Proton Exchange

Complexes of the type (R 2 Cyclam)ZrCl 2 (where R = CH 2 C(H)CH 2 (All), CH 2 C(Me)CH 2 ( Me All), and PhCH 2 (Bn)) react with suitable Grignard reagents to produce the corresponding alkyl derivatives (R 2 Cyclam)ZrR′ 2 (R′ = Me, CH 2 Ph). Thermally induced double metalation of the pending arms of the cyclam ligand led to the formation of complexes ((CH C(H)CH 2 ) 2 Cyclam)Zr, 14, ((CHC(Me)CH 2 ) 2 Cyclam)Zr, 15, or ((C 6 H 4 CH 2 ) 2 Cyclam)Zr, 16. These reactions proceed through C(sp 2 )-H bond activation and R′H elimination and convert the original dianionic tetracoordinated cyclam-based ligands in tetraanionic hexacoordinated ligands that establish two new Zr-C bonds. The cleavage of the Zr-C bonds may be readily achieved by treatment of the bis(ortho-metalated) species 16 with protic substrates (tert-butanol, phenol, thiophenol, aniline, benzophenone hydrazone, pyrazole, and N,N′-diphenylhydrazine), to give rise to (Bn 2 Cyclam)ZrX 2 complexes (X = O t Bu, OPh, SPh, NHPh, NHNCPh 2 , C 3 H 3 N 2 , N,N′-PhNNPh). In catalytic conditions, complexes (All 2 Cyclam)Zr(NMe 2 ) 2 , 14, 15, or 16 convert 2,2-diphenyl-pent-4-enylamine to 2-methyl-4,4diphenylpyrrolidine with 100% selectivity and conversion values varying between 61 and 88% in 4.5 h, at 115 °C. Complexes 14 and 15, which display metalated allyl and methallyl pending groups on the cyclam ring, are the most active species (1.7 < TOF < 2.0 h -1 ). The mechanism of this reaction was studied by density functional theory that revealed two competitive paths, one proceeding through an imido intermediate and another that occurs via an amido ligand. Both cases represent cooperative mechanisms with active participation of the cyclam, as proton exchange between the coordinated substrate and the ligand side arm with reversible C-H activation is a crucial feature of the mechanism.

The hydration of the collagen-like Ac-(Gly-Pro-Hyp) 6 -NH 2 triple-helical peptide in solution was investigated using an integrated set of high-resolution NMR hydration experiments, including different recently developed exchange-network editing methods. This approach was designed to explore the hydration dynamics in the proximity of labile groups, such as the hydroxyproline hydroxyl group, and revealed that the ®rst shell of hydration in collagen-like triple helices is kinetically labile with upper limits for water molecule residence times in the nanosecond to sub-nanosecond range. This result is consistent with a ``hopping'' hydration model in which solvent molecules are exchanged in and out of solvation sites at a rate that is not directly correlated to the degree of site localization. The hopping model thus reconciles the dynamic view of hydration revealed by NMR with the previously suggested partially ordered semi-clathratelike cylinder of hydration. In addition, the nanosecond to sub-nanosecond upper limits for water molecule residence times imply that hydration-dehydration events are not likely to be the rate-limiting step for triple helix self-recognition, complementing previous investigations on water dynamics in collagen ®bers. This study has also revealed labile proton features expected to facilitate the characterization of the structure and folding of triple helices in collagen peptides.

The interaction of humang alectin-1 with av ariety of oligosaccharides, from di-(N-acetyllactosamine) to tetrasaccharides (blood Bt ype-II antigen)h as been scrutinized by using ac ombined approacho fd ifferent NMR experiments, molecular dynamics (MD) simulations, and isothermal titration calorimetry.L igand-and receptor-based NMR experiments assisted by computational methods allowed proposing three-dimensional structures for the different complexes, which explainedt he lack of enthalpy gain when increasing the chemical complexityo ft he glycan. Interestingly,a nd independently of the glycan ligand, the entropy term does not oppose the binding event, ar ather unusual feature for protein-sugar interactions. CLEANEX-PM and relaxation dispersion experiments revealed that sugar binding affected residues far from the binding site and described significant changes in the dynamics of the protein. In particular, motions in the microsecond-millisecond timescale in residues at the protein dimer interface were identified in the presence of high affinity ligands. The dynamic process was furthere xplored by extensive MD simulations, which provided additional support fort he existence of allostery in glycan recognition by human galectin-1.

Complementary oligonucleotides with 5' overhanging deoxyguanosine or deoxycytidine stretches, respectively, of the general form 5'-d(GGGCAARAAC)-5'-d(C-CCGTTYTTG), where R represents the bases adenine (A), hypoxanthine (base of inosine nucleoside, I), purine (M),

Anomalous cross-peaks observed in the NOESY spectra of 2,4-disubstituted thiazolidines and oxazolidines that cannot be attributed to classical dipolar NOE or chemical exchange peaks have been investigated experimentally and computationally and have been shown to arise from scalar cross-relaxation of the first kind. This process is stimulated by the relatively slow modulation of scalar couplings and, for the systems studied, arises from slow on−off proton exchange of the amino nitrogen, a process influenced by solution temperature, acidity, and concentration. The mechanism is likely to be significant for many systems in which proton exchange occurs on the millisecond time scale, and misinterpretation of these cross-peaks may lead to erroneous conclusions should their true origins not be recognized.

