A cytogenetic study of men environmentally and occupationally exposed to airborne pollutants.

The level of sister-chromatid exchanges (SCE), high-frequency cells (HFC), chromosomal aberrations (CA) as well as the proliferation rate index (PRI) were measured in peripheral blood lymphocytes from three groups of volunteers. The environmentally exposed donors were residents from the vicinity of a coke factory; the occupationally exposed persons were cokery workers, while rural region inhabitants served as a control group. Compared with the control group, statistically significant increases of SCE and HFC, as well as decreased cell kinetics (PRI) were observed for both occupationally and environmentally exposed groups. The effect was especially pronounced when only smokers were taken into account. A statistically significant increase of CA was observed in the environmentally exposed group when CA including gaps (CA + G) were evaluated. The proportion of HFC was found to be the most sensitive method to detect genetic effects on the tested human population. This study demonstrates the usefulness of all 4 biomarkers (SCE, HFC, CA and PRI) in monitoring populations exposed to ambient pollution and clearly indicates effects from residential as well as occupational exposure to industrial air pollutants.     

HaCaT keratinocytes secrete lysosomal cysteine proteinases during migration

Cathepsin B, a lysosomal cysteine proteinase, was detected within vesicles of cellular protrusions forming cell-cell contact sites between keratinocytes of the stratum spinosum of human skin. This observation suggested the possibility that secretion of the protease into the pericellular spaces could be involved in the dissociation of cell-cell contacts to enable intraepidermal keratinocyte migration. To determine whether cathepsin B is indeed secreted from migrating keratinocytes, we first used subconfluent HaCaT cells as a culture model to study spontaneous keratinocyte migration. A cathepsin B-specific fluorescent affinity label proved the association of mature cathepsin B with the surfaces of HaCaT cells at the leading edges of growing cells. Second, we used scratch-wounds of confluent HaCaT monolayers as a model of induced keratinocyte migration. Cathepsin B was detected within lysosomes, i.e. vesicles within the perinuclear region of non-wounded cells. Expression of cathepsin B was up-regulated and cathepsin B-positive vesicles showed a redistribution from perinuclear to peripheral regions of keratinocytes at the wound margins within 4 h after wounding. Enzyme cytochemistry further showed that cell surface-associated cathepsin B was proteolytically active at the leading fronts of migrating keratinocytes. In addition, increased amounts of mature forms of cathepsin B were detected within the conditioned media of HaCaT cells during the first 4 h after scratch-wounding. In contrast, and as a control, the activity of the cytosolic enzyme lactate dehydrogenase was not significantly higher in media of wounded cells as compared with non-wounded controls, arguing for a specific induction of cathepsin B secretion upon wounding and migration of the cells. This was further substantiated by applying various cathepsin B-specific inhibitors after wounding. These experiments showed that the migration ability of keratinocytes was reduced due to the blockage of functional cathepsin B. Thus, our results strongly suggest that cell surface-associated cathepsin B is a protease that contributes to the remodelling of the extracellular matrix and thereby promotes keratinocyte migration during wound healing.     

Crystal structure of isoaspartyl aminopeptidase in complex with L-aspartate

The crystal structure of Escherichia coli isoaspartyl aminopeptidase/asparaginase (EcAIII), an enzyme belonging to the N-terminal nucleophile (Ntn)-hydrolases family, has been determined at 1.9-A resolution for a complex obtained by cocrystallization with l-aspartate, which is a product of both enzymatic reactions catalyzed by EcAIII. The enzyme is a dimer of heterodimers, (alphabeta)(2). The (alphabeta) heterodimer, which arises by autoproteolytic cleavage of the immature protein, exhibits an alphabetabetaalpha-sandwich fold, typical for Ntn-hydrolases. The asymmetric unit contains one copy of the EcAIII.Asp complex, with clearly visible l-aspartate ligands, one bound in each of the two active sites of the enzyme. The l-aspartate ligand is located near Thr(179), the N-terminal residue of subunit beta liberated in the autoproteolytic event. Structural comparisons with the free form of EcAIII reveal that there are no major rearrangements of the active site upon aspartate binding. Although the ligand binding mode is similar to that observed in an l-aspartate complex of the related enzyme human aspartylglucosaminidase, the architecture of the EcAIII active site sheds light on the question of substrate specificity and explains why EcAIII is not able to hydrolyze glycosylated asparagine substrates.     

The Influence of Conspecific Males on Spermatophore Deposition in the Eriophyid Mite Aculus Fockeui

Similarly to males of Aculus fockeui on plum, males inhabiting peach showed interest in quiescent female nymphs and behaved non-aggressively in contact with each other. When maintained on fresh leaf arenas for 5h, males in groups of seven deposited significantly fewer spermatophores per male than solitary ones. The inhibitory effect of the presence of conspecific males on spermatophore deposition rate is indicated and discussed in the context of previous opposite observations on this mite.     

