On the origin of the temperature dependence of the supercoiling free energy.

Monte Carlo simulations using temperature-invariant torsional and bending rigidities fail to predict the rather steep decline of the experimental supercoiling free energy with increasing temperature, and consequently fail to predict the correct sign and magnitude of the supercoiling entropy. To illustrate this problem, values of the twist energy parameter (E(T)), which governs the supercoiling free energy, were simulated using temperature-invariant torsion and bending potentials and compared to experimental data on pBR322 over a range of temperatures. The slope, -dE(T)/dT, of the simulated values is also compared to the slope derived from previous calorimetric data. The possibility that the discrepancies arise from some hitherto undetected temperature dependence of the torsional rigidity was investigated. The torsion elastic constant of an 1876-bp restriction fragment of pBR322 was measured by time-resolved fluorescence polarization anisotropy of intercalated ethidium over the range 278-323 K, and found to decline substantially over that interval. Simulations of a 4349-bp model DNA were performed using these measured temperature-dependent torsional rigidities. The slope, -dE(T)/dT, of the simulated data agrees satisfactorily with the slope derived from previous calorimetric measurements, but still lies substantially below that of Duguet's data. Models that involve an equilibrium between different secondary structure states with different intrinsic twists and torsion constants provide the most likely explanation for the variation of the torsion constant with T and other pertinent observations.     

Latent heat loss of dairy cows in an equatorial semi-arid environment

The present study aimed to evaluate evaporative heat transfer of dairy cows bred in a hot semi-arid environment. Cutaneous (E(S)) and respiratory (E(R)) evaporation were measured (810 observations) in 177 purebred and crossbred Holstein cows from five herds located in the equatorial semi-arid region, and one herd in the subtropical region of Brazil. Rectal temperature (T(R)), hair coat surface temperature (T(S)) and respiratory rate (F(R)) were also measured. Observations were made in the subtropical region from August to December, and in the semi-arid region from April to July. Measurements were done from 1100 to 1600 hours, after cows remained in a pen exposed to the sun. Environmental variables measured in the same locations as the animals were black globe temperature (T(G)), air temperature (T(A)), wind speed (U), and partial air vapour pressure (P(V)). Data were analysed by mixed models, using the least squares method. Results showed that average E(S) and E(R) were higher in the semi-arid region (117.2 W m(-2) and 44.0 W m(-2), respectively) than in the subtropical region (85.2 W m(-2) and 30.2 W m(-2), respectively). Herds and individual cows were significant effects (P < 0.01) for all traits in the semi-arid region. Body parts did not affect T(S) and E(S) in the subtropical region, but was a significant effect (P < 0.01) in the semi-arid region. The average flank T(S) (42.8°C) was higher than that of the neck and hindquarters (39.8°C and 41.6°C, respectively). Average E(S) was higher in the neck (133.3 W m(-2)) than in the flank (116.2 W m(-2)) and hindquarters (98.6 W m(-2)). Coat colour affected significantly both T(S) and E(S) (P < 0.01). Black coats had higher T(S) and E(S) in the semi-arid region (41.7°C and 117.2 W m(-2), respectively) than white coats (37.2°C and 106.7 W m(-2), respectively). Rectal temperatures were almost the same in both subtropical and semi-arid regions. The results highlight the need for improved management methods specific for semi-arid regions.     