The interaction of humang alectin-1 with av ariety of oligosaccharides, from di-(N-acetyllactosamine) to tetrasaccharides (blood Bt ype-II antigen)h as been scrutinized by using ac ombined approacho fd ifferent NMR experiments, molecular dynamics (MD) simulations, and isothermal titration calorimetry.L igand-and receptor-based NMR experiments assisted by computational methods allowed proposing three-dimensional structures for the different complexes, which explainedt he lack of enthalpy gain when increasing the chemical complexityo ft he glycan. Interestingly,a nd independently of the glycan ligand, the entropy term does not oppose the binding event, ar ather unusual feature for protein-sugar interactions. CLEANEX-PM and relaxation dispersion experiments revealed that sugar binding affected residues far from the binding site and described significant changes in the dynamics of the protein. In particular, motions in the microsecond-millisecond timescale in residues at the protein dimer interface were identified in the presence of high affinity ligands. The dynamic process was furthere xplored by extensive MD simulations, which provided additional support fort he existence of allostery in glycan recognition by human galectin-1.

An extensive set of water-1 H magnetic relaxation dispersion (MRD) data are presented for aqueous agarose and gelatin gels. It is demonstrated that the EMOR model, which was developed in a companion paper to this study (see Halle, this issue), accounts for the dependence of the water-1 H spin-lattice relaxation rate on resonance frequency over more than four decades and on pH. The parameter values deduced from analysis of the 1 H MRD data are consistent with values derived from 2 H MRD profiles from the same gels and with small-molecule reference data. This agreement indicates that the water-1 H relaxation dispersion in aqueous biopolymer gels is produced directly by exchange-mediated orientational randomization of internal water molecules or labile biopolymer protons, with little or no role played by collective biopolymer vibrations or coherent spin diffusion. This ubiquitous mechanism is proposed to be the principal source of water-1 H spin-lattice relaxation at low magnetic fields in all aqueous systems with rotationally immobile biopolymers, including biological tissue. The same mechanism also contributes to transverse and rotating-frame relaxation and magnetization transfer at high fields. Magn Reson Med 56:73-81, 2006.

Water productivity need to be enhanced in view of diminishing water supplies. A study was conducted to assess the existing water productivity status in cotton-wheat cropping zone in Punjab Province of Pakistan and identifying factors for its improvement. Six watercourses on four distributaries taking off from Lower Bari Doab Canal (LBDC) of river Ravi were selected in tehsil Kabirwala, district Khanewal. The data regarding crop yield, irrigation water, fertilizer and pesticides was recorded from farmers and cross checked with information collected from Patwaris. The results indicated that mean yield, apparent water productivity (yield/irrigation water) and real water productivity (yield/water evapotranspired) for wheat was 3210 kg ha-1 , 0.43 kg m-3 and 1.12 kg m-3 , respectively. The corresponding values for cotton were 2675 kg ha-1 , 0.22 kg m-3 and 0.26 kg m-3 , respectively. It was concluded that all the three considered factors (irrigation water, fertilizer and pesticide) improved real water productivity (yield/water evapotranspired). Further pesticide in wheat and fertilizer in cotton was the most effective. However, water productivity studies on the larger scale and inclusion of factors (irrigation method, soil fertility, crop variety, irrigation scheduling etc.) influencing water productivity are recommended for future to improve water productivity.

Direct observation of multiple reactive sites in the zeolite HZSM-5, a member of the MFI family of zeolite structures, contradicts the traditional view of only one type of active protonic species in industrially important zeolites. In addition to the well-known Brönsted acid site proton, two other protonic species undergo room-temperature hydrogen-deuterium exchange with an alkane hydrocarbon reagent, including one zeolite moiety characterized by a broad H chemical shift at ca. 12-15 ppm that is reported here for the first time. Although the ca. 13 ppm chemical shift value is consistent with computational predictions from the literature for a surface-stabilized hydroxonium ion in a zeolite, data suggest that the signal does not arise from hydroxonium species but rather from hydroxyls on extra-lattice aluminol species proximate to Brönsted lattice sites, i.e., a small population of highly deshielded acid sites. Double-resonance experiments show that this species is proximate to Al at...

The presence of an abasic site in duplex DNA lowers the thermodynamic stability, as monitored by the optical melting temperature, and decreases the rate of imino proton exchange with water, by about an order of magnitude, as monitored by direct measurement of both the exchange lifetimes and the imino proton TIS. The exchange lifetimes of the imino protons with water as a function of base catalyst concentration were analyzed to determine the origin of the effect of the abasic site on imino exchange lifetimes. Analysis of the results showed that the helix opening rate is not significantly changed by the presence of an abasic site. The differences in exchange lifetimes are attributed to a faster helix closing rate in the presence of an abasic site. The faster rate of helix closing may be an important contribution to the stability of abasic sites in duplex DNA to base-catalyzed elimination reaction. It is noted that duplex DNAs containing analogues of the aldehydic abasic site apparently do not exhibit these exchange lifetime effects.

Water productivity need to be enhanced in view of diminishing water supplies. A study was conducted to assess the existing water productivity status in cotton-wheat cropping zone in Punjab Province of Pakistan and identifying factors for its improvement. Six watercourses on four distributaries taking off from Lower Bari Doab Canal (LBDC) of river Ravi were selected in tehsil Kabirwala, district Khanewal. The data regarding crop yield, irrigation water, fertilizer and pesticides was recorded from farmers and cross checked with information collected from Patwaris. The results indicated that mean yield, apparent water productivity (yield/irrigation water) and real water productivity (yield/water evapotranspired) for wheat was 3210 kg ha-1 , 0.43 kg m-3 and 1.12 kg m-3 , respectively. The corresponding values for cotton were 2675 kg ha-1 , 0.22 kg m-3 and 0.26 kg m-3 , respectively. It was concluded that all the three considered factors (irrigation water, fertilizer and pesticide) improved real water productivity (yield/water evapotranspired). Further pesticide in wheat and fertilizer in cotton was the most effective. However, water productivity studies on the larger scale and inclusion of factors (irrigation method, soil fertility, crop variety, irrigation scheduling etc.) influencing water productivity are recommended for future to improve water productivity.

