Impact of mismatches in HbA1c vs glucose values on the diagnostic classification of diabetes and prediabetes.

AIMS: To determine whether HbA1c mismatches (HbA1c levels that are higher or lower than expected for the average glucose levels in different individuals) could lead to errors if diagnostic classification is based only on HbA1c levels. METHODS: In a cross-sectional study, 3106 participants without known diabetes underwent a 75-g oral glucose tolerance test (fasting glucose and 2-h glucose) and a 50-g glucose challenge test (1-h glucose) on separate days. They were classified by oral glucose tolerance test results as having: normal glucose metabolism; prediabetes; or diabetes. Predicted HbA1c was determined from the linear regression modelling the relationship between observed HbA1c and average glucose (mean of fasting glucose and 2-h glucose from the oral glucose tolerance test, and 1-h glucose from the glucose challenge test) within oral glucose tolerance test groups. The haemoglobin glycation index was calculated as [observed - predicted HbA1c ], and divided into low, intermediate and high haemoglobin glycation index mismatch tertiles. RESULTS: Those participants with higher mismatches were more likely to be black, to be men, to be older, and to have higher BMI (all P<0.001). Using oral glucose tolerance test criteria, the distribution of normal glucose metabolism, prediabetes and diabetes was similar across mismatch tertiles; however, using HbA1c criteria, the participants with low mismatches were classified as 97% normal glucose metabolism, 3% prediabetes and 0% diabetes, i.e. mostly normal, while those with high mismatches were classified as 13% normal glucose metabolism, 77% prediabetes and 10% diabetes, i.e. mostly abnormal (P<0.001). CONCLUSIONS: Measuring only HbA1c could lead to under-diagnosis in people with low mismatches and over-diagnosis in those with high mismatches. Additional oral glucose tolerance tests and/or fasting glucose testing to complement HbA1c in diagnostic classification should be performed in most individuals.

Ras transformation requires metabolic control by 6-phosphofructo-2-kinase.

Neoplastic cells transport large amounts of glucose in order to produce anabolic precursors and energy within the inhospitable environment of a tumor. The ras signaling pathway is activated in several cancers and has been found to stimulate glycolytic flux to lactate. Glycolysis is regulated by ras via the activity of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFK2/FBPase), which modulate the intracellular concentration of the allosteric glycolytic activator, fructose-2,6-bisphosphate (F2,6BP). We report herein that sequential immortalization and ras-transformation of mouse fibroblasts or human bronchial epithelial cells paradoxically decreases the intracellular concentration of F2,6BP. This marked reduction in the intracellular concentration of F2,6BP sensitizes transformed cells to the antimetabolic effects of PFK2/FBPase inhibition. Moreover, despite co-expression of all four mRNA species (PFKFB1-4), heterozygotic genomic deletion of the inducible PFKFB3 gene in ras-transformed mouse lung fibroblasts suppresses F2,6BP production, glycolytic flux to lactate, and growth as soft agar colonies or tumors in athymic mice. These data indicate that the PFKFB3 protein product may serve as an essential downstream metabolic mediator of oncogenic ras, and we propose that pharmacologic inhibition of this enzyme should selectively suppress the high rate of glycolysis and growth by cancer cells.

Solution conformation of a parallel DNA triple helix with 5' and 3' triplex-duplex junctions.

BACKGROUND: Polypurine x polypyrimidine sequences of DNA can form parallel triple helices via Hoogsteen hydrogen bonds with a third DNA strand that is complementary to the purine strand. The triplex prevents transcription and could therefore potentially be used to regulate specific genes. The determination of the structures of triplex-duplex junctions can help us to understand the structural basis of specificity, and aid in the design of optimal antigene oligonucleotides. RESULTS: The solution structures of the junction triplexes d(GAGAGACGTA)-X-(TACGTCTCTC)-X-(CTCTCT) and d(CTCTCT)-X-(TCTCTCAGTC)-X-(GACTGAGAGA) (where X is bis(octylphosphate) and nucleotides in the triplex regions are underlined) have been solved using nuclear magnetic resonance (NMR) spectroscopy. The structure is characterised by significant changes in the conformation of the purine residues, asymmetry of the 5' and 3' junctions, and variations in groove widths associated with the positive charge of the protonated cytosine residues in the third strand. The thermodynamic stability of triplexes with either a 5' or a 3'CH+ is higher than those with a terminal thymidine. CONCLUSIONS: The observed sequence dependence of the triplex structure, and the distortions of the DNA at the 5' and 3' termini has implications for the design of optimal triplex-forming sequences, both in terms of the terminal bases and the importance of including positive charges in the third strand. Thus, triplex-stabilising ligands might be designed that can discriminate between TA x T-rich and CG x C+-rich sequences that depend not only on charge, but also on local groove widths. This could improve the stabilisation and specificity of antigene triplex formation.