Effect of polyethylene glycol on the supercoiling free energy of DNA

The supercoiling free energy of pUC19 DNA [2686 base pairs (bp)] was measured in various concentrations of PEG 8000 (polyethylene glycol; molecular weight 8000) by the topoisomer distribution method. The effective twist energy parameter (E(T)) that governs the supercoiling free energy declined linearly by 1.9-fold with increasing w/v % PEG from 0 to 7.5%, which lies below the threshold for intermolecular condensation. In principle, PEG could affect E(T) either via an osmotic exclusion mechanism or by altering the torsion elastic constant, bending rigidity, or self-repulsions of the DNA. Possible alterations of the DNA secondary structure and torsion elastic constant were assessed by CD spectroscopy and time-resolved fluorescence polarization anisotropy of intercalated ethidium. Up to 7.5% PEG, the secondary structure of the DNA remained largely unaltered, as evidenced by (1) the absence of any significant change in the CD spectrum, (2) an extremely small relative decrease (-0.0013) in intrinsic twist, and (3) a negligibly small change in the torsion elastic constant. The observed reduction in E(T) cannot be ascribed primarily to a decrease in torsion elastic constant, and most likely does not stem from a decrease in bending rigidity either. The decrease in medium dielectric constant due to PEG should increase the self-repulsions, and thereby increase E(T), which is opposite to the observed trend. Instead, the observed decline in E(T) is attributed to an osmotic exclusion mechanism. The change in molar volume excluded to the PEG (Delta V(ex)), when the linking difference converts from Delta l = 0 to Delta l = +/-1, was determined from the observed E(T) value and PEG osmotic pressure at each concentration. The experimental Delta V(ex) values agree well with theoretical estimates reckoned for a simple osmotic exclusion model, in which PEG is excluded by hard-core interactions from a concentric cylindrical volume around every duplex segment. The difference in volume excluded to PEG between the Delta l = 0 and the Delta l = +/-1 topoisomers is attributed entirely to the approximately 0.7 additional writhe "crossing" of two duplex strands at roughly 90 degrees, which is known to occur in the latter species. When the separation between the duplex centers at the "crossing" was adjusted so that the theoretical estimate of Delta V(ex) matched the experimental value at each PEG concentration, a value near 5.7 nm was obtained in each case. The invariance and plausible magnitude of this mean separation at the crossing provide strong support for this simple osmotic exclusion model. An alternative model, in which the PEG is excluded from the entire coil envelope of the DNA out to its radius of gyration, perhaps because it decreases the local dielectric constant, was also considered. The estimated difference in excluded volume in that case exceeds the experimental value by a factor of nearly 10(4), and could be ruled out on that basis.     

Structural studies of N- and C-terminally truncated human apolipoprotein A-I

Apolipoprotein A-I (apoA-I) plays an important structural and functional role in lipid transport and metabolism. This work is focused on the central region of apoA-I (residues 60-183) that is predicted to contain exclusively amphipathic alpha-helices. Six N- and/or C-terminally truncated mutants, delta(1-41), delta(1-59), delta(198-243), delta(209-243), delta(1-41,185-243), and delta(1-59,185-243), were analyzed in their lipid-free state in solution at pH 4.7-7.8 by far- and near-UV CD spectroscopy. At pH 7.8, all mutants show well-defined secondary structures consisting of 40-52% alpha-helix. Comparison of the alpha-helix content in the wild type and mutants suggests that deletion of either the N- or C-terminal region induces helical unfolding elsewhere in the structure, indicating that the terminal regions are important for the integrity of the solution conformation of apoA-I. Near-UV CD spectra indicate significant tertiary and/or quaternary structural changes resulting from deletion of the N-terminal 41 residues. Reduction in pH from 7.8 to 4.7 leads to an increase in the mutant helical content by 5-20% and to a large increase in thermal unfolding cooperativity. Van't Hoff analysis of the mutants at pH 4.7 indicates melting temperatures T(m) ranging from 51 to 59 degrees C and effective enthalpies deltaH(v)(T(m)) = 35 +/- 5 kcal/mol, similar to the values for plasma apoA-I at pH 7.8 (T(m) = 57 degrees C, deltaH(v) = 32 kcal/mol). Our results provide the first report of the pH effects on the secondary, tertiary, and/or quaternary structure of apoA-I variants and indicate the importance of the electrostatic interactions for the solution conformation of apoA-I.     