Photodeposition of metallic nanostructures onto ferroelectric surfaces is typically based on patterning local surface reactivity via electric field poling. Here, we demonstrate metal deposition onto substrates which have been chemically patterned via proton exchange (i.e., without polarization reversal). The chemical patterning provides the ability to tailor the electrostatic fields near the surface of lithium niobate crystals and these engineered fields are used to fabricate metallic nanostructures. The effect of the proton exchange process on the 2 piezoelectric and electrostatic properties of the surface is characterized using voltage modulated atomic force microscopy techniques, which combined with modeling of the electric fields at the surface of the crystal, reveal that the deposition occurs preferentially along the boundary between ferroelectric and proton exchanged regions. The metallic nanostructures have been further functionalized with a target probe molecule, 4-aminothiophenol, from which surface enhanced Raman scattering (SERS) signal is detected, demonstrating the suitability of chemically patterned ferroelectrics as SERS-active templates.

Water productivity need to be enhanced in view of diminishing water supplies. A study was conducted to assess the existing water productivity status in cotton-wheat cropping zone in Punjab Province of Pakistan and identifying factors for its improvement. Six watercourses on four distributaries taking off from Lower Bari Doab Canal (LBDC) of river Ravi were selected in tehsil Kabirwala, district Khanewal. The data regarding crop yield, irrigation water, fertilizer and pesticides was recorded from farmers and cross checked with information collected from Patwaris. The results indicated that mean yield, apparent water productivity (yield/irrigation water) and real water productivity (yield/water evapotranspired) for wheat was 3210 kg ha-1 , 0.43 kg m-3 and 1.12 kg m-3 , respectively. The corresponding values for cotton were 2675 kg ha-1 , 0.22 kg m-3 and 0.26 kg m-3 , respectively. It was concluded that all the three considered factors (irrigation water, fertilizer and pesticide) improved real water productivity (yield/water evapotranspired). Further pesticide in wheat and fertilizer in cotton was the most effective. However, water productivity studies on the larger scale and inclusion of factors (irrigation method, soil fertility, crop variety, irrigation scheduling etc.) influencing water productivity are recommended for future to improve water productivity.

Water productivity need to be enhanced in view of diminishing water supplies. A study was conducted to assess the existing water productivity status in cotton-wheat cropping zone in Punjab Province of Pakistan and identifying factors for its improvement. Six watercourses on four distributaries taking off from Lower Bari Doab Canal (LBDC) of river Ravi were selected in tehsil Kabirwala, district Khanewal. The data regarding crop yield, irrigation water, fertilizer and pesticides was recorded from farmers and cross checked with information collected from Patwaris. The results indicated that mean yield, apparent water productivity (yield/irrigation water) and real water productivity (yield/water evapotranspired) for wheat was 3210 kg ha-1 , 0.43 kg m-3 and 1.12 kg m-3 , respectively. The corresponding values for cotton were 2675 kg ha-1 , 0.22 kg m-3 and 0.26 kg m-3 , respectively. It was concluded that all the three considered factors (irrigation water, fertilizer and pesticide) improved real water productivity (yield/water evapotranspired). Further pesticide in wheat and fertilizer in cotton was the most effective. However, water productivity studies on the larger scale and inclusion of factors (irrigation method, soil fertility, crop variety, irrigation scheduling etc.) influencing water productivity are recommended for future to improve water productivity.

A method is proposed to measure global bending in DNA and RNA structures. It relies on a properly defined averaging of base-fixed coordinate frames, computes mean frames of suitably chosen groups of bases and uses these mean frames to evaluate bending. The method is applied to DNA A-tracts, known to induce considerable bend to the double helix. We performed atomistic molecular dynamics simulations of sequences containing the A 4 T 4 and T 4 A 4 tracts, in a single copy and in two copies phased with the helical repeat. Various temperature and salt conditions were investigated. Our simulations indicate bending by roughly 10 per A 4 T 4 tract into the minor groove, and an essentially straight structure containing T 4 A 4 , in agreement with electrophoretic mobility data. In contrast, we show that the published NMR structures of analogous sequences containing A 4 T 4 and T 4 A 4 tracts are significantly bent into the minor groove for both sequences, although bending is less pronounced for the T 4 A 4 containing sequence. The bending magnitudes obtained by frame averaging are confirmed by the analysis of superhelices composed of repeated tract monomers.