Role of Right Ventricular Global Longitudinal Strain in Predicting Early and Long-Term Mortality in Cardiac Resynchronization Therapy Patients

Background

Right ventricular (RV) dysfunction has been associated with poor prognosis in chronic heart failure (HF). However, less data is available about the role of RV dysfunction in patients with cardiac resynchronization therapy (CRT). We aimed to investigate if RV dysfunction would predict outcome in CRT.

Design

We enrolled prospectively ninety-three consecutive HF patients in this single center observational study. All patients underwent clinical evaluation and echocardiography before CRT and 6 months after implantation. We assessed RV geometry and function by using speckle tracking imaging and calculated strain parameters. We performed multivariable Cox regression models to test mortality at 6 months and at 24 months.

Results

RV dysfunction, characterized by decreased RVGLS (RV global longitudinal strain) [10.2 (7.0–12.8) vs. 19.5 (15.0–23.9) %, p<0.0001] and RVFWS (RV free wall strain) [15.6 (10.0–19.3) vs. 17.4 (10.5–22.2) %, p = 0.04], improved 6 months after CRT implantation. Increasing baseline RVGLS and RVFWS predicted survival independent of other parameters at 6 months [hazard ratio (HR) = 0.37 (0.15–0.90), p = 0.02 and HR = 0.42 (0.19–0.89), p = 0.02; per 1 standard deviation increase, respectively]. RVGLS proved to be a significant independent predictor of mortality at 24 months [HR = 0.53 (0.32–0.86), p = 0.01], and RVFWS showed a strong tendency [HR = 0.64 (0.40–1.00), p = 0.05]. The 24-month survival was significantly impaired in patients with RVGLS below 10.04% before CRT implantation [area under the curve = 0.72 (0.60–0.84), p = 0.002, log-rank p = 0.0008; HR = 5.23 (1.76–15.48), p = 0.003].

Conclusions

Our findings indicate that baseline RV dysfunction is associated with poor short-term and long-term prognosis after CRT implantation.     

HIF-1α activation results in actin cytoskeleton reorganization and modulation of Rac-1 signaling in endothelial cells

Background

Increasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins.

Findings

Here we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus.

Conclusions

Given both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated.     

Malate plays a crucial role in starch metabolism, ripening, and soluble solid content of tomato fruit and affects postharvest softening

Despite the fact that the organic acid content of a fruit is regarded as one of its most commercially important quality traits when assessed by the consumer, relatively little is known concerning the physiological importance of organic acid metabolism for the fruit itself. Here, we evaluate the effect of modifying malate metabolism in a fruit-specific manner, by reduction of the activities of either mitochondrial malate dehydrogenase or fumarase, via targeted antisense approaches in tomato (Solanum lycopersicum). While these genetic perturbations had relatively little effect on the total fruit yield, they had dramatic consequences for fruit metabolism, as well as unanticipated changes in postharvest shelf life and susceptibility to bacterial infection. Detailed characterization suggested that the rate of ripening was essentially unaltered but that lines containing higher malate were characterized by lower levels of transitory starch and a lower soluble sugars content at harvest, whereas those with lower malate contained higher levels of these carbohydrates. Analysis of the activation state of ADP-glucose pyrophosphorylase revealed that it correlated with the accumulation of transitory starch. Taken together with the altered activation state of the plastidial malate dehydrogenase and the modified pigment biosynthesis of the transgenic lines, these results suggest that the phenotypes are due to an altered cellular redox status. The combined data reveal the importance of malate metabolism in tomato fruit metabolism and development and confirm the importance of transitory starch in the determination of agronomic yield in this species.     

Key amino acid residues of ankyrin-sensitive phosphatidylethanolamine/phosphatidylcholine-lipid binding site of βI-spectrin

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that "opening" of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton.     

Engineering the respiratory complex I to energy-converting NADPH:ubiquinone oxidoreductase

The respiratory complex I couples the electron transfer from NADH to ubiquinone with a translocation of protons across the membrane. Its nucleotide-binding site is made up of a unique Rossmann fold to accommodate the binding of the substrate NADH and of the primary electron acceptor flavin mononucleotide. Binding of NADH includes interactions of the hydroxyl groups of the adenosine ribose with a conserved glutamic acid residue. Structural analysis revealed that due to steric hindrance and electrostatic repulsion, this residue most likely prevents the binding of NADPH, which is a poor substrate of the complex. We produced several variants with mutations at this position exhibiting up to 200-fold enhanced catalytic efficiency with NADPH. The reaction of the variants with NAD(P)H is coupled with proton translocation in an inhibitor-sensitive manner. Thus, we have created an energy-converting NADPH:ubiquinone oxidoreductase, an activity so far not found in nature. Remarkably, the oxidation of NAD(P)H by the variants leads to an enhanced production of reactive oxygen species.