Non-nearest-neighbor dependence of the stability for RNA bulge loops based on the complete set of group I single-nucleotide bulge loops

Fifty-nine RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters DeltaH degrees, DeltaS degrees, DeltaG 37 degrees, and TM for each sequence were determined. Sequences from this study were combined with sequences from previous studies [Longfellow, C. E., et al. (1990) Biochemistry 29, 278-285; Znosko, B. M., et al. (2002) Biochemistry 41, 10406-10417], thus examining all possible group I single-nucleotide bulge loop and nearest-neighbor sequence combinations. The free energy increments at 37 degrees C for the introduction of a group I single-nucleotide bulge loop range between 1.3 and 5.2 kcal/mol. The combined data were used to develop a model for predicting the free energy of a RNA duplex containing a single-nucleotide bulge. For bulge loops with adjacent Watson-Crick base pairs, neither the identity of the bulge nor the nearest-neighbor base pairs had an effect on the influence of the bulge loop on duplex stability. The proposed model for prediction of the stability of a duplex containing a bulged nucleotide was primarily affected by non-nearest-neighbor interactions. The destabilization of the duplex by the bulge was related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. The stability of a duplex containing a bulged nucleotide adjacent to a wobble base pair also was primarily affected by non-nearest-neighbor interactions. Again, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. However, when one or both of the bulge nearest neighbors was a wobble base pair, the free energy increment for insertion of a bulge loop is dependent upon the position and orientation of the wobble base pair relative the bulged nucleotide. Bulge sequences of the type ((5'UBX)(3'GY)), ((5'GBG)(3'UU)) and ((5'UBU)(3'GG)) are less destabilizing by 0.6 kcal/mol, and bulge sequences of the type ((5'GBX)(3'UY)) and ((5'XBU)(3'YG)) are more destabilizing by 0.4 kcal/mol than bulge loops adjacent to Watson-Crick base pairs.     

Comparison of mechanical properties of four large, wave-exposed seaweeds

Seaweeds have a simple structural design compared to most terrestrial plants. Nonetheless, some species have adapted to the severe mechanical conditions of the surf zone. The material properties of either tissue sections or the whole stipe of four wave-exposed seaweeds, Durvillaea antarctica, D. willana, Laminaria digitata, and L. hyperborea, were tested in tension, bending, and torsion. Durvillaea has a very low modulus of elasticity in tension (E(tension) = 3-7 MN·m(-2)) and in bending (E(bending) = 9-12 MN · m(-2)), torsion modulus (G = 0.3 MN · m(-2)) and strength (σ(b)rk = 1-2 MN · m(-2)), combining a compliable and twistable stipe "material" with a comparatively high breaking strain (ε(brk) = 0.4-0.6). In comparison, the smaller stipes of Laminaria have a higher modulus of elasticity in tension (E(tension) = 6-28 MN·m(-2)) and in bending (E(bending) = 84-109 MN·m(-2)), similar strength (σ(brk) = 1-3 MN·m(-2)), and a higher torsion modulus (G = 0.7-10 MN·m(-2)), combined with a lower breaking strain (ε(brk) = 0.2-0.3) than Durvillaea. Time-dependent, viscoelastic reactions were investigated with cycling tests. The tested species dissipated 42-52% of the loading energy in tension through plastic-viscoelastic processes, a finding that bears important ecological implications. Overall, there seems to be no correlation between single material properties and the size or habitat position of the tested seaweed species.     

Simultaneous recognition of nucleobase and sites of DNA damage: effect of tethered cation on the binding affinity

BACKGROUND: The 3,5-diamino-N-(3-aminopropyl)-6-chloropyrazine-2-carboxamide (DCPC-NH(2)) has been synthesized and characterized by Mass and (1)H NMR. The selective binding of the ligand to thymine (T) target base is investigated by the melting temperature (T(m)) and fluorescence measurements. METHODS: Thermal denaturation study of DNA duplex containing T target base revealed the DeltaT(m) of 5.1 degrees C, while least influence was observed for other target bases. The fluorescence of the ligand DCPC-NH(2) is quenched only upon adding the DNA containing T target base. RESULTS: The binding constant for the interaction of the ligand to T target base containing DNA duplex was determined to be 4.7 (+/-0.3)x10(6) M(-1). The tethered cation in the ligand is found to enhance the binding constant. The ligand binds to both a target nucleotide and an AP site on the complimentary strand for the target strand in a DNA duplex. GENERAL SIGNIFICANCE: Interestingly, the electronic behavior of the ligand depends on the bases flanking the AP site. Its fluorescence is quenched with guanine flanking bases, while it is enhanced with DNA duplex containing T bases flanking an AP site. Finally, the binding modes were visualized by molecular modeling.     