To determine the origin of the UV-specific CC to TT tandem mutation at the CC site, we made a duplex DNA decamer containing a uracil cis-syn cyclobutane dimer (CBD) as the deaminated model of a cytosine dimer. Two-dimensional 1 H-NMR spectroscopy studies were performed on this sequence where two adenines (Ade) were opposite to the uracil dimer. Two imino protons of the uracil dimer were found to retain Watson-Crick hydrogen bonding with the opposite Ade, although the 59-U(NH) of the dimer site showed unusual upfield shift like that of the 59-T(NH) of the TT dimer, which seemed to be associated with deshielding by the flanking base rather than with reduced hydrogen bonding. (McAteer et al. 1998, J. Mol. Biol. 282:1013-1032). Hydrogen bondings at the dimer site were also supported by detecting typical strong nuclear Overhauser effects (NOE) between two imino protons and the opposite Ade H2 or NH 2. But sequential NOE interactions of base protons with sugar protons were absent at the two flanking nucleotides of the 59 side of the uracil dimer and at the intradimer site, contrasting with its thymine analog where sequential NOE was absent only at the A4-T5 step. In addition, NOE cross peak for U5(NH) $ A4(H2) was detected, although the NOE interactions of U6(NH) with A7(H2) and A17(H2) were not observed in contrast to the thymine dimer duplex. This different local structural alteration may be affected by the induced right-hand twisted puckering mode of cis-syn cyclobutane ring of the uracil dimer in the B-DNA duplex, even though the isolated uracil dimer had left-hand twisted puckering rigidly. In parallel, these observations may be correlated with observed differences in mutagenic properties between cis-syn UU dimer and cis-syn TT dimer.

A two-year cave monitoring study at Westcave Preserve in central Texas provides insight into the controls on the rate of calcite growth and drip water Mg/Ca, Sr/Ca, and Ba/Ca variations. The cave is shallow and has a large ratio of its opening area to its volume, which results in year-round ventilation of the cave. Unlike larger and deeper caves in the region that ventilate seasonally, cave-air temperature and CO 2 concentrations at Westcave are near atmospheric throughout the year and calcite growth is continuous. Changes in the rate of calcite growth positively correlate with seasonal temperature variations at all six drip sites studied (r 2 = 0.12-0.76; mean r 2 = 0.47). Average monthly surface air temperature is positively correlated with drip-water Sr/Ca at five of six drip sites studied (r 2 = 0.21-0.80; mean r 2 = 0.44), and Ba/Ca at all six sites (r 2 = 0.41-0.85; mean r 2 = 0.57); whereas this correspondence is only seen in one of six drip sites for Mg/Ca. Applying geochemical modeling of mineralsolution reactions to the Sr/Ca and Ba/Ca time series at Westcave indicates that the evolution of drip-water Sr/Ca and Ba/Ca can be accounted for by two mechanisms: (1) prior calcite precipitation and/or incongruent calcite dissolution (PCP/ICD), which dominate drip-water evolution at one site; and (2) a combination of PCP/ICD and water-rock interaction (WRI) at the other five drip sites. The results suggest a possible seasonality in the operation of the mechanisms of drip-water evolution, whereby PCP/ICD plays a larger role than WRI during the warmer months of the year. Understanding drip-water seasonal Sr/Ca and Ba/Ca variations has implications for paleoclimate studies using speleothems. It is important to first determine if seasonal geochemical variations in drip waters can be identified. One can then determine if these variations are preserved as geochemical laminae in speleothems, which may then provide seasonal temperature variations and thus seasonal age constraints for speleothems. Determining the proportional contributions of the mineral-solution reactions that drive drip-water trace element variations for different drip sites, as well as the extent to which trace element concentrations vary seasonally, will help inform speleothem sample selection and interpretation of geochemical data for paleoclimate study. Our results indicate that speleothems near the well-ventilated entrances of many larger and deeper caves may warrant further consideration for paleoclimate studies.

To determine the origin of the UV-specific CC to TT tandem mutation at the CC site, we made a duplex DNA decamer containing a uracil cis-syn cyclobutane dimer (CBD) as the deaminated model of a cytosine dimer. Two-dimensional 1 H-NMR spectroscopy studies were performed on this sequence where two adenines (Ade) were opposite to the uracil dimer. Two imino protons of the uracil dimer were found to retain Watson-Crick hydrogen bonding with the opposite Ade, although the 59-U(NH) of the dimer site showed unusual upfield shift like that of the 59-T(NH) of the TT dimer, which seemed to be associated with deshielding by the flanking base rather than with reduced hydrogen bonding. (McAteer et al. 1998, J. Mol. Biol. 282:1013-1032). Hydrogen bondings at the dimer site were also supported by detecting typical strong nuclear Overhauser effects (NOE) between two imino protons and the opposite Ade H2 or NH 2. But sequential NOE interactions of base protons with sugar protons were absent at the two flanking nucleotides of the 59 side of the uracil dimer and at the intradimer site, contrasting with its thymine analog where sequential NOE was absent only at the A4-T5 step. In addition, NOE cross peak for U5(NH) $ A4(H2) was detected, although the NOE interactions of U6(NH) with A7(H2) and A17(H2) were not observed in contrast to the thymine dimer duplex. This different local structural alteration may be affected by the induced right-hand twisted puckering mode of cis-syn cyclobutane ring of the uracil dimer in the B-DNA duplex, even though the isolated uracil dimer had left-hand twisted puckering rigidly. In parallel, these observations may be correlated with observed differences in mutagenic properties between cis-syn UU dimer and cis-syn TT dimer.

