A comparison of the reproducibility of two cine-derived strain software programmes in disease states.

BACKGROUND: Systolic strain and peak-early diastolic strain rate (PEDSR) measure subclinical cardiac dysfunction. These parameters can be derived from cardiovascular magnetic resonance (CMR) cine images using new software packages, but the comparative test-retest reproducibility of these software in disease states is unknown. This study compared the test-retest reproducibility of strain measures derived from two software packages (feature-tracking software (FT) and tissue-tracking (TT)) in disease populations with preserved ejection fractions. METHODS: This was a prospective study of 10 patients with aortic stenosis (AS), 10 haemodialysis patients and 10 diabetic patients at 1.5 and 3-Tesla. 30 subjects underwent test-retest reproducibility scans of global circumferential strain (GCS), global longitudinal strain (GLS), circumferential-PEDSR and longitudinal-PEDSR calculated using TT and FT software. RESULTS: Test-retest reproducibility of GCS and GLS were similar for FT and TT across patient groups. Coefficient of variability (CoV) for FT-derived GCS 8.1%, 5% and 7.9% for AS, diabetic and haemodialysis patients, compared to 3.3%, 9.2% and 5.4% for TT-derived GCS, with CoV for FT-derived GLS 8%, 6.4% and 8.2% for AS, diabetic and haemodialysis patients, compared to 5.3%, 4.8% and 7% for TT-derived GLS). Reproducibility of FT-derived circumferential and longitudinal-PEDSR was worse than TT-derived circumferential and longitudinal-PEDSR (CoV for FT-derived circumferential-PEDSR 18.2%, 18% and 17.4% for AS, diabetic and haemodialysis patients, compared to 6.1%, 11.7% and 11% for TT-derived circumferential-PEDSR with CoV for FT-derived longitudinal PEDSR 18.2%, 18.9%, 18.3% for AS, diabetic and haemodialysis patients, compared to 8.9%, 9.1% and 11.4% for TT-derived longitudinal-PEDSR). Bland-Altman analysis revealed no systematic bias with tighter limits of agreement for TT-derived strain measures. CONCLUSIONS: Reproducibility of GCS and GLS are excellent with FT and TT software across diseases. TT had superior test-retest reproducibility for quantification of longitudinal and circumferential-PEDSR than FT-derived PEDSR across diseases.

GH replacement in patients with non-functioning pituitary adenoma (NFA) treated solely by surgery is not associated with increased risk of tumour recurrence.

BACKGROUND: Subjects with non-functioning pituitary adenomas (NFAs) frequently develop GH deficiency due to tumour expansion or as a consequence of tumour therapy. The safety of GH replacement (GHR) in these individuals remains unclear. OBJECTIVE: To assess the effect of GHR on tumour recurrence in patients with NFAs solely treated by surgical removal. PATIENTS AND METHODS: The study involved all patients with NFA who presented to the Department of Endocrinology in Oxford between January 1989 and July 2005 and were treated solely by surgical removal of the tumour. Patients with follow up < 1 year were excluded. Recurrence was diagnosed on the basis of radiological appearances (detectable tumour after gross total removal or regrowth of pre-existing residue) on regular imaging surveillance. RESULTS: One hundred and thirty patients were included in the study, and were followed up for a mean period of 6.8 +/- 4.2 years (median 5.7, range 1.2-17.6). Twenty-three patients received GHR [16 male, 7 female, mean age at tumour diagnosis 53.7 +/- 14.6 years (range 20-80)]. The mean duration of GHR was 4.6 +/- 2.5 years (median 5.3, range 0.4-8.7). One hundred and seven subjects did not receive GH therapy [61 male, 46 female, mean age at tumour diagnosis 56.2 +/- 14.0 years (range 20-87)]. Tumour regrowth occurred in 38 non-GH treated subjects (36%) and 8 GHR subjects (35%). Regrowth was detected at a mean of 4.8 +/- 2.8 years (range 1-11 years) in the non-GH treated group, and at 6.5 +/- 2.3 years in the GHR group. In the GHR group, recurrence occurred after a mean of 2.9 +/- 2.2 years (range 0.4-5.9 years) following commencement of GH treatment. The Cox regression analysis showed that after adjusting for sex, age at tumour diagnosis, cavernous sinus invasion at diagnosis and type of tumour removal (partial or complete based on postoperative scan), GH treatment was not a significant independent predictor of recurrence (P = 0.09; hazard ratio = 0.51; 95% CI, 0.24-1.12). CONCLUSION: GH replacement in patients with NFA treated by surgery alone is not associated with an increased risk of tumour recurrence.