The determination of the conformational properties of nucleic acids in solution from NMR data.

A program, NUCFIT, has been written for simulating the effects of conformational averaging on nuclear Overhauser enhancement (NOE) intensities for the spin systems found in nucleic acids. Arbitrary structures can be generated, and the NOE time courses can be calculated for truncated one-dimensional NOEs, two-dimensional NOE and rotating frame NOE spectroscopy (NOESY and ROESY) experiments. Both isotropic and anisotropic molecular rotation can be treated, using Woessner's formalism (J. Chem. Phys. (1962) 37, 647-654). The effects of slow conformational averaging are simulated by taking population-weighted means of the conformations present. Rapid motions are allowed for by using order parameters which can be supplied by the user, or calculated for specific motional models using the formalism of Tropp (J. Chem. Phys. (1980) 72, 6035-6043). NOE time courses have been simulated for a wide variety of conformations and used to determine the quality of structure determinations using NMR data for nucleic acids. The program also allows grid-searching with least-squares fitting of structures to experimental data, including the effects of spin-diffusion, conformational averaging and rapid internal motions. The effects of variation of intra and internucleotide conformational parameters on NOE intensities has been systematically explored. It is found that (i) the conformation of nucleotides is well determined by realistic NOE data sets, (ii) some of the helical parameters, particularly the base pair roll, are poorly determined even for extensive, noise-free data sets, (iii) conformational averaging of the sugars by pseudorotation has at most second-order influence on the determination of other parameters and (iv) averaging about the glycosidic torsion bond also has, in most cases, an insignificant effect on the determination of the conformation of nucleotides.

Solution conformations of d(CGTACG)2 determined from NMR experiments.

The conformations of all the nucleotides in the hexamer d(CGTACG)2 have been determined using time-dependent one- and two-dimensional nuclear Overhauser enhancements (NOEs) and the program NUCFIT (see previous article). The glycosidic torsion angles are well determined, the fraction of the C2' endo state for the sugar puckers is less well determined, and the pseudorotation phase angle is poorly determined by the NOEs. The average glycosidic torsion angle is -107 +/- 9 degrees, and the deoxyriboses of the purine residues have a higher fraction of the C2' endo state than those of the pyrimidine residues. There is good agreement between the one- and two-dimensional NOE data. Of the helical parameters, the local rise and twist are moderately well determined, but the roll and tilt of the bases are not well described. The overall structure belongs to the B family of conformations, as previously described by Gronenborn et al. (Biochem. J. (1984) 221, 723-736), but there are significant differences which can be ascribed to the improved treatment of the spin-diffusion and motional averaging possible with the program NUCFIT. The results obtained using NUCFIT are compared with those from restrained energy minimisation calculations using distance restraints obtained from NUCFIT.

AT selectivity and DNA minor groove binding: modelling, NMR and structural studies of the interactions of propamidine and pentamidine with d(CGCGAATTCGCG)2.