To determine the origin of the UV-specific CC to TT tandem mutation at the CC site, we made a duplex DNA decamer containing a uracil cis-syn cyclobutane dimer (CBD) as the deaminated model of a cytosine dimer. Two-dimensional 1 H-NMR spectroscopy studies were performed on this sequence where two adenines (Ade) were opposite to the uracil dimer. Two imino protons of the uracil dimer were found to retain Watson-Crick hydrogen bonding with the opposite Ade, although the 59-U(NH) of the dimer site showed unusual upfield shift like that of the 59-T(NH) of the TT dimer, which seemed to be associated with deshielding by the flanking base rather than with reduced hydrogen bonding. (McAteer et al. 1998, J. Mol. Biol. 282:1013-1032). Hydrogen bondings at the dimer site were also supported by detecting typical strong nuclear Overhauser effects (NOE) between two imino protons and the opposite Ade H2 or NH 2. But sequential NOE interactions of base protons with sugar protons were absent at the two flanking nucleotides of the 59 side of the uracil dimer and at the intradimer site, contrasting with its thymine analog where sequential NOE was absent only at the A4-T5 step. In addition, NOE cross peak for U5(NH) $ A4(H2) was detected, although the NOE interactions of U6(NH) with A7(H2) and A17(H2) were not observed in contrast to the thymine dimer duplex. This different local structural alteration may be affected by the induced right-hand twisted puckering mode of cis-syn cyclobutane ring of the uracil dimer in the B-DNA duplex, even though the isolated uracil dimer had left-hand twisted puckering rigidly. In parallel, these observations may be correlated with observed differences in mutagenic properties between cis-syn UU dimer and cis-syn TT dimer.

A new method for the determination of the kinetics of exchange of the imino protons of DNA duplex is reported using a combination NOESY and ROESY experiments at short mixing times (<20 ms). These results have been compared with the commonly used longitudinal relaxation approach through the T1 measurement. To calculate kex and Tex by ROESY-NOESY experiment, the volume of the cross-peaks between imino protons and water in the NOESY and ROESY spectra have been measured separately from the magnetization term. This work shows that the present approach for the measurement of the kinetics of slow exchanging imino protons of DNA duplex is comparable to the saturation recovery experiment in which the exchange rate can be accelerated by the addition of a base catalyst. The present ROESY-NOESY approach has been found to be particularly useful and reasonably accurate for the measurement of exchange kinetics of both the fastand slow-exchanging imino protons in DNA duplex both under non-physiological and physiological condition where the saturation recovery method can not be used.

The 5-methyl group of thymidine residues protrudes into the major groove of double helical DNA. The structural in¯uence of this exocyclic substituent has been examined using a PCR-made 160 bp fragment in which thymidine residues were replaced with uridine residues. We show that the dT 3 dU substitution and the consequent deletion of the methyl group affects the cleavage of DNA by deoxyribonuclease I and micrococcal nuclease. Analysis of the DNase I cleavage sites, in terms of di and trinucleotides, indicates that homopolymeric tracts of d(AT) become signi®cantly more susceptible to DNase I cleavage when uridine is substituted for thymidine residues. The results indicate that removal of the thymidine methyl groups from the major groove at AT tracts induces structural perturbations that transmit into the opposite minor groove, where they can be detected by endonuclease probing. In contrast, DNase I footprinting experiments with different mono and bis-intercalating drugs reveal that dT 3 dU substitution does not markedly affect sequence-speci®c drug-DNA recognition in the minor or major groove of the double helix. The consequences of demethylation of thymidine residues are discussed in terms of changes in the minor groove width connected to variations in the¯exibility of DNA and the intrinsic curvature associated with AT tracts. The study identi®es the methyl group of thymine as an important molecular determinant controlling the width of the minor groove and/or the¯exibility of the DNA.

Complementary oligonucleotides with 5' overhanging deoxyguanosine or deoxycytidine stretches, respectively, of the general form 5'-d(GGGCAARAAC)-5'-d(C-CCGTTYTTG), where R represents the bases adenine (A), hypoxanthine (base of inosine nucleoside, I), purine (M),

Water productivity need to be enhanced in view of diminishing water supplies. A study was conducted to assess the existing water productivity status in cotton-wheat cropping zone in Punjab Province of Pakistan and identifying factors for its improvement. Six watercourses on four distributaries taking off from Lower Bari Doab Canal (LBDC) of river Ravi were selected in tehsil Kabirwala, district Khanewal. The data regarding crop yield, irrigation water, fertilizer and pesticides was recorded from farmers and cross checked with information collected from Patwaris. The results indicated that mean yield, apparent water productivity (yield/irrigation water) and real water productivity (yield/water evapotranspired) for wheat was 3210 kg ha-1 , 0.43 kg m-3 and 1.12 kg m-3 , respectively. The corresponding values for cotton were 2675 kg ha-1 , 0.22 kg m-3 and 0.26 kg m-3 , respectively. It was concluded that all the three considered factors (irrigation water, fertilizer and pesticide) improved real water productivity (yield/water evapotranspired). Further pesticide in wheat and fertilizer in cotton was the most effective. However, water productivity studies on the larger scale and inclusion of factors (irrigation method, soil fertility, crop variety, irrigation scheduling etc.) influencing water productivity are recommended for future to improve water productivity.

Crystal engineering techniques that mimic triple helical fragments in the crystal lattice are able to reveal interesting features of short triple Pu 3 PuPy helical fragments. The nonamer d(GCGAATTCG) and decamer d(GGCCAATTGG), containing respectively one and two overhanging guanines, were designed to form G 3 GC triplets in the crystal packing. To introduce a third subsequent T 3 AT triplet and thus a Pu 3 PuPy to Py 3 PuPy triplet transition, the decamer was further extended with one overhanging thymine residue. The resulting undecamer d(TGGCCTTAAGG) forms an extra parallel Hoogsteen T 3 AT triplet at one end of the duplex and an unexpected (swung out over 270°), antiparallel reverse-Hoogsteen T 3 AT triplet at the other end of the duplex, providing detailed X-ray structural models of parallel and antiparallel T 3 AT triplets. The derived parameters allow for the construction of larger parallel and antiparallel triple helical models.