Fluorescence spectroscopic and (19)f NMR studies of human thymidylate synthase with its cognate RNA.

In order to investigate the interaction between hTS protein and its cognate mRNA, a 29nt fragment of TS mRNA was synthesized. This region has been suggested as a putative stem-loop involved in translational autoregulation. The melting temperature of the 29ntRNA was 65 degrees C, suggesting that this region does indeed form a stem-loop. Fluorescence spectroscopy was used to monitor the RNA: hTS protein interaction [dissociation constant (K(d)) 3.9 +/- 0.8 nM; stoichiometry of binding 1dimeric hTS: 1RNA]. When hTS was titrated against FdUMP, this gave the expected stoichiometry of 1dimeric hTS: 1.7 FdUMP but in the presence of the 29ntRNA, the stoichiometry of binding changed to 1dimeric hTS: 1RNA: 1FdUMP. Experiments using methotrexate (MTX) gave a stoichiometry of 1dimeric hTS: 1MTX and in the presence of 29ntRNA, the stoichiometry was unchanged. (19)F-NMR spectra of human TS: FdUMP complexes were found to be strikingly similar to analogous NMR spectra of complexes formed by L.casei TS and mouse TS. In the presence of FdUMP, spectra exhibited two additional resonances (-1.50 ppm and -34.4 ppm). The resonance at -1.50 ppm represents non-covalently bound FdUMP, the peak at -34.4 ppm represents covalently bound FdUMP. The addition of methotrexate to the binary TS-FdUMP complex caused a displacement of the internal equilibrium, with only the covalently-bound form seen, and with a slightly disturbed (19)F chemical shift (-36.5 ppm). Similar results were found when MTX was replaced by folinic or folic acid. The addition of 29ntRNA caused no changes to the (19)F spectra of either the binary or ternary complexes.

Distribution of hydrogen in the near surface of Mars: evidence for subsurface ice deposits.

Using the Gamma-Ray Spectrometer on the Mars Odyssey, we have identified two regions near the poles that are enriched in hydrogen. The data indicate the presence of a subsurface layer enriched in hydrogen overlain by a hydrogen-poor layer. The thickness of the upper layer decreases with decreasing distance to the pole, ranging from a column density of about 150 grams per square centimeter at -42 degrees latitude to about 40 grams per square centimeter at -77 degrees. The hydrogen-rich regions correlate with regions of predicted ice stability. We suggest that the host of the hydrogen in the subsurface layer is ice, which constitutes 35 +/- 15% of the layer by weight.

Design, docking, and evaluation of multiple libraries against multiple targets.

We present a general approach to the design, docking, and virtual screening of multiple combinatorial libraries against a family of proteins. The method consists of three main stages: docking the scaffold, selecting the best substituents at each site of diversity, and comparing the resultant molecules within and between the libraries. The core "divide-and-conquer" algorithm for side-chain selection, developed from an earlier version (Sun et al., J Comp Aided Mol Design 1998;12:597-604), provides a way to explore large lists of substituents with linear rather than combinatorial time dependence. We have applied our method to three combinatorial libraries and three serine proteases: trypsin, chymotrypsin, and elastase. We show that the scaffold docking procedure, in conjunction with a novel vector-based orientation filter, reproduces crystallographic binding modes. In addition, the free-energy-based scoring procedure (Zou et al., J Am Chem Soc 1999;121:8033-8043) is able to reproduce experimental binding data for P1 mutants of macromolecular protease inhibitors. Finally, we show that our method discriminates between a peptide library and virtual libraries built on benzodiazepine and tetrahydroisoquinolinone scaffolds. Implications of the docking results for library design are explored.

Telomerase inhibitors for the treatment of cancer: the current perspective.

Telomerase is a holoenzyme responsible for the maintenance of telomeres, the protein-nucleic acid complexes at the ends of eukaryotic chromosomes that serve to maintain chromosomal stability and integrity. Telomerase activity is essential for the sustained proliferation of most immortal cells, including cancer cells. Since the discovery that telomerase activity is detected in 85-90% of all human tumours and tumour-derived cell lines but not in most normal somatic cells, telomerase has become the focus of much attention as a novel and potentially highly-specific target for the development of new anticancer chemotherapeutics. Herein we review the current perspective for the development of telomerase inhibitors as cancer chemotherapeutics. These include antisense strategies, reverse transcriptase inhibitors and compounds capable of interacting with high-order telomeric DNA tetraplex ("G-quadruplex") structures, so as to prevent enzyme access to the necessary linear telomere substrate. Critical appraisal of each individual approach is provided together with highlighted areas of likely future development.

