The impact of selected planned motorways and expressways on the potential accessibility of the Polish-Slovak borderland with respect to tourism development

Further tourism development in the Polish-Slovak borderland, as well as its overall economic development, depends on the construction of a motorway and expressway network. This paper analyses the impact of selected planned motorways and expressways (D1, A4, D3/S69, R1/R3/S7, and R4/S19) on the potential accessibility the Polish-Slovak borderland with respect to the development of tourism. The most important investment project in Slovakia is the completion of the (started) D1 motorway. The R4/S19 and the R1/R3/S7 expressways and the D3 motorway/S69 expressway are expected to contribute to improved cross-border connections.132

The impact of selected planned motorways and expressways on the potential accessibility of the Polish-Slovak borderland with respect to tourism development

Further tourism development in the Polish-Slovak borderland, as well as its overall economic development, depends on the construction of a motorway and expressway network. This paper analyses the impact of selected planned motorways and expressways (D1, A4, D3/S69, R1/R3/S7, and R4/S19) on the potential accessibility of the Polish-Slovak borderland with respect to the development of tourism. The most important investment project in Slovakia is the completion of the (started) D1 motorway. The R4/S19 and the R1/R3/S7 expressways and the D3 motorway/S69 expressway are expected to contribute to improved cross-border connections

Characterization of the molecular chaperone ClpB from the pathogenic spirochaete Leptospira interrogans.

Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that an AAA+ chaperone ClpB (a member of the Hsp100 family) from L. interrogans (ClpBLi) is not only essential for survival of Leptospira under the thermal and oxidative stresses, but also during infection of a host. The aim of this study was to provide further insight into the role of ClpB in the pathogenic spirochaetes and explore its biochemical properties. We found that a non-hydrolysable ATP analogue, ATPγS, but not AMP-PNP induces the formation of ClpBLi hexamers and stabilizes the associated form of the chaperone. ADP also induces structural changes in ClpBLi and promotes its self-assembly, but does not produce full association into the hexamers. We also demonstrated that ClpBLi exhibits a weak ATPase activity that is stimulated by κ-casein and poly-lysine, and may mediate protein disaggregation independently from the DnaK chaperone system. Unexpectedly, the presence of E. coli DnaK/DnaJ/GrpE did not significantly affect the disaggregation activity of ClpBLi and ClpBLi did not substitute for the ClpBEc function in the clpB-null E. coli strain. This result underscores the species-specificity of the ClpB cooperation with the co-chaperones and is most likely due to a loss of interactions between the ClpBLi middle domain and the E. coli DnaK. We also found that ClpBLi interacts more efficiently with the aggregated G6PDH in the presence of ATPγS rather than ATP. Our results indicate that ClpB's importance during infection might be due to its role as a molecular chaperone involved in reactivation of protein aggregates

Structural characteristics of ClpB_Li used in this study.

<p>(A) Comparison of the domain organization of ClpB from <i>L</i>. <i>interrogans</i> and <i>E</i>. <i>coli</i>. Bacterial ClpB proteins are composed of the following domains: N-terminal domain (ND), nucleotide binding domain 1 (NBD1), middle coiled-coil domain (MD), and nucleotide binding domain 2 (NBD2). The functions of the domains are indicated at the top. The amino acid residue numbers are shown for each chaperone and the amino acid sequence identity between ClpB_Ec and ClpB_Li is indicated for each domain. (B) CD spectra of ClpB_Li at 20°C (folded form) and 75°C (unfolded form) are shown. The CD signal was expressed as mean molar residue ellipticity (θ). (C) Temperature-induced changes in the CD signal at 222 nm for ClpB_Li.</p

ATPase activity of ClpB_Li and ClpB_Ec.

<p>The rate of ATP hydrolysis was determined at 37°C in the absence of other proteins (basal activity), in the presence of κ-casein (0.1 mg/ml), poly-lysine (0.04 mg/ml) (polyLys), or aggregated G6PDH (2.1 μM) (aggG6PDH). The average values from three independent experiments are shown with the standard deviations.</p

Summary of sedimentation coefficients for ClpB_Li.

<p>Summary of sedimentation coefficients for ClpB_Li.</p

Effect of the <i>clpB</i>_Li gene expression on the growth and survival of <i>E</i>. <i>coli</i> <i>Δ</i><i>clpB</i> mutant under heat shock.

<p>(A) Immunodetection of ClpB_Li with specific antibodies in <i>E</i>. <i>coliΔclpB</i> cells grown at 30°C and after 2h of heat shock at 45°C. An asterisk indicates ClpB_Li. The position of ClpB_Ec (control of heat-inducible expression) was marked by a circle. (B) Growth curves of <i>E</i>. <i>coliΔclpB</i> cells carrying empty pGB2 (control 1), pGB2-ClpB_Ec (control 2) or pGB2-ClpB_Li exposed to a mild heat shock at 45°C for the indicated times. (C) Survival of the same bacterial strains as in (B) after exposure to a severe heat shock at 50°C for the indicated times. The average values from three independent experiments are shown in (B) and (C).</p

Reactivation of the aggregated substrates in the presence of ClpB_Li and ClpB_Ec.

<p>The reactivation of aggregated enzymes, G6PDH (A) and Fda (B) in the presence of DnaK/DnaJ/GrpE (KJE) from <i>E</i>. <i>coli</i> without ClpB and with ClpB_Ec or ClpB_Li. The native activity of G6PDH or Fda determined before the chemical denaturation or the heat treatment at 55°C, respectively, corresponds to 100%; the fraction of the enzyme activity remaining after the denaturation and also corresponding to the reactivation extent in the absence of chaperones (control) is marked by the broken line. (C) The effect of ClpB_Li and ClpB_Ec on the reactivation of β-galactosidase sequestered into IBs (VP1LAC) isolated from <i>E</i>. <i>coliΔclpB</i> mutant cells. A statistically significant difference in the β-galactosidase activity regain in the absence and presence of ClpB_Li assessed by the paired t-test (using GraphPad Prism software) is indicated as **, p<0.01. The results are presented as the average of three (A, C) or four (B) independent experiments with the standard deviations indicated.</p