TatB functions as an oligomeric binding site for folded Tat precursor proteins

Twin-arginine-containing signal sequences mediate the transmembrane transport of folded proteins. The cognate twin-arginine translocation (Tat) machinery of Escherichia coli consists of the membrane proteins TatA, TatB, and TatC. Whereas Tat signal peptides are recognized by TatB and TatC, little is known about molecular contacts of the mature, folded part of Tat precursor proteins. We have placed a photo-cross-linker into Tat substrates at sites predicted to be either surface-exposed or hidden in the core of the folded proteins. On targeting of these variants to the Tat machinery of membrane vesicles, all surface-exposed sites were found in close proximity to TatB. Correspondingly, incorporation of the cross-linker into TatB revealed multiple precursor-binding sites in the predicted transmembrane and amphipathic helices of TatB. Large adducts indicative of TatB oligomers contacting one precursor molecule were also obtained. Cross-linking of Tat substrates to TatB required an intact twin-arginine signal peptide and disappeared upon transmembrane translocation. Our collective data are consistent with TatB forming an oligomeric binding site that transiently accommodates folded Tat precursors.     

Biodegradation Processes in a Laboratory-Scale Groundwater Contaminant Plume Assessed by Fluorescence Imaging and Microbial Analysis

Flow reactors containing quartz sand colonized with biofilm were set up as physical model aquifers to allow degrading plumes of acetate or phenol to be formed from a point source. A noninvasive fluorescent tracer technique was combined with chemical and biological sampling in order to quantify transport and biodegradation processes. Chemical analysis of samples showed a substantial decrease in carbon concentration between the injection and outflow resulting primarily from dilution but also from biodegradation. Two-dimensional imaging of the aqueous oxygen [O₂(aq)] concentration field quantified the depletion of O₂(aq) within the contaminant plume and provided evidence for microbial respiration associated with biodegradation of the carbon source. Combined microbiological, chemical, and O₂(aq) imaging data indicated that biodegradation was greatest at the plume fringe. DNA profiles of bacterial communities were assessed by temperature gradient gel electrophoresis, which revealed that diversity was limited and that community changes observed depended on the carbon source used. Spatial variation in activity within the plume could be quantitatively accounted for by the changes observed in active cell numbers rather than differences in community structure, the total biomass present, or the increased enzyme activity of individual cells. Numerical simulations and comparisons with the experimental data were used to test conceptual models of plume processes. Results demonstrated that plume behavior was best described by growth and decay of active biomass as a single functional group of organisms represented by active cell counts.     

Decreased E‐Cadherin in MCF7 Human Breast Cancer Cells Forming Multicellular Spheroids Exposed to Simulated Microgravity

MCF7 human breast cancer cells were cultured under normal gravity (1 g) and on a random positioning machine (RPM) preventing sedimentation. After 2 weeks, adherent 1 g‐control and adherent RPM cells (AD) as well as multicellular spheroids (MCS) were harvested. AD and MCS had been exposed to the RPM in the same culture flask. In a subsequent proteome analysis, the majority of the proteins detected showed similar label‐free quantification (LFQ) scores in each of the respective subpopulations, but in both AD or MCS cultures, proteins were also found whose LFQs deviated at least twofold from their counterparts in the 1 g‐control cells. They included the cell junction protein E‐cadherin, which was diminished in MCS cells, where proteins of the E‐cadherin autodegradation pathway were enhanced and c‐Src (proto‐oncogene tyrosine‐protein kinase c‐Src) was detected. Spheroid formation was prevented by inhibition of c‐Src but promoted by antibodies blocking E‐cadherin activity. An interaction analysis of the detected proteins that are involved in forming and regulating junctions or adhesion complexes and in E‐cadherin autodegradation indicated connections between the two protein groups. This suggests that the balance of proteins that up‐ or downregulate E‐cadherin mediates the tendency of MCF7 cells to form MCS during RPM exposure.     

Antiprotozoal and cysteine proteases inhibitory activity of dipeptidyl enoates

A family of dipeptidyl enoates has been prepared and tested against the parasitic cysteine proteases rhodesain, cruzain and falcipain-2 related to sleeping sickness, Chagas disease and malaria, respectively. They have also been tested against human cathepsins B and L1 for selectivity. Dipeptidyl enoates resulted to be irreversible inhibitors of these enzymes. Some of the members of the family are very potent inhibitors of parasitic cysteine proteases displaying k_2nd (M^?1s^?1) values of seven orders of magnitude. In vivo antiprotozoal testing was also performed. Inhibitors exhibited IC₅₀ values in the micromolar range against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and even more promising lower values against Leishmania donovanii.     

Invariant characteristics of horizontal-plane minimum-torque-change movements with one mechanical degree of freedom

 Uno et al. (1989) suggested that movements are organized such that the squared change of torque is minimized over time. Although influential, this theory has attracted much less attention from experimental researchers than the competing minimum-jerk model (Flash and Hogan 1985). One reason for this relative neglect has been the lack of general quantitative predictions, which results from the belief that minimum-torque-change trajectories have to be computed numerically for individual movements and arm-dynamical parameters. In the present paper, we show that for an important special case, that of planar horizontal movements with one mechanical degree of freedom (DOF), it is actually possible to find an analytic expression for the predicted minimum-torque-change trajectories. Based on this mathematical result, we derive a set of properties which are characteristic of these trajectories and compare them to experimental data which have not previously been related to the minimum-torque-change model. Certain discrepancies between these experimental data and minimum-torque-change model predictions are revealed. Received: 3 September 1996 / Accepted in revised form: 7 January 1997     

Evidence of divergent selection for drought and cold tolerance at landscape and local scales in Abies alba Mill. in the French Mediterranean Alps

Understanding local adaptation in forest trees is currently a key research and societal priority. Geographically and ecologically marginal populations provide ideal case studies, because environmental stress along with reduced gene flow can facilitate the establishment of locally adapted populations. We sampled European silver fir (Abies alba Mill.) trees in the French Mediterranean Alps, along the margin of its distribution range, from pairs of high‐ and low‐elevation plots on four different mountains situated along a 170‐km east–west transect. The analysis of 267 SNP loci from 175 candidate genes suggested a neutral pattern of east–west isolation by distance among mountain sites. F_ST outlier tests revealed 16 SNPs that showed patterns of divergent selection. Plot climate was characterized using both in situ measurements and gridded data that revealed marked differences between and within mountains with different trends depending on the season. Association between allelic frequencies and bioclimatic variables revealed eight genes that contained candidate SNPs, of which two were also detected using F_ST outlier methods. All SNPs were associated with winter drought, and one of them showed strong evidence of selection with respect to elevation. Q_ST–F_ST tests for fitness‐related traits measured in a common garden suggested adaptive divergence for the date of bud flush and for growth rate. Overall, our results suggest a complex adaptive picture for A. alba in the southern French Alps where, during the east‐to‐west Holocene recolonization, locally advantageous genetic variants established at both the landscape and local scales.