A molecular modelling strategy has been developed to identify potential binding sites for bis(amidine) ligands in the minor groove of duplex DNA. Calculations of interaction energy for propamidine and pentamidine with d(CGCGAAT TCGCG)2 show that this duplex contains two symmetrically equivalent binding sites of identical affinity, each displaced by 0.3-0.4 bp from the centre of the AT segment. The ligands occupy groove sites spanning approximately 4 and 4-5 bp, respectively with asymmetric binding to the 5'-AATT sequence. The DNA-bis(amidine) interactions have been examined by high-resolution 1H-NMR. The patterns of induced changes in DNA proton chemical shift and the DNA-ligand NOEs confirm that both agents bind in the AT minor groove in a non-centrosymmetric fashion. Detailed structures were determined for each complex using a NOE-restrained simulated annealing procedure, showing that the B-type DNA conformation is not significantly altered upon complexation with either ligand. The free DNA duplex has previously been shown to be extensively hydrated in the minor groove [Kubinec, M.G. and Wemmer, D.E. (1992) J. Am, Chem. Soc. 114, 8739-8740 Liepinsh, E. Otting, G. and Wüthrich, K. (1992) Nucleic Acids Res. 20. 6549-6553]. We detect hydration water close to the A(H2) protons in the presence of propamidine, which may stabilise certain waters against exchange. This conclusion supports recent crystallographic analyses, suggesting that such ligands may use water molecules to bridge between amidinium protons and host DNA bases Details of the ligand interactions with AT-tract DNA duplexes can now be compared for the subsequences 5'-AAT, 5'-AATT and 5'-AAATTT.

The effect of the trp repressor from Escherichia coli on the structure and dynamics of dA20dT20.

We have determined the effect of the tryptophan (trp) repressor from Escherichia coli on the structure and dynamics of dA20dT20. The structure was determined using time-dependent nuclear Overhauser effects and spin-lattice relaxation times. The deoxyribose conformation is near C3' endo for the thymine residues, and a mixture of about 30% C3' endo and 70% C2' endo for the adenine residues. The glycosidic torsion angles are -50 degrees for T and -60 degrees for A. The roll is 20 degrees and the propellor twist is about 29 degrees. The conformation is consistent with recent calculations (Rao, K. and Kollman, P.A. (1985) J. Am. Chem. Soc. 107, 1507-1511). The rate constant for exchange of the imino protons is similar to that usually found for AT base-pairs, with an activation energy of 20 +/- 2 kcal/mol, and an activation entropy of 17 +/- 7 cal/mol per K. The repressor greatly retards the exchange of imino protons, and the activation energy increases to 38 kcal/mol. There are small changes in the structure of the DNA on forming the complex, with the adenine and thymidine residues becoming more similar in conformation.

The interaction of the trp repressor from Escherichia coli with the trp operator.

We have examined the interaction of the trp repressor from Escherichia coli with a 20 base-pair synthetic operator. Nonspecific binding was relatively strong (Kd = 2 microM), but only weakly sensitive to the concentration of added salt [d log Kd)/(d log [Na]) = -1). 1H-NMR studies indicate that the structure of the repressor is not greatly altered on forming the complex, and that few if any of the lysine and arginine residues make direct contact with the DNA. However, the mobility of one of the two tyrosine residues is significantly decreased in the complex. The repressor makes close contact with the major grooves of the operator such that the base protons are broadened much more than expected on the basis of increased correlation time. There are large, differential changes in chemical shifts of the imino protons on forming the complex, as well as changes in the rate constants for exchange. The fraying of the ends is greatly diminished, consistent with a target size of about 20 base-pairs. The effects of the repressor on the NMR spectra and relaxation rate constants can be interpreted as a change in the conformation of the operator, possibly a kinking in the centre of the molecule.

Combined use of 1H-NMR and GC-MS for metabolite monitoring and in vivo 1H-NMR assignments.

Thirty-three metabolites were observed in perchloric acid extracts of four different tissues by in vitro 1H-NMR, GC-MS and alcohol dehydrogenase assay, and the information was used to interpret an in vivo two-dimensional nuclear Overhauser effect 1H-NMR spectrum. The metabolite profiles of the different tissues indicate a number of potential tissue-specific markers: N-acetylaspartate and gamma-aminobutyric acid for rat brain, glutamine/glutamic acid ratio for dog heart, arginine and sucrose for carrot, and t-aconitate, sucrose, asparagine/aspartic acid concentration ratios for corn roots. gamma-Aminobutyric acid and malate can be regarded as metabolic indicators for stressed corn roots. Concentrations of threonine and valine in corn roots were constant under hypoxic and salt stress, and can serve as internal standards for both in vivo and in vitro NMR studies. The in vitro information was further used to identify 12 compounds from the in vivo 1H-NMR spectra (including the two-dimensional nuclear Overhauser effect spectrum) of a carrot cylinder by correlating the chemical shift and nuclear Overhauser effect information. Thus, our choice of methods with a capability for structural determination allows the characterization of complex tissue extracts with minimum sample preparation, and supports, as well as complements, in vivo 1H-NMR investigations of metabolism.