2-Amino-7-deazaadenine forms stable base pairs with cytosine and thymine.

2-Amino-7-deazaadenine ((AD)A) was incorporated into oligodeoxynucleotides (ODN) and their base-pairing properties with natural nucleobases were investigated. In melting temperature (T(m)) experiments, the duplex containing an (AD)A/C base pair showed a high stability comparable to that containing (AD)A/T base pair. Destabilization of the duplex usually observed for existing degenerate bases was not observed. However, the incorporation efficiency of dCTP was only 1.8% for TTP in single-nucleotide insertion reactions using polymerase.     

Sweat rate prediction equations for outdoor exercise with transient solar radiation

We investigated the validity of employing a fuzzy piecewise prediction equation (PW) [Gonzalez et al. J Appl Physiol 107: 379-388, 2009] defined by sweat rate (m(sw), g·m(-2)·h(-1)) = 147 + 1.527·(E(req)) - 0.87·(E(max)), which integrates evaporation required (E(req)) and the maximum evaporative capacity of the environment (E(max)). Heat exchange and physiological responses were determined throughout the trials. Environmental conditions were ambient temperature (T(a)) = 16-26°C, relative humidity (RH) = 51-55%, and wind speed (V) = 0.5-1.5 m/s. Volunteers wore military fatigues [clothing evaporative potential (i(m)/clo) = 0.33] and carried loads (15-31 kg) while marching 14-37 km over variable terrains either at night (N = 77, trials 1-5) or night with increasing daylight (N = 33, trials 6 and 7). PW was modified (Pw,sol) for transient solar radiation (R(sol), W) determined from measured solar loads and verified in trials 6 and 7. PW provided a valid m(sw) prediction during night trials (1-5) matching previous laboratory values and verified by bootstrap correlation (r(bs) of 0.81, SE ± 0.014, SEE = ± 69.2 g·m(-2)·h(-1)). For trials 6 and 7, E(req) and E(max) components included R(sol) applying a modified equation Pw,sol, in which m(sw) = 147 + 1.527·(E(req,sol)) - 0.87·(E(max)). Linear prediction of m(sw) = 0.72·Pw,sol + 135 (N = 33) was validated (R(2) = 0.92; SEE = ±33.8 g·m(-2)·h(-1)) with PW β-coefficients unaltered during field marches between 16°C and 26°C T(a) for m(sw) ≤ 700 g·m(-2)·h(-1). PW was additionally derived for cool laboratory/night conditions (T(a) < 20°C) in which E(req) is low but E(max) is high, as: PW,cool (g·m(-2)·h(-1)) = 350 + 1.527·E(req) - 0.87·E(max). These sweat prediction equations allow valid tools for civilian, sports, and military medicine communities to predict water needs during a variety of heat stress/exercise conditions.     

Effects of GA mismatches on the structure and thermodynamics of RNA internal loops

