Psychophysiological reactivity and personality traits of left- and right-handers during mental stress

The aim of the present study was to establish whether there is dependence of vascular reactivity to mental stress on the levels of neuroticism and anxiety in left- and right-handers. Thirty-two left-handed and 32 righthanded volunteers (16 males and 16 females in each group) between 18 and 30 years of age were studied. During mental stress both groups demonstrated a "standard" type of response, indicating an increase of the sympathetic-adrenal activity. In left-handers, however, the magnitude of the stress vascular reactivity was significantly higher (p<0.05) in comparison with that of right-handers. In left-handed females a moderate negative correlation between vascular reactivity and the levels of neuroticism (r = -0.39) and trait anxiety (r = -0.47, F = 4.04, p = 0.06) was established. In right-handed males a moderate positive correlation between neuroticism and vascular reactivity (r = 0.35) and significant positive correlation between trait anxiety and vascular reactivity (r = 0.60, F = 7.92, p = 0.01) was found. The results obtained show that vascular reactivity to mental stress is not influenced in a specific to left- and right-handers way by the levels of negative emotionality (neuroticism and trait anxiety).     

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na⁺] and [K⁺] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na⁺‐selective class I‐HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single‐nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near‐isogenic lines (NILs): 157‐14 (double homozygote for the cheesmaniae alleles) and 157‐17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157‐14 in comparison with 157‐17. A significant difference between NILs was also found for [K⁺] and the [Na⁺]/[K⁺] ratio in leaf and stem but not for [Na⁺] arising a disagreement with the corresponding RIL population. Their association with leaf [Na⁺] and salt tolerance in tomato is also discussed.     

Molecular basis of the attenuated phenotype of human APOBEC3B DNA mutator enzyme

The human APOBEC3A and APOBEC3B genes (A3A and A3B) encode DNA mutator enzymes that deaminate cytidine and 5-methylcytidine residues in single-stranded DNA (ssDNA). They are important sources of mutations in many cancer genomes which show a preponderance of CG->TA transitions. Although both enzymes can hypermutate chromosomal DNA in an experimental setting, only A3A can induce double strand DNA breaks, even though the catalytic domains of A3B and A3A differ by only 9% at the protein level. Accordingly we sought the molecular basis underlying A3B attenuation through the generation of A3A-A3B chimeras and mutants. It transpires that the N-terminal domain facilitates A3B activity while a handful of substitutions in the catalytic C-terminal domain impacting ssDNA binding serve to attenuate A3B compared to A3A. Interestingly, functional attenuation is also observed for the rhesus monkey rhA3B enzyme compared to rhA3A indicating that this genotoxic dichotomy has been selected for and maintained for some 38 million years. Expression of all human ssDNA cytidine deaminase genes is absent in mature sperm indicating they contribute to somatic mutation and cancer but not human diversity.     

Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis

The KEAP1/Nrf2 pathway senses and responds to changes in intracellular oxidative stress. Mutations that result in constitutive activation of Nrf2 are present in several human tumors, especially non-small cell lung cancer. Therefore, compounds that inhibit Nrf2 activity might be beneficial in treating patients whose tumors show activation of this pathway. Recent reports suggest that the natural product brusatol can potently and selectively inhibit Nrf2 activity, resulting in cell cytotoxicity, and can be effectively combined with chemotherapeutic agents. Here, we analyzed the effects of brusatol on the cellular proteome in the KEAP1 mutant non-small cell lung cancer cell line A549. Brusatol was found to rapidly and potently decrease the expression of the majority of detected proteins, including Nrf2. The most dramatically decreased proteins are those that display a short half-life, like Nrf2. This effect was confirmed by restricting the analysis to newly synthesized proteins using a labeled methionine analogue. Moreover, brusatol increased the expression of multiple components of the ribosome, suggesting that it regulates the function of this macromolecular complex. Finally, we show that brusatol induces its potent cellular cytotoxicity effects on multiple cancer cell lines in a manner independent of KEAP1/Nrf2 activity and with a profile similar to the protein translation inhibitor silvestrol. In conclusion, our data show that the activity of brusatol is not restricted to Nrf2 but, rather, functions as a global protein synthesis inhibitor.The KEAP1¹/Nrf2 pathway is one of the most important cellular mechanisms to react and respond to oxidative stresses. Under normal physiological conditions, Nrf2 protein is maintained at low levels due to constitutive ubiquitination and degradation. This is achieved through recruitment to the Cul3 ubiquitin ligase complex by the substrate adaptor protein KEAP1. KEAP1 exists as a dimer, which binds a single Nrf2 molecule through two peptide motifs at its N terminus, a high affinity GluThrGlyGLU motif and a lower affinity AspLeuGly motif. This positions Nrf2 for effective ubiquitin transfer. Polyubiquitinated Nrf2 is subsequently degraded by the proteasome. In response to oxidative stress or electrophiles, key cysteine residues in KEAP1 become oxidized, thereby impairing its ability to recruit and ubiquitinate Nrf2 (1). This results in Nrf2 stabilization, allowing it to translocate to and accumulate in the nucleus (reviewed in (2)). Nrf2 binds specific DNA sequences in complex with the small Maf proteins, which were originally identified as viral oncogenes containing leucine zipper motifs (3). Hundreds of target genes have been identified using ChIP-seq and RNAseq experiments (4–6), highlighting the importance of Nrf2 in reducing levels of oxidative stress by increasing glutathione and NADPH synthesis, and up-regulating drug efflux pumps. Therefore, this pathway senses and responds to oxidative stress to maintain cellular redox homeostasis.When dysregulated, this pathway also contributes to many human pathologies including cardiovascular and neurodegenerative diseases, inflammation and cancer. Large-scale genome sequencing efforts have identified mutations in both KEAP1 and Nrf2 in several human tumor indications, but especially in non-small cell lung cancers (NSCLC). Mutations in KEAP1 are distributed across the gene and occur in ∼20 and ∼12% of the adenomatous tumors and squamous NSCLC, respectively (7). In addition to KEAP1, ∼15% of squamous NSCLC cases present with mutations in Nrf2 (8), supporting the hypothesis that Nrf2 degradation is frequently dysregulated in this indication (9). Nrf2 mutations cluster in two hotspot regions, ∼10 amino acids each, near the DLG and ETGE KEAP1 interacting regions, resulting in constitutive Nrf2 activation. A reasonable expectation is that these tumors become addicted to the high levels of Nrf2 activity, and some data have been presented that supports this hypothesis (10). Identification of a selective inhibitor of this pathway would be of great interest for potential therapeutic applications, which led us to explore the use of brusatol as an Nrf2 inhibitor. Brusatol is a representative compound from a family of natural product quassinoids. Recent publications have suggested that it can decrease the levels of Nrf2 and sensitize cells to various chemotherapy agents (11, 12).To identify and characterize the cellular targets of brusatol, we chose to employ a mass-spectrometry-based strategy. The binding of small molecules to their target proteins can induce conformational changes and alter their thermal stability compared with the apo form. This fact has been recently exploited to demonstrate engagement of known protein targets by small molecules in cells using Western blotting in an approach termed the cellular thermal shift assay (CETSA) (13). More recently, this assay was coupled with multiplexed mass spectrometry to yield an assay capable of surveying small molecule–protein interactions across a large fraction of the proteome (14). Our work using this approach, and follow up studies, suggest that brusatol regulates Nrf2 through an indirect mechanism involving global inhibition of protein synthesis.     

Regulation of GM2 ganglioside metabolism in cultured cells

GM2-ganglioside (II3NeuAcGgOse3Cer) is a minor component of adult nervous tissue, but is probably an oncofetal antigen. Its biological role is unknown, but several lines of evidence indicate its potential role in cell adhesion both in the retina and in oligodendrocytes. The biosynthesis of GM2-ganglioside appears to be tightly regulated, since it is a key intermediate in complex ganglioside synthesis. The specific GM3: hexosaminyl-transferase is activated under conditions which activate cyclic AMP-dependent protein kinase, and cell transformation with retroviruses inactivates it. Catabolism of GM2 requires the concerted action of three gene products (alpha-chain, beta-chain and activator protein in a thermolabile alpha beta 2 AP complex referred to as HexA). Defects in either three components results in the neuronal storage of GM2 ganglioside and the manifestations of Tay-Sachs Disease in children or motor neuron disease in adults.     

Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800)

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.     

Long-term effects of ungulates on phytophagous insects

Abstract.  1. Most plants interact with a diverse suite of herbivores, allowing the opportunity for the existence of positive and negative interactions between highly dissimilar organisms. However, most studies on herbivorous interactions have been performed under the assumption that they occur mainly between similar species. Consequently, ecologists are still far from a full understanding of the ecological factors that determine insect population dynamics.
2. In this study, a 7-year field experiment was conducted that manipulated the presence of ungulates to evaluate their effects on the abundance, attack rate, and survival of four guilds of co-occurring herbivorous insects living on the same host plant: seed predators, stem borers, gall makers and sap suckers. These four guilds differed in habits and behaviour, the first three being sessile and endophytic and the last being free-living.
3. This study shows that the abundance of all four guilds was negatively affected by ungulates. However, the effect on attack rate differed among guilds, as mammals do not affect the seed predator attack rate. Ungulates also differentially affected insect survival, ingesting only seed predators and gall makers.
4. In summary, this study suggests that diverse mechanisms may affect different insect guilds in different ways. Therefore, competition between disparate herbivores appears to be complex and can be provoked by multiple mechanisms.