Temperature dependence of arginine kinase reaction in the tail muscle of live Sycionia ingentis as measured in vivo by 31P-NMR driven saturation transfer.

We have employed the driven 31P-NMR saturation transfer method to measure in vivo the temperature dependence of the forward and reverse unidirectional fluxes of the arginine kinase reaction in the tail muscle of a live shrimp, Sycionia ingentis. Our results indicated that neither the forward nor the reverse rate constants of this reaction were significantly temperature-dependent between 8 and 16 degrees C, in contrast to the kinetic characteristics of isolated arginine kinases.

Hydration and solution structure of d(CGCAAATTTGCG)2 and its complex with propamidine from NMR and molecular modelling.

The hydration of the d(CGCAAATTTGCG)2 duplex and its complex with a propamidine reporter ligand has been examined in aqueous solution by two-dimensional NMR at two spectrometer frequencies and three temperatures. Quantitative analysis of ROESY and NOESY cross-peaks showed effective correlation times of approximately 0.5 ns at 283 K for DNA-water interactions in the major groove. In some cases the sign of the NOE inverts on changing either the temperature or spectrometer frequency. Larger effective correlation times of approximately 1 ns were observed for water interactions with A5(H2) and A6(H2) atoms located in the minor groove. Interproton NOEs and changes in chemical shifts showed that propamidine binds in the minor groove 5'-AATTT region of the host duplex, but does not displace waters adjacent to either A5(H2) or A6(H2). In the complex, the effective correlation times of these waters increase more than two-fold, possibly as a result of stabilisation due to H-bonded interaction with the amidine groups of the ligand. Hydration of the bound molecule was also found, suggesting that water may contribute to the DNA binding process for bis(amidine) drugs. Structure refinement by a NOE-restrained dynamic annealing procedure revealed that ligand binding is non-centrosymmetric with respect to the duplex, in accordance with the energetically favoured 5'-ATT (= 5'-AAT) sites predicted by analytical molecular modelling. In particular, the bound propamidine spans 3-4 base pairs in the A6-T7-T8 tract and makes close H-bonded contacts with A(N3/O4) acceptors positioned close to the minor groove floor. The refined NMR structure for the DNA-propamidine complex is compared with that determined recently using X-ray crystallographic methods.

Nuclear magnetic resonance study of the proton exchange rate in the operator-promoter DNA sequence of the trp operon of Escherichia coli.

The dynamic behavior of a palindromic oligonucleotide (C-G-T-A-C-T-A-G-T-T-A-A-C-T-A-G-T-A-C-G) representative of the operator sequence and containing the Pribnow box of the trp operon of Escherichia coli was investigated. The resonances of the imino protons and of the H2 protons of the adenosine residues were all assigned. The opening rate constants of the base-pairs were calculated by monitoring the exchange rate of the observable imino protons (nine out of ten), using selective temperature (T1) measurements, which avoid the complication of cross-relaxation and spin diffusion. These measurements have to be performed in conditions where the exchange process is much faster than the opening and closing of the base-pairs, so that the observed exchange rate is equal to the opening rate. It is shown that the catalysis of the exchange process by phosphate dianions is not very efficient (kB approximately equal to 7 X 10(4) M-1 S-1). Hence, in phosphate buffer, the necessary opening-rate limiting condition is met only at high pH values (approximately equal to 9.5) where efficient catalysis by OH- takes place, or at very high buffer concentration. While G X C base-pairs show very little exchange, acting in the sequence as molecular "staples", the A X T base-pairs that are protected from the fraying have very different opening and closing rates, depending on the sequence. Although it seems possible that the opening process could occur at the base-pair level, it is localized at most to two base-pairs in that particular sequence. The activation energies for the opening process of all non-fraying base-pairs are very similar (19 +/- 1 kcal mol-1; 1 cal = 4.184 J), and the differences in the opening rates are essentially due to differences in the activation entropies. With regard to the role of this sequence in the promoter, it is observed that the end of the Pribnow box exchanges relatively easily, and that the activation parameters for the "breathing" process and for the isomerization step of the promoter--RNA polymerase are not very different.