UV melting, CD and NMR studies indicate rGCGAGCG and rGCAGGCG from unusually stable duplexes of type a and b. The observed delta G degree 37's in 1 M NaCl are -6.7 and -6.3 kcal/mol, respectively. For the related duplex, c, delta G degree 37 is -4.2 kcal/mol. The predicted delta G degree 37 from nearest-neighbor parameters (formula; see text) for all three duplexes is -4.7 kcal/mol (Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373-9377). The results suggest a special interaction in the duplexes containing GA mismatches. Presumably, this is hydrogen bonding between G and A. While the thermodynamics for (rGCGAGCG)2 and (rGCAGGCG)2 are similar, CD and the imino region of the proton NMR spectra indicate their structures are different. In particular, (rGCAGGCG)2 exhibits a CD spectrum typical of A-form geometry with a weak negative band at 280 nm. In contrast, the CD spectrum for (rGCGAGCG)2 has an intense positive band at 285 nm. The NMR spectrum of (rGCAGGCG)2 has a resonance corresponding to a hydrogen-bonded GA mismatch, while for (rGCGAGCG)2 no hydrogen-bonded imino proton is observed for the mismatch. The glycosidic torsion angles of the bases in the GA mismatches of (rGCAGGCG)2 and (rCGCAGGCG)2 are anti. Duplexes of type d, where X is A, G, or U, are more stable than e, and the stability differences are similar to those (formula; see text) observed for f versus g. Thus, 3'-dangling ends in this system make contributions to duplex stability that are similar to contributions observed with fully paired duplexes.     

Structure of the 1,4-bis(2'-deoxyadenosin-N6-yl)-2R,3R-butanediol cross-link arising from alkylation of the human N-ras codon 61 by butadiene diepoxide

The solution structure of the 1,4-bis(2'-deoxyadenosin-N(6)-yl)-2R,3R-butanediol cross-link arising from N(6)-dA alkylation of nearest-neighbor adenines by butadiene diepoxide (BDO(2)) was determined in the oligodeoxynucleotide 5'-d(CGGACXYGAAG)-3'.5'-d(CTTCTTGTCCG)-3'. This oligodeoxynucleotide contained codon 61 (underlined) of the human N-ras protooncogene. The cross-link was accommodated in the major groove of duplex DNA. At the 5'-side of the cross-link there was a break in Watson-Crick base pairing at base pair X(6).T(17), whereas at the 3'-side of the cross-link at base pair Y(7).T(16), base pairing was intact. Molecular dynamics calculations carried out using a simulated annealing protocol, and restrained by a combination of 338 interproton distance restraints obtained from (1)H NOESY data and 151 torsion angle restraints obtained from (1)H and (31)P COSY data, yielded ensembles of structures with good convergence. Helicoidal analysis indicated an increase in base pair opening at base pair X(6).T(17), accompanied by a shift in the phosphodiester backbone torsion angle beta P5'-O5'-C5'-C4' at nucleotide X(6). The rMD calculations predicted that the DNA helix was not significantly bent by the presence of the four-carbon cross-link. This was corroborated by gel mobility assays of multimers containing nonhydroxylated four-carbon N(6),N(6)-dA cross-links, which did not predict DNA bending. The rMD calculations suggested the presence of hydrogen bonding between the hydroxyl group located on the beta-carbon of the four-carbon cross-link and T(17) O(4), which perhaps stabilized the base pair opening at X(6).T(17) and protected the T(17) imino proton from solvent exchange. The opening of base pair X(6).T(17) altered base stacking patterns at the cross-link site and induced slight unwinding of the DNA duplex. The structural data are interpreted in terms of biochemical data suggesting that this cross-link is bypassed by a variety of DNA polymerases, yet is significantly mutagenic [Kanuri, M., Nechev, L. V., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580].     

Thermodynamic and structural properties of pentamer DNA.DNA, RNA.RNA, and DNA.RNA duplexes of identical sequence

Four pentamers with the general sequence 5'CU(T)GU(T)G/5'CACAG have been prepared by chemical synthesis in order to generate duplex structures with common sequences. The four duplexes studied include the DNA.DNA duplex (5'dCACAG/5'dCTGTG) and the RNA.RNA duplex (5'rCUGUG/5'rCACAG) as well as the two corresponding DNA.RNA heteroduplexes (5'rCUGUG/5'dCACAG and 5'CACAG/5'dCTGTG). The measured entropy, enthalpy, and free energy changes upon melting are reported for each pentamer and compared to the predicted values where possible. Results show that the two DNA.RNA heteroduplexes are destabilized (delta G degrees 25 = -4.2 +/- 0.4 kcal/mol) relative to either the DNA.DNA duplex (delta G degrees 25 = -4.8 +/- 0.5 kcal/mol) or the RNA.RNA duplex (delta G degrees 25 = -5.8 +/- 0.6 kcal/mol). Circular dichroism spectra indicate that the RNA and the two heteroduplexes adopt an A-form conformation, while the DNA conformation is B-form. Imino proton NMR spectra also show that the heteroduplex structures resemble the RNA.RNA duplex.     