The opening pathway of wobble pairs dG‚T and rG‚U has been investigated in four DNA and two RNA duplexes. Using NMR spectroscopy, we measured the imino proton exchange of both G(H1) and T/U(H3), catalyzed by ammonia, tris, and OH -, and we calculated the free energy surface related to G‚T/U opening by molecular dynamics simulations. Taken together the experimental and theoretical results, we suggest that wobble pairs open through a coupled rotation of the bases toward the major groove where exchange of both imino protons takes place with the surrounding water. † CNRS UPR 9080. Present address:

The effects of substitutions on various NMR parameters, which may influence the hydrogen bond strengths of Watson-Crick base pairs, were investigated for DNA dodecamers containing 5-substituted-2&#39;-deoxyuridine derivatives and 2&#39;-deoxynebularine in the oligomers, 5&#39;-d(CGCGNA TX CGCG)-3&#39;; where N and X are 2&#39;-deoxy nebularine and [N3-(15)N]-2&#39;-deoxyuridine derivatives. The substitution effects on NMR parameters were linearly correlated with the pKa values of the 2&#39;-dexoyuridine derivatives.

The effects on various NMR parameters of substitutions, which may influence the hydrogen bond strengths of Watson-Crick-type base pairs, were investigated for DNA dodecamers containing 5-substituted-2&#39;-deoxyuridine derivatives in oligomers, 5&#39;-d(CGCGAATXCGCG)-3&#39;, where A and X were [ul-15N]-2&#39;-deoxyadenosine and [3(-15)N]-2&#39;-deoxyuridine derivatives. The substitution effects on the NMR parameters were linearly correlated with the pKa values of the 2&#39;-deoxyuridine derivatives.

Thanks to rather large (5-9 Hz) long-range imino proton-13 C J-couplings, heteronuclear correlation experiments in H 2 O provide unambiguous assignment of imino protons by intranucleotide through-bond connectivities to guanosine H8 and thymidine CH 3 protons, or sequence-specific assignment of non-exchangeable protons when the imino protons are identified independently. This method is demonstrated in the Dickerson dodecamer [d(CGCGAATTCGCG)] 2 and in a human telomeric fragment of 22 nucleotides.

Nuclear Overhauser effect-derived distances between adenine H2 protons and anomeric Hi' protons on the same strand or on the complementary strand are presented for several different DNA duplexes. The cross-strand (n)AH2 to (m + 1)Hl' distances [designated as x, where (u) and (m) are complementary residues] vary by up to 1 A depending on the sequence. In all possible A-containing pyrimidine-purine steps (CA, TG, and TA), x is >4.5 A. In GA steps, x varies within rather wide limits in the range 3.8-4.5 A, whereas in AA steps the lower limit is 3.7 A and the upper limit is ==4.2 A. In purine-purine steps, x is affected by at least three factors: (i) adjacent pyrimidine-purine steps at the 5' end [e.g., YRA sequences (where Y = T or C and R = G or A)], or a pyrimidine-purine step at the 3' end of the pyrimidinepyrimidine step on the complementary strand, cause x to increase, (fi) an AT step at the 3' end of a purine-purine step (e.g., RAT) causes x to decrease, and (ii) substitution of bases at the next-nearest neighbor position leads to changes in x at GA and AA steps. The latter factor seems to be due to a cooperative effect arising from formation of the "anomalous" B' structure when the substitution produces an A.T1, tract (which always produces a decrease in x). The data indicate that Abbreviation: NOESY, nuclear Overhauser effect spectroscopy. §Present address:

Starting from a model first developed by W. Li [44], an expression for the correlation function of a model is obtained. We also obtain an asymptotic expression for the exponents as a function of the probability of expansion. The relationship with DNA evolution is also established.

Studies of 1H NMR selective saturation recovery were performed to determine the imino proton exchange with solvent water of the base pairs in the Eco RI endonuclease recognition sequence GAATTC, placed at the center of selfcomplementary decamer and dodecamer oligonucleotides. In one oligonucleotide the innermost adenine was replaced by the fluorescent base analogue 2-aminopurine (2AP). From the measurements at different concentrations of TRIS buffer acting as proton exchange catalyst, base pair lifetimes were evaluated. The results at 250 show that the AT base pairs have lifetimes of the order of a few ms, whereas the surrounding CC base pairs in a dodecamer have lifetimes of about 100 ms. The (2AP)T base pair has a shorter lifetime than the corresponding AT base pair. The temperature dependent optical absorption, and for the 2AP containing oligonucleotide fluorescence, were used to study the single strand-duplex equilibrium of the decamers. The results indicate that NMR and the optical techniques, although applied at very different concentrations, monitor the same conformational transition of the oligonucleotide.

The structure of parallel-stranded duplexes of DNA-containing a mixture of guanines (G) and adenines (A) is studied by means of molecular dynamics (MD) simulation, as well as NMR and circular dichroism (CD) spectroscopy. Results demonstrate that the structure is based on the Hoogsteen motif rather than on the reverse Watson-Crick one. Molecular dynamics coupled to thermodynamic integration (MD/TI) calculations and melting experiments allowed us to determine the effect of 8-amino derivatives of A and G and of 8-amino-2′-deoxyinosine on the stability of parallel-stranded duplexes. The large stabilization of the parallel-stranded helix upon 8-amino substitution agrees with a Hoogsteen pairing, confirming MD, NMR, and CD data, and suggests new methods to obtain DNA triplexes for antigene and antisense purposes.

