealed a high homology (92–99%) between them.     

Histone Deacetylase Inhibitor Upregulates Peroxisomal Fatty Acid Oxidation and Inhibits Apoptotic Cell Death in Abcd1-Deficient Glial Cells

In X-ALD, mutation/deletion of ALD gene (ABCD1) and the resultant very long chain fatty acid (VLCFA) derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD). The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal β-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs) (1 and 3) in both cell types. However, higher induction of ELOVL''s in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL) and cell survival (phospho-Erk1/2) proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid) leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.     

Multiparametric magnetic resonance imaging-guided salvage radiotherapy in prostate cancer

AimTo analyse the efficacy and toxicity of postprostatectomy SRT in patients with a BCR evaluated with mpMRI.BackgroundMultiparametric magnetic resonance imaging (mpMRI) has the ability to detect the site of pelvic recurrence in patients with biochemical recurrence (BCR) after radical prostatectomy (RP). However, we do not know the oncological outcomes of mpMRI-guided savage radiotherapy (SRT).ResultsLocal, lymph node, and pelvic bone recurrence was observed in 13, 4 and 2 patients, respectively. PSA levels were significantly lower in patients with negative mpMRI (0.4 ng/mL [0.4]) vs. positive mpMRI (2.2 ng/mL [4.1], p = 0.003). Median planning target volume doses in patients with visible vs. non-visible recurrences were 76 Gy vs. 70 Gy. Overall, mean follow-up was 41 months (6–81). Biochemical relapse-free survival (bRFS) at 3 years was 82.3% and 82.5%, respectively, for the negative and positive mpMRI groups (p = 0.800). Three-year rates of late grade ≥2 urinary and rectal toxicity were 14.8% and 1.9%, respectively; all but one patient recovered without sequelae.ConclusionSRT to the macroscopic recurrence identified by mpMRI is a feasible and well-tolerated option. In this study, there were no differences in bRFS between MRI-positive and MRI-negative patients, indicating effective targeting of MRI-positive lesions.     

Sex chromosomes of basal placental mammals

Placental (eutherian) mammals are currently classified into four superordinal clades (Afrotheria, Xenarthra, Laurasiatheria and Supraprimates) of which one, the Afrotheria (a unique lineage of African origin), is generally considered to be basal. Therefore, Afrotheria provide a pivotal evolutionary link for studying fundamental differences between the sex chromosomes of human/mouse (both representatives of Supraprimates and the index species for studies of sex chromosomes) and those of the distantly related marsupials. In this study, we use female fibroblasts to investigate classical features of X chromosome inactivation including replication timing of the X chromosomes and Barr body formation. We also examine LINE-1 accumulation on the X chromosomes of representative afrotherians and look for evidence of a pseudoautosomal region (PAR). Our results demonstrate that asynchronous replication of the X chromosomes is common to Afrotheria, as with other mammals, and Barr body formation is observed across all Placentalia, suggesting that mechanisms controlling this evolved before their radiation. Finally, we provide evidence of a PAR (which marsupials lack) and demonstrate that LINE1 is accumulated on the afrotherian and xenarthran X, although this is probably not due to transposition events in a common ancestor, but rather ongoing selection to retain recently inserted LINE1 on the X.     

Shortcut predictions for fitness properties at the mutation-selection-drift balance and for its buildup after size reduction under different management strategies

For populations at the mutation-selection-drift (MSD) balance, I develop approximate analytical expressions giving expectations for the number of deleterious alleles per gamete, the number of loci at which any individual is homozygous for deleterious alleles, the inbreeding depression rate, and the additive and dominant components of fitness variance. These predictions are compared to diffusion ones, showing good agreement under a wide range of situations. I also give approximated analytical predictions for the changes in mean and additive variance for fitness when a population approaches a new equilibrium after its effective size is reduced to a stable value. Results are derived for populations maintained with equal family contribution or with no management after size reduction, when selection acts through viability or fertility differences. Predictions are compared to previously published results obtained from transition matrices or stochastic simulations, a good qualitative fit being obtained. Predictions are also obtained for populations of various sizes under different sets of plausible mutational parameters. They are compared to available empirical results for Drosophila, and conservation implications are discussed.     