The contribution of cytosine protonation to the stability of parallel DNA triple helices.

The influence of the position of the CG.C+ triplet and the contribution of protonation at the N3 of the Hoogsteen cytosine residue on the stability of various sequences of parallel triple helices having the general composition d[(A5G)-x-(T5C)-x-(T5C)] and d[(A4G2)-x-(T4C2)-x-(T4C2)], where x is the hexaethylene glycol linker, has been determined by NMR, ultraviolet melting and absorbance spectrophotometry. The apparent pK value, i.e. the pH at which the observable has changed by 50% of its range, was typically in the range 6 to 7. However, the NMR spectra unequivocally showed that the pK of the protonated cytosine residue must be at least 9.5 for internal positions. This is five units above the pK of the free nucleotide, and represents a free energy of stabilisation from protonation of >11.5 RT. The pK of terminal cytosine residues is much lower, in the range 6.2 to 7.2, accounting for a free energy of stabilisation from protonation of 3.6 to 6 RT. The van't Hoff enthalpies were determined for the dissociation of the protonated triplex into the duplex+strand, and for the duplex to strand transition. The mean value for the duplexes were 23 to 27 kJ mol-1 base-pair, and 25 to 30 kJ mol-1 for the triplexes containing internal CG.C+ triplets. Good agreement was obtained for the thermodynamic parameters by the different methods. Free energy differences for the transition between the protonated triplex and the duplex+protonated strand were calculated at 298 K. The DeltaG of stabilisation of an internal CG.C triplet compared with a terminal CG.C triplet was about 6 kJ mol-1 ; a similar stabilisation was observed for the triplexes containing two CG.C triplets compared with those containing a single CG.C triplet. The very large stabilisation from protonation is too large to be accounted for by a single hydrogen bond, and is likely to include contributions from electrostatic interactions of the positive charge with the phosphate backbone, and more favourable interactions between neighbouring bases owing to the very different electronic properties of the protonated C.

Crystal structure of the transcription elongation/anti-termination factor NusA from Mycobacterium tuberculosis at 1.7 A resolution.

Mycobacterium tuberculosis is the cause of tuberculosis in humans, a disease that affects over a one-third of the world's population. This slow-growing pathogen has only one ribosomal RNA operon, thus making its transcriptional apparatus a fundamentally interesting target for drug discovery. NusA binds to RNA polymerase and modulates several of the ribosomal RNA transcriptional processes. Here, we report the crystal structure of NusA, and reveal that the molecule consists of four domains. They are organised as two distinct entities. The N-terminal domain (residues 1 to 99) that resembles the B chain of the Rad50cd ATP binding cassette-ATPase (ABC-ATPase) and a C-terminal module (residues 108 to 329) consisting of a ribosomal S1 protein domain followed by two K homology domains. The S1 and KH domains are tightly integrated together to form an extensive RNA-binding structure, but are flexibly tethered to the N-terminal domain. The molecule's surfaces and architecture provide insights into RNA and polymerase interactions and the mechanism of pause site discrimination. They also allow us to rationalize certain termination-defective and cold shock-sensitive mutations in the nusA gene that have been studied in Escherichia coli.

Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from Antarctic cod.

The 1H and 13C NMR spectra of a 14-residue antifreeze glycopeptide from Antarctic cod (Tetramatomnus borchgrevinki) containing two proline residues have been assigned. 13C NMR relaxation experiments indicate motional anisotropy of the peptide, with a tumbling time in water at 5 degrees C of 3-4 ns. The relaxation data and lack of long-range NOEs are consistent with a linear peptide undergoing significant segmental motion. However, extreme values of some coupling constants and strong sequential NOEs indicate regions of local order, which are most evident at the two ATPA subsequences. Similar spectroscopic properties were observed in the 16-residue analogue containing an Arg-Ala dipeptide added to the C-terminus. Molecular modeling also showed no evidence of long-range order, but the two ATPA subsequences were relatively well determined by the experimental data. These motifs were quite distinct from helical structures or beta turns commonly found in proteins, but rather resemble sections of an extended polyproline helix. Thus, the NMR data provide a description of the local order, which is of relevance to the mechanism of action of the antifreeze activity of the antifreeze glycopeptides as well as their ability to protect cells during hypothermic storage.

