In-Cell NMR Spectroscopy of Functional Riboswitch Aptamers in Eukaryotic Cells.

We report here the in-cell NMR-spectroscopic observation of the binding of the cognate ligand 2'-deoxyguanosine to the aptamer domain of the bacterial 2'-deoxyguanosine-sensing riboswitch in eukaryotic cells, namely Xenopus laevis oocytes and in human HeLa cells. The riboswitch is sufficiently stable in both cell types to allow for detection of binding of the ligand to the riboswitch. Most importantly, we show that the binding mode established by in vitro characterization of this prokaryotic riboswitch is maintained in eukaryotic cellular environment. Our data also bring important methodological insights: Thus far, in-cell NMR studies on RNA in mammalian cells have been limited to investigations of short (<15 nt) RNA fragments that were extensively modified by protecting groups to limit their degradation in the intracellular space. Here, we show that the in-cell NMR setup can be adjusted for characterization of much larger (≈70 nt) functional and chemically non-modified RNA.

Microenvironmental interactions between endothelial and lymphoma cells: a role for the canonical WNT pathway in Hodgkin lymphoma.

The interaction between vascular endothelial cells (ECs) and cancer cells is of vital importance to understand tumor dissemination. A paradigmatic cancer to study cell-cell interactions is classical Hodgkin Lymphoma (cHL) owing to its complex microenvironment. The role of the interplay between cHL and ECs remains poorly understood. Here we identify canonical WNT pathway activity as important for the mutual interactions between cHL cells and ECs. We demonstrate that local canonical WNT signaling activates cHL cell chemotaxis toward ECs, adhesion to EC layers and cell invasion using not only the Wnt-inhibitor Dickkopf, tankyrases and casein kinase 1 inhibitors but also knockdown of the lymphocyte enhancer binding-factor 1 (LEF-1) and ß-catenin in cHL cells. Furthermore, LEF-1- and ß-catenin-regulated cHL secretome promoted EC migration, sprouting and vascular tube formation involving vascular endothelial growth factor A (VEGF-A). Importantly, high VEGFA expression is associated with a worse overall survival of cHL patients. These findings strongly support the concept that WNTs might function as a regulator of lymphoma dissemination by affecting cHL cell chemotaxis and promoting EC behavior and thus angiogenesis through paracrine interactions.

An A-type double helix of DNA having B-type puckering of the deoxyribose rings.

DNA usually adopts structure B in aqueous solution, while structure A is preferred in mixtures of trifluoroethanol (TFE) with water. However, the octamer d(CCCCGGGG) and other d(C(n)G(n)) fragments of DNA provide CD spectra that suggest that the base-pairs are stacked in an A-like fashion even in aqueous solution. Yet, d(CCCCGGGG) undergoes a cooperative TFE-induced transition into structure A, indicating that an important part of the aqueous duplex retains structure B. NMR spectroscopy shows that puckering of the deoxyribose rings is of the B-type. Hence, combination of the information provided by CD spectroscopy and NMR spectroscopy suggests an unprecedented double helix of DNA in which A-like base stacking is combined with B-type puckering of the deoxyribose rings. In order to determine whether this combination is possible, we used molecular dynamics to simulate the duplex of d(CCCCGGGG). Remarkably, the simulations, completely unrestrained by the experimental data, provided a very stable double helix of DNA, exhibiting just the intermediate B/A features described above. The double helix contained well-stacked guanine bases but almost unstacked cytosine bases. This generated a hole in the double helix center, which is a property characteristic for A-DNA, but absent from B-DNA. The minor groove was narrow at the double helix ends but wide at the central CG step where the Watson-Crick base-pairs were buckled in opposite directions. The base-pairs stacked tightly at the ends but stacking was loose in the duplex center. The present double helix, in which A-like base stacking is combined with B-type sugar puckering, is relevant to replication and transcription because both of these phenomena involve a local B-to-A transition.

A-like guanine-guanine stacking in the aqueous DNA duplex of d(GGGGCCCC).