Allelochemical Stress Can Trigger Oxidative Damage in Receptor Plants: Mode of Action of Phytotoxicity

Plants can interact with other plants through the release of chemical compounds or allelochemicals. These compounds released by donor plants influence germination, growth, development, and establishment of receptor plants; having an important role on the pattern of vegetation, i.e as invasive strategy, and on crop productivity. This phytotoxic or negative effect of the released allelochemicals (allelochemical stress) is caused by modifying or altering diverse metabolic processes, having many molecular targets in the receptor plants. Recently, using an aggressive and allelopathic plant Sicyos deppei as the donor plant, and Lycopersicon esculentum as the receptor plant, we showed that the allelochemicals released by S. deppei caused oxidative damage through an increase in reactive oxygen species (ROS) and activation or modification of antioxidant enzymes. Based on this study, we proposed that oxidative stress is one of the mechanisms, among others, by which an allelopathic plant causes phytotoxicity to other plants.Key Words: allelochemical stress, Sicyos deppei, Lycopersicon esculentum, plant allelochemicals, phytotoxicity, ROS, lipid peroxidationIt is well known that plants interact with many organisms, including co-habitation with other plants. Among these relations are the ones referred to as allelochemical interactions. Allelopathy can be defined as a mechanism of interference in plant growth and development mediated by the addition of plant-produced secondary products (allelochemicals) to the soil rhizosphere. Allelochemicals are present in all types of plants and tissues and are released into the soil rhizosphere by a variety of mechanisms, including decomposition of residues, volatilization, and root exudation.^1–3 These released allelochemicals become stressful only when they are toxic or when they affect the growth and development of surrounding plants (phytotoxicity). Studies on allelochemical stress have been expanding; recently the phenomenon has taken on increased importance, since it can help explain plant growth inhibition in interspecies interactions and in structuring the plant community. It appears to be one mechanism or strategy used by invasive plants to become successful and replace other native ones.^4–6On the other hand, the chemical diversity of the organic compounds that mediate these allelochemical interactions is as diverse as their modes of action. Many studies have shown that allelochemicals interfere with several physiological processes in the receptor organism.^3,7,8 The physiological effects on receptor plants or other organisms are useful in determining the role of the allelochemicals in the system. Recently, it has been proposed that allelochemicals can cause oxidative stress in target plants and therefore activate the antioxidant mechanism.^3,8–12 In particular; our studies have been focused on knowing the physiological targets of the phytotoxic compounds released by a noxious and endemic weed Sicyos deppei G. Don (Cucurbitaceae). We have taken as the model the receptor or damaged plant Lycopersicon esculentum Mill (Solanaceae), since in Mexican crop-fields, it is common to find both plants. We have observed the strong allelopathic potential of S. deppei and are exploring the potential metabolic target that could be involved in the strong phytotoxic effect of this weed.^13–16 We recently documented the oxidative damage that an aqueous leachate of S. deppei caused in the target plant L. esculentum.¹⁶ In this work we explored in seeds and in primary roots the antioxidant mechanism of tomato to determine whether or not the inhibitory effect of S. deppei was due to oxidative damage. We analyzed the activity and expression of some antioxidant enzymes involved in the detoxification of ROS, and found an imbalance in its activity as well as an increase in the levels of H₂O₂ at 24 h of treatment. Additional studies on the levels of ROS, including hydrogen peroxide, were monitored in primary roots from germinating seeds under allelochemical stress by imaging the ROS-sensitive fluorescent dye dichlorofluorescein (_H2DCFDA, carboxy-2′, 7′-diclhlorofluorescein diacetate) in a confocal microscope (BIORAD 1024, 488 nm dichroic and 510–560 nm emission). DCFDA fluorescence increases as the dye is oxidized by ROS to dichlorofluorescein (DCF). Figure 1 shows a marked increase in fluorescence at 48 h and 72 h of treatment (Fig. 1A–C) compared with the same treatment at 24 h, and with the corresponding control. This fluorescence was more evident at the root cap and at the zone of root hairs in treated seeds.Open in a separate windowFigure 1Allelochemical stress caused by S. deppei elicits ROS generation in tomato germinating seeds. Panels show control (left) and treatment (right) at 24 h (A), 48 h (B), and 72 h (C). Lower panels show higher magnification (40X) of the corresponding time. Seedlings with primary roots were stained for 10–15 minutes with 25 µM DCFDA in distilled water.Clearly, allelochemical stress caused by S. deppei is producing an oxidative imbalance as evidenced by generation of ROS and alteration of activity of antioxidant enzymes. Another result that supports this observation is the high level of lipid peroxidation that we observed at 48 and 72 h, which correlates with the inhibition of two membrane-associated enzymes, H⁺-ATPase¹⁵ and NADPH oxidase.¹⁶ We believe, however, that the oxidative damage we observed is not solely responsible for the phytotoxic effect of S. deppei on tomato growth. In other words, we suggest that its inhibitory effect represents the sum of many metabolic processes affected at different times. Currently we are studying the dynamics of carbohydrate mobilization, cell wall loosing of the endosperm to allow the protrusion of the radicle, and ABA content. Preliminary results have shown that there is a delay in expression of some enzyme activities and a high content of ABA.