Effects of ethylene glycol on the torsion elastic constant and hydrodynamic radius of p30delta DNA

Upon increasing the concentration of ethylene glycol (EG) at 37 degrees C, the twist energy parameter, E(T), which governs the supercoiling free energy, was recently found to undergo a decreasing (or reverse) sigmoidal transition with a midpoint near 20 w/v % EG. In this study, the effects of adding 20 w/v % EG on the torsion elastic constant (alpha) of linear p30delta DNA and on the hydrodynamic radius (R(H)) of a synthetic 24 bp duplex DNA were examined at both 40 and 20 degrees C. The time-resolved fluorescence intensity and fluorescence polarization anisotropy (FPA) of intercalated ethidium were measured in order to assess the effects of 20 w/v % EG on: (1) alpha; (2) R(H); (3) the lifetimes of intercalated and non-intercalated dye; (4) the amplitude of dye wobble in its binding site; and (5) the binding constant for intercalation. The effects of 20 w/v % EG on the circular dichroism (CD) spectrum of the DNA and on the emission spectrum of the free dye were also measured. At 40 degrees C, addition of 20 w/v % EG caused a substantial (1.27- to 1.35-fold) increase in alpha, a significant change in the CD spectrum, and a very small, marginally significant increase in R(H), but little or no change in the amplitude of dye wobble in its binding site or the lifetime of intercalated dye. Together with previously reported measurements of E(T), these results imply that the bending elastic constant of DNA is significantly decreased by 20 w/v % EG at 40 degrees C. At 20 degrees C, addition of 20 w/v % EG caused a marginally significant decrease in alpha and very little change in any other measured properties. Also at 20 degrees C, addition of 30 w/v % betaine caused a marginally significant increase in alpha and significant but modest change in the CD spectrum, but very little change in any other properties.     

Interactions of the cbbII promoter-operator region with CbbR and RegA (PrrA) regulators indicate distinct mechanisms to control expression of the two cbb operons of Rhodobacter sphaeroides

In a previous study (Dubbs, J. M., Bird, T. H., Bauer, C. E., and Tabita, F. R. (2000) J. Biol. Chem. 275, 19224-19230), it was demonstrated that the regulators CbbR and RegA (PrrA) interacted with both promoter proximal and promoter distal regions of the form I (cbb(I)) promoter operon specifying genes of the Calvin-Benson-Bassham cycle of Rhodobacter sphaeroides. To determine how these regulators interact with the form II (cbb(II)) promoter, three cbbF(II)::lacZ translational fusion plasmids were constructed containing various lengths of sequence 5' to the cbb(II) operon of R. sphaeroides CAC. Expression of beta-galactosidase was monitored under a variety of growth conditions in both the parental strain and knock-out strains that contain mutations that affect synthesis of CbbR and RegA. The binding sites for both CbbR and RegA were determined by DNase I footprinting. A region of the cbb(II) promoter from +38 to -227 bp contained a CbbR binding site and conferred low level regulated cbb(II) expression. The region from -227 to -1025 bp contained six RegA binding sites and conferred enhanced cbb(II) expression under all growth conditions. Unlike the cbb(I) operon, the region between -227 and -545 bp that contains one RegA binding site, was responsible for the majority of the observed enhancement. Both RegA and CbbR were required for maximal cbb(II) expression. Two potentially novel and specific cbb(II) promoter-binding proteins that did not interact with the cbb(I) promoter region were detected in crude extracts of R. sphaeroides. These results, combined with the observation that chemoautotrophic expression of the cbb(I) operon is RegA independent, indicated that the mechanisms controlling cbb(I) and cbb(II) operon expression during chemoautotrophic growth are quite different.     