We have used CD spectroscopy, NMR spectroscopy and unrestrained molecular dynamics to study conformational properties of a DNA duplex formed by the self-complementary octamer d(GGGGCCCC). Its unusual CD spectrum contains features indicating A-like stacking of half of the bases, whereas the other half stack in a B-like fashion. Unrestrained molecular dynamics simulations converged to a stable B-like double-helix of d(GGGGCCCC). However, the double-helix contained a central hole whose size was half of that occurring in structure A. In the canonical structure B, the hole does not exist at all because the base-pairs cross the double-helix centre. The cytosine bases were stacked in the duplex of d(GGGGCCCC) as in structure B, while stacking of the guanine bases displayed features characteristic for structure A. NMR spectroscopy revealed that the A-like guanine-guanine stacking was accompanied by an increased tendency of the deoxyribose rings attached to the guanine bases to be puckered in an A-like fashion. Otherwise, the duplex of d(GGGGCCCC) showed no clash, no bend and no other significant deviation from structure B. The present analysis demonstrates a remarkable propensity of the guanine runs to stack in an A-like fashion even within the B-DNA framework. This property explains why the oligo(dG). oligo(dC) tracts switch into structure A so easily. Secondly, this property may influence replication, because structure A is replicated more faithfully than structure B. Thirdly, the oligo(dG) runs might have played an important role in early evolution, when DNA took on functions that originally evolved on RNA. Fourthly, the present study extends the vocabulary of DNA secondary structures by the heteronomous duplex of d(GGGGCCCC) in which the B-like strand of oligo(dC) is bound to the A-like strand of oligo(dG).

Effect of the carbon source on assessment of degrading bacteria with the spread-plating technique during in situ bioremediation.

Spread-plating belongs to traditional microbiological methods employed for quantification of subsurface microflora during bioremediation projects in the Czechia. Concentration of degrading organisms is estimated from the number of colonies grown on agar plates supplied with contaminant as the sole carbon source. The data obtained during in situ bioremediation of the Dacice site contaminated by cutting oil suggests that changes in the composition of the carbon source in the subsurface may cause a discrepancy between laboratory data and situation in subsurface.

Reaction mechanism and stereochemistry of gamma-hexachlorocyclohexane dehydrochlorinase LinA.

gamma-Hexachlorocyclohexane dehydrochlorinase (LinA) catalyzes the initial steps in the biotransformation of the important insecticide gamma-hexachlorocyclohexane (gamma-HCH) by the soil bacterium Sphingomonas paucimobilis UT26. Stereochemical analysis of the reaction products formed during conversion of gamma-HCH by LinA was investigated by GC-MS, NMR, CD, and molecular modeling. The NMR spectra of 1,3,4,5,6-pentachlorocyclohexene (PCCH) produced from gamma-HCH using either enzymatic dehydrochlorination or alkaline dehydrochlorination were compared and found to be identical. Both enantiomers present in the racemate of synthetic gamma-PCCH were converted by LinA, each at a different rate. 1,2,4-trichlorobenzene (1,2,4-TCB) was detected as the only product of the biotransformation of biosynthetic gamma-PCCH. 1,2,4-TCB and 1,2,3-TCB were identified as the dehydrochlorination products of racemic gamma-PCCH. delta-PCCH was detected as the only product of dehydrochlorination of delta-HCH. LinA requires the presence of a 1,2-biaxial HCl pair on a substrate molecule. LinA enantiotopologically differentiates two 1,2-biaxial HCl pairs present on gamma-HCH and gives rise to a single PCCH enantiomer 1,3(R),4(S),5(S),6(R)-PCCH. Furthermore, LinA enantiomerically differentiates 1,3(S),4(R),5(R),6(S)-PCCH and 1,3(R),4(S),5(S),6(R)-PCCH. The proposed mechanism of enzymatic biotransformation of gamma-HCH to 1,2,4-TCB by LinA consists of two 1,2-anti conformationally dependent dehydrochlorinations followed by 1,4-anti dehydrochlorination.