31P NMR investigation of the backbone conformation and dynamics of the hexamer duplex d(5'-GCATGC)2 in its complex with the antibiotic nogalamycin.

Heteronuclear chemical shift correlation experiments confirm that the two down-field shifted 31P resonances in the spectrum of the (nogalamycin)2-d(GCATGC)2 complex correspond to the phosphodiesters CpA and TpG at the intercalation sites. 31P relaxation measurements (R1, R2 and [1H]-31P NOE) at 4.7 and 9.4 T permit the correlation time of each phosphate to be determined together with their chemical shift anisotropies. Significant differences in deoxyribose H3'-31P coupling constants and chemical shift anisotropy contributions are observed, consistent with an asymmetric DNA backbone conformation for the phosphate groups at the intercalation sites. Large amplitude internal motions of the phosphates do not appear to contribute significantly to relaxation.

A temperature dependent transition in the Pribnow box of the trp promoter.

Proton NMR spectra of the trp operator-promoter (sequence CGTACTAGTT.AACTAGTACG) show selective changes in chemical shift and relaxation rates over the range of temperature 0-45 degrees C for the non-exchangeable protons of A11 and A12 only. These bases are in the centre of the Pribnow box. The changes imply that at least three conformational states become significantly populated in this range of temperature, and probably involve a change in the propellor twists of A11 and A12 for one transition, and changes in the helical twist and local pitch for the other. As (1) mutations in the Pribnow box that destroy the TAA sequence impair promoter activity, and (2) the abortive initiation assay for RNA polymerase shows a transition near 20 degrees C, we propose that the observed conformational transitions in the trp promoter are an essential feature of good promoters.

NMR assignments and secondary structure of the UvrC binding domain of UvrB.

The 55 residue C-terminal domain of UvrB that interacts with UvrC during excision repair in Escherichia coli has been expressed and purified as a (His)6 fusion construct. The fragment forms a stable folded domain in solution. Heteronuclear NMR experiments were used to obtain extensive 15N, 13C and 1H NMR assignments. NOESY and chemical shift data showed that the protein comprises two helices from residues 630 to 648 and from 652 to 670. 15N relaxation data also show that the first 11 and last three residues are unstructured. The effective rotational correlation time within the structured region is not consistent with a monomer. This oligomerisation may be relevant to the mode of dimerisation of UvrB with the homologous domain of UvrC.

Determination of fast dynamics of nucleic acids by NMR.

Double-stranded oligonucleotides of < 10 base pairs are adequately described as an isotropic rotor, using the correlation time for the cytosine H6-H5 vector. For longer fragments, the cylindrical model should be used for detailed analysis of NOEs. The appropriate correlation times can be calculated using the formulae of Tirado and Garcia de la Torre or derived from measurements of the cross-relaxation rate constants for cytosine (or uridine) H6-H5. Order parameters describing the degree of motion of different vectors on the subnanosecond time scale vary substantially, with typical values of S2 > 0.8 for base vectors and 0.5-0.8 for intrasugar and base-sugar vectors. Order parameters for terminal nucleotides are typically significantly smaller than for internal nucleotides, which may also mean that their conformation will be less well determined in the formalism of a unique structure. The CSA relaxation rates of the phosphodiesters appear to be insensitive to internal motions and may, therefore, provide the most accurate estimate of the overall tumbling time in nucleic acid fragments. Using a combination of relaxation data for different nuclei and different spectrometer frequencies may be expected to yield detailed information about fast motions in nucleic acid fragments.

Nuclear magnetic resonance measurements of slow conformational dynamics in macromolecules.

The combined use of rotating-frame relaxation methods, temperature-dependent measurements of line shapes and magnetization transfer experiments allows in favorable cases the examination in some detail of exchange processes that occur on the millisecond time scale. It is possible to determine not only the rate constants, but also the activation parameters and chemical shifts even for events that are in fast exchange on the chemical shift time scale. Such measurements complement the information obtainable from heteronuclear relaxation methods that probe mainly the fast librational motions in macromolecules and may provide information important for functional studies of biological macromolecules.