Pyrimidine.pyrimidine base-pair mismatches in DNA. A nuclear magnetic resonance study of T.T pairing at neutral pH and C.C pairing at acidic pH in dodecanucleotide duplexes

Structural features of pyrimidine.pyrimidine mismatches in the interior of oligonucleotide duplexes have been investigated by high resolution two-dimensional proton nuclear magnetic resonance (n.m.r.) spectroscopy. These studies were conducted on the self-complementary d(C-G-C-T-A-G-C-T-T-G-C-G) duplex (designated T.T 12-mer) and the self-complementary d(C-G-C-C-A-G-C-T-C-G-C-G) duplex (designated C.C 12-mer) containing T.T and C.C pairs located at identical positions four base-pairs from either end of the duplex. Proton n.m.r. studies on the T.T 12-mer duplex were undertaken in the neutral pH range, while studies on the C.C 12-mer duplex were recorded at acidic pH. The proton spectra narrowed considerably on lowering the pH below neutrality for the C.C 12-mer duplex. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) data sets have been recorded on the T.T 12-mer and C.C 12-mer duplexes in high salt H2O and D2O solution. The magnitude of the NOE crosspeaks and the directionality of the NOE connectivities demonstrate that both duplexes are right-handed with all bases, including those at the mismatch site, adopting an anti configuration about the glycosidic bond. The observed base and sugar proton chemical shifts suggest structural similarities for the trinucleotide segments centered about the T.T and C.C mismatches. A NOE is detected between the resolved imino protons of T4 and T9 at the mismatch site, consistent with formation of a stacked "wobble" T4(anti).T9(anti) pair in the T.T 12-mer duplex. A comparison of the imino proton chemical shift and NOE data suggests that the imino-carbonyl hydrogen bonds in the wobble T.T mismatch are weaker than the corresponding imino-carbonyl hydrogen bonds in the wobble G.T mismatch. The 4-amino protons of C4 and C9 at the mismatch site in the C.C 12-mer duplex do not exhibit the pattern of hydrogen-bonded and exposed protons separated by approximately 1.5 parts per million characteristic of cytidine amino protons involved in Watson-Crick G.C pairing. The experimental data are insufficient to differentiate between wobble C(anti).C+(anti) and other pairing possibilities for the mismatch in the C.C 12-mer duplex at acidic pH.     

Structure of the capsular polysaccharide of Escherichia coli O9:K32(A):H19

The capsular polysaccharide of the bacterium Escherichia coli O9:K32(A):H19 was analyzed using chemical methods (hydrolysis, sequential Smith degradation, methylation analysis) together with 1H- and 13C-n.m.r. spectroscopy. 13C-N.m.r. spectroscopy and chemical analyses indicated that the K32 polysaccharide is composed of equimolar proportions of glucose, galactose, rhamnose, and glucuronic acid, and carries O-acetyl groups. 1H-N.m.r. analysis of native K32 polysaccharide revealed five resonances in the anomeric region (delta 5.52, 5.16, 5.12, 5.02, and 4.73) and the presence of an acetyl group (delta 2.18). O-Deacetylation of the polysaccharide resulted in the loss of the resonance at delta 2.18 and one of the resonances (delta 5.52) in the anomeric region. The "extra" anomeric resonance in the 1H-n.m.r. spectrum of the native K32 polymer was assigned to H-2 of rhamnose, which experiences a large downfield shift when the 2-position is O-acetylated. This was confirmed by a 2D-COSY n.m.r. experiment and studies of model compounds. The K32 capsular polysaccharide is of the "2 + 2" type, comprised of the following repeating unit: (sequence; see text) This structure is identical to that of Klebsiella K55 capsular polysaccharide.     

Supercoiled DNA-directed knotting by T4 topoisomerase.