Nuclear magnetic resonance spectroscopy has been used to characterize the kinetics and energetics of opening of base pairs in the DNA dodecamer [d(CGCAAATTTGCG)12. The dodecamer contains an A3T3 tract that induces intrinsic curvature of the helix axis. Previous studies from this and other laboratories have shown that the kinetics of base pair opening in AnTn tracts is unique: the opening rates of the A.T base pairs in the interior of the tract are much lower than that of the A-T base pair at the 5'-end of the tract. In the present work, we have investigated the energetics of the pathways for opening of the A.T base pairs in the A3T3 tract. The energetic parameters of the activated state(s) are obtained from the temperature dependence of the opening rate constants. The lower opening rates for the A.T base pairs situated in the interior of the tract are shown to originate from higher activation enthalpies which are compensated, in part, by increases in the activation entropies. We have also obtained an energetic characterization of the open state(s) of the A.T base pairs In the dodecamer by measuring the equilibrium constants for base pair opening and their temperature dependence. The results suggest that the transitions from closed to open state(s) in the A.T base pairs of the A3T3 tract are energetically similar.

Nuclear magnetic resonance spectroscopy has been used to characterize the kinetics and energetics of opening of base pairs in the DNA dodecamer [d(CGCAAATTTGCG)12. The dodecamer contains an A3T3 tract that induces intrinsic curvature of the helix axis. Previous studies from this and other laboratories have shown that the kinetics of base pair opening in AnTn tracts is unique: the opening rates of the A.T base pairs in the interior of the tract are much lower than that of the A-T base pair at the 5'-end of the tract. In the present work, we have investigated the energetics of the pathways for opening of the A.T base pairs in the A3T3 tract. The energetic parameters of the activated state(s) are obtained from the temperature dependence of the opening rate constants. The lower opening rates for the A.T base pairs situated in the interior of the tract are shown to originate from higher activation enthalpies which are compensated, in part, by increases in the activation entropies. We have also obtained an energetic characterization of the open state(s) of the A.T base pairs In the dodecamer by measuring the equilibrium constants for base pair opening and their temperature dependence. The results suggest that the transitions from closed to open state(s) in the A.T base pairs of the A3T3 tract are energetically similar.

We have designed programmable DNA-based nanoswitches whose closing/opening can be triggered over specific different pH windows. These nanoswitches form an intramolecular triplex DNA structure through pH-sensitive parallel Hoogsteen interactions. We demonstrate that by simply changing the relative content of TAT/CGC triplets in the switches, we can rationally tune their pH dependence over more than 5 pH units. The ability to design DNA-based switches with tunable pH dependence provides the opportunity to engineer pH nanosensors with unprecedented wide sensitivity to pH changes. For example, by mixing in the same solution three switches with different pH sensitivity, we developed a pH nanosensor that can precisely monitor pH variations over 5.5 units of pH. With their fast response time (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;200 ms) and high reversibility, these pH-triggered nanoswitches appear particularly suitable for applications ranging from the real-time monitoring of pH changes in vivo to the development of pH sensitive smart nanomaterials.

1 D and 2D NMR investigations of the 15 residue deoxynucleotide sequence d(TCTCTC-TTT-GAGAGA) show that above pH = 6.5 the molecule adopts a B-form hairpin conformation. As the pH is lowered below 6.5 molecules progressively associate in pairs to form a partially triple helical, partially single stranded structure in which the bases of the oligopyrimidine d(TC)3 tract from one molecule form Hoogsteen pairs with the d(G-A)3 tract of the other. Imino protons of protonated cytosines can be observed at very low field (= 15 ppm). The enthalpy of triplex formation was estimated by NMR techniques to be -16 kcal mol-1. Intense H6 to H3' cross peaks from residues in all three strands suggest the presence of N-type sugars at some but not at all possible sites. Surprisingly strong cross peaks between H5' or H5" and non-exchangeable base protons are also observed. These suggest that certain of the 05'-C5'-C4'-C3' phosphate backbone torsion angles (e) are unusual.

Pots used for experiments conducted on plants grown in them create rooting environments that are affected by limited soil volume, which can affect various physiological processes, including transpiration, and plant growth. However, the applicability of results from pot experiments to the field has received limited attention. The objective of this study was to compare the growth and transpiration of maize (Zea mays L.) and winter wheat (Triticum aestivum L.) when grown in pots and field plots under various constant water deficits. The experiments were conducted under similar environmental conditions for both pots and plots. Transpirational responses at both transient (RTTr) and daily (RDTr) time scales to a decreasing fraction of available soil water (FASW) were analyzed. For a comparable FASW, there was a significant reduction in shoot dry weight and total transpiration for plants in pots compared to those in plots. A parabolic relationship between shoot dry weight and total transpiration existed and was not influenced by soil volume or crop type. The plot experiment data for both crops was consistent with pot data for the response of RDTr and RTTr to changes in FASW, which was represented by a linearplateau function. However, the threshold values were significantly different for the two time scales. The threshold values and slopes of the linear-plateau function for maize and wheat were not significantly different in the response of RTTr to FASW, but were significantly different in the response of RDTr to FASW. Therefore, the transpirational responses of the selected maize and winter wheat hybrids to soil drought were different at the daily and transient time scales.

Abstract A method is proposed to measure global bending in DNA and RNA structures. It relies on a properly defined averaging of base-fixed coordinate frames, computes mean frames of suitably chosen groups of bases and uses these mean frames to evaluate bending. The method is applied to DNA A-tracts, known to induce considerable bend to the double helix.