The mechanism by which the type 2 topoisomerase from bacteriophage T4 mediates knotting of negatively supercoiled DNA was deduced from an analysis of product topology. The knotted products were nicked and then subjected to electrophoresis in order to separate species on the basis of the minimum number of crossings in the knotted form. Knots with defined numbers of crossings were purified and the configuration of these crossings determined in the electron microscope by the RecA coating method. The product knots were exclusively of the twist form, in which an interwound region is entrapped by a single interlock of two looped ends. The interwound region was of negative sign in greater than 98% of the molecules examined, whereas the single interlock was equally likely to be positive or negative. These results are interpreted in terms of a model for knot formation in which random strand passage mediated by the topoisomerase links bent or branched portions of a superhelix that has a specific interwound geometry. Superhelix interwinding and DNA contacts stabilized by excess enzyme molecules explain the very high frequency of knotting.     

Thermodynamic and kinetic determinants of Thermotoga maritima cold shock protein stability: a structural and dynamic analysis

The cold shock protein (CSP) from hyperthermophile Thermotoga maritima (TmCSP) is only marginally stable (DeltaG(T(opt)) = 0.3 kcal/mol) at 353 K, the optimum environmental temperature (T(opt)) for T. maritima. In comparison, homologous CSPs from E. coli (DeltaG(T(opt)) = 2.2 kcal/mol) and B. subtilis (DeltaG(T(opt)) = 1.5 kcal/mol) are at least five times more stable at 310 K, the T(opt) for the mesophiles. Yet at the room temperature, TmCSP is more stable (DeltaG(T(R)) = 4.7 kcal/mol) than its homologues (DeltaG(T(R)) = 3.0 kcal/mol for E. coli CSP and DeltaG(T(R)) = 2.1 kcal/mol for B. subtilis CSP). This unique observation suggests that kinetic, rather than thermodynamic, barriers toward unfolding might help TmCSP native structure at high temperatures. Consistently, the unfolding rate of TmCSP is considerably slower than its homologues. High temperature (600 K) complete unfolding molecular dynamics (MD) simulations of TmCSP support our hypothesis and reveal an unfolding scheme unique to TmCSP. For all the studied homologues of TmCSP, the unfolding process first starts at the C-terminal region and N-terminal region unfolds in the end. But for TmCSP, both the terminals resist unfolding for consistently longer simulation times and, in the end, unfold simultaneously. In TmCSP, the C-terminal region is better fortified and has better interactions with the N-terminal region due to the charged residues, R2, E47, E49, H61, K63, and E66, being in spatial vicinity. The electrostatic interactions among these residues are unique to TmCSP. Consistently, the room temperature MD simulations show that TmCSP is more rigid at its N- and C-termini as compared to its homologues from E. coli, B. subtilis, and B. caldolyticus.     

Effects of diving and swimming behavior on body temperatures of pacific leatherback turtles in tropical seas

Mathematical models and recordings of cloacal temperature suggest that leatherback turtles (Dermochelys coriacea) maintain core body temperature higher than ambient water temperature (T(W)) while freely swimming at sea. We investigated the thermoregulatory capabilities of free-ranging leatherbacks and, specifically, the effect that changes in diving patterns and ambient temperatures have on leatherback body temperatures (T(B)). Data loggers were used to record subcarapace and gastrointestinal tract temperatures (T(SC) and T(GT), respectively), T(W), swim speed, dive depth, and dive times of female leatherback turtles during internesting intervals off the coast of Guanacaste, Costa Rica. Mean T(SC) (28.7 degrees -29.0 degrees C) was significantly higher than mean T(W) (25.0 degrees -27.5 degrees C). There was a significant positive relationship between T(SC) and T(W) and a significant negative correlation between T(SC) and dive depth and T(GT) and dive depth. Rapid fluctuations in T(GT) occurred during the first several days of the internesting interval, which suggests that turtles were ingesting prey or water during this time. Turtles spent 79%-91% of the time at sea swimming at speeds greater than 0.2 m s(-1), and the average swim speed was 0.7 +/- 0.2 m s(-1). Results from this study show that alterations in diving behavior and T(W) affect T(B) of leatherback turtles in the tropics. Body temperatures of free-ranging leatherback turtles correspond well with values for T(B) predicted by mathematical models for tropical conditions.