Nucleic acid triplexes are formed by sequence-specific interactions between single-stranded polynucleotides and the double helix. These triplexes are implicated in genetic recombination in vivo and have application to areas that include genome analysis and antigene therapy. Despite the importance of the triple helix, only limited high-resolution structural information is available. The x-ray crystal structure of the oligonucleotide d(GGCCAATTGG) is described; it was designed to contain the d(G middle dotGC)2 fragment and thus provide the basic repeat unit of a DNA triple helix. Parameters derived from this crystal structure have made it possible to construct models of both parallel and antiparallel triple helices.

Using 1H NMR spectroscopy, the base-pair opening dynamics of an antiparallel foldback DNA triplex and the corresponding duplex has been characterized via catalyzed imino proton exchange. The triplex system was found to be in an equilibrium between a duplex and a triplex form. The exchange rate between the two forms (i.e., the on/off-rate of the third strand) was measured to be 5 s-1 at 1 °C, and the base-pair dynamics of both forms were investigated separately. Both Watson-Crick and reverse Hoogsteen base pairs were found to have base-pair lifetimes in the order of milliseconds. The stability of the Watson-Crick base pairs was, however, substantially increased in the presence of the third strand. In the DNA triplex, the opening dynamics of the reverse Hoogsteen base pairs was significantly faster than the dynamics of the Watson-Crick pairs. We were able to conclude that, for both Watson-Crick and reverse Hoogsteen base pairs, spontaneous and individual opening from within the closed base triplet is the dominating opening pathway.

Optimisation of DNA triplex stability is of fundamental importance in the anti-gene strategy. In the present work, thermal denaturation studies by UV-spectrophotometry and structural and dynamical characterizations by NMR spectroscopy have been used systematically to investigate the effects on triplex stability of isolated insertions of different base triplets into an otherwise homogeneous 15-mer dT∙dA-dT oligo-triplex. It is found that insertion of a single central C+∙G-C or T∙D-T triplet (D = 2,6-diaminopurine) leads to a pronounced stabilization (up to 20 deg. C if the cytosine base is C5 methylated) at acidic as well as neutral pH. To a smaller degree, this is the case also for a C+∙I-C triplet insertion. Using imino proton exchange measurements, it is shown that insertion of a DT base-pair in the underlying duplex perturbs the intrinsic A-tract structure in the same way as has been shown for a GC insert. We propose that the intrinsic properties of A-tract duplex DNA (e. g. high propeller twist and rigidity) are unfavourable for triplex formation and that GC- or DT-inserts stabilize the triplex by interfering with the A-tract features of the underlying duplex. The C+∙I-C triplet without the N2 amino group in the minor groove is readily accommodated within the typical, highly propeller-twisted A-tract structure. This might be related to its smaller effect on the stability of the corresponding triplex. These results may be valuable for understanding DNA triplex formation in vivo as well as for the design of efficient triplex-forming oligonucleotides and in choosing suitable target sequences in the anti-gene strategy.

The base-pair opening kinetics of the self-complementary oligomer d(CGCGAATTCGCG)2 has been derived from NMR measurements of the imino proton exchange. In general, it has previously been found that imino proton exchange in duplex DNA is limited by the proton-transfer step from the open state and that the dependence of the exchange times on the inverse concentration of an added exchange catalyst is linear. In the present study, a curvature is observed for, in particular, the innermost AT base pair with both ammonia and trimethylamine (TMA) as exchange catalysts. The two catalysts act on the same open states, but the accessibility of TMA is reduced by a factor of 2-3 compared to ammonia. Assuming that ammonia accesses the imino proton equally in the open state of the base pair and in the mononucleoside, the curvature is consistent with 7-9% of the openings ending in open states with lifetimes of about 1 ís while the bulk of open-state lifetimes fall in the nanosecond range. A curvature is also found for the exchange times of the imino protons in the A-tract sequence CGCA8CGC/GCGT8GCG. This curvature becomes increasingly pronounced from the 5¢-end toward the center of the tract and hereby seems to be correlated with the contraction of the minor groove. Thus, while the base-pair lifetimes deduced from the present study are in accordance with previous measurements, a substantial fraction of the open states formed by the central AT-base pairs in the two oligomers exhibits microsecond lifetimes in contrast to previous estimates in the nanosecond range. These findings may be of relevance for the way sequence specific recognition is accomplished by proteins and ligands.

""Sequences of four or more AT base pairs without a 5΄-TA-3΄ step, so-called A-tracts, influence the global properties of DNA by causing curvature of the helix axis if phased with the helical repeat and also influence nucleosome packaging. Hence it is interesting to understand this phenomenon on the molecular level, and numerous studies have been devoted to investigations of dynamical and structural features of A-tract DNA. It was early observed that anomalously slow base pair-opening kinetics were a striking physical property unique to DNA A-tracts (Leroy, J. L., Charretier, E., Kochoyan, M.,
and Gueron, M. (1988) Biochemistry 27, 8894–8898). Furthermore, a strong correlation between DNA curvature and anomalously slow base pair-opening dynamics was found. In the present work it is shown, using imino proton exchange measurements by NMR spectroscopy that the main contribution to the dampening of the base pair-opening fluctuations in A-tracts comes from the C5 methylation of the thymine base. Because the methyl group has been shown to have a very limited effect on the DNA curvature as well as the structure of the DNA helix, the thymine C5 methyl group stabilizes the helix
directly. Empirical potential energy calculations show that methylation of the tract improves the stacking energy of a base pair with its neighbors in the tract by 3–4 kcal/mol.""