Chemical modification of a variant of human MIP-1alpha; implications for dimer structure.

A sequence variant of human MIP-1alpha, in which Asp26 has been replaced by Al alpha, has been chemically modified by the addition of 13C-labeled methyl groups at each of the lysine residues and the N-terminus. The sites of methylation have been verified by a combination of MALDI-TOF mass spectrometric experiments and tryptic digestion followed by N-terminal mapping. The effect of the modification on the structure and activity of the protein have been determined by analytical ultra-centrifugation, 13C NMR spectroscopy and receptor binding studies. The results of these experiments suggest that huMIP-alpha D26A (BB10010), when present as a dimer, adopts a globular structure, like MCP-3, rather than the elongated or cylindrical structure determined for dimers of huMIP-1beta and RANTES.

Structural equilibria in RNA as revealed by 19F NMR.

We have incorporated 5-fluorouridine into several sites within a 19-mer RNA modelled on the translational operator of the MS2 bacteriophage. The 19F NMR spectra demonstrate the different chemical shifts of helical and loop fluorouridines of the hairpin secondary structure. Addition of salt gives rise to a species in which the loop fluorouridine gains the chemical shift of its helical counterparts, due to the formation of the alternative bi-molecular duplex form. This is supported by UV thermal melting behaviour which becomes highly dependent on the RNA concentration. Distinct 19F NMR signals for duplex and hairpin forms allow the duplex-hairpin equilibrium constant to be determined under a range of conditions, enabling thermodynamic characterisation and its salt dependence to be determined. Mg2+ also promotes duplex formation, but more strongly than Na+, such that at 25 degrees C, 10 mM MgCl2 has a comparable duplex-promoting effect to 300 mM NaCl. A similar effect is observed with Sr2+, but not Ca2+ or Ba2+. Additional hairpin species are observed in the presence of Na+ as well as Mg2+, Ca2+, Sr2+ and Ba2+ ions. The overall, ensemble average, hairpin conformation is therefore salt-dependent. Electrostatic considerations are thus involved in the balance between different hairpin conformers as well as the duplex-hairpin equilibrium. The data presented here demonstrate that 19F NMR is a powerful tool for the study of conformational heterogeneity in RNA, which is particularly important for probing the effects of metal ions on RNA structure. The thermodynamic characterisation of duplex-hairpin equilibria will also be valuable in the development of theoretical models of nucleic acid structure.

Internal mammary artery perfusing the Leriche's syndrome.

Two cases of collateral perfusion of a lower extremity, by way of an internal mammary artery, in the presence of Leriche's syndrome are described. The importance of recognizing this condition prior to coronary artery bypass grafting is emphasized.

The H(2)(18)O solvent-induced isotope shift in (19)F NMR.

The H(2)(16)O/H(2)(18)O solvent-induced isotope shifts ((18)O SIIS) of the (19)F NMR signals of a number of fluorine compounds have been measured. These isotope shifts are observed to be upfield, downfield, or zero, depending on the specific compound and the precise solution conditions. At 25 degrees C and with an (18)O enrichment of 86%, the (18)O SIIS of several fluorinated amino acids were in the range of 0.0014-0.0018 ppm downfield. 5-Fluorouridine displays a significantly wider range of (18)O SIIS values. A 5-fluorouridine-labeled 16-mer RNA also displayed observable (18)O SIIS values, but the characteristics of these were significantly modified from those of free 5-fluorouridine. The experimental observations are consistent with the (18)O SIIS being composed of upfield and downfield components, with the relative contributions of these determining the size and direction of the overall isotope shift. This is discussed in terms of a combination of van der Waals interactions between the fluorine atom and the solvent, electrical and hydrogen bonding effects, and the perturbations to these due to (18)O substitution in the solvent water. This isotope effect promises to be a highly useful tool in a range of (19)F NMR studies.

Internal mammary artery perfusing Leriche's syndrome in association with significant coronary arteriosclerosis: four case reports and review of literature.

Four cases of collateral perfusion of a lower extremity by way of an internal mammary artery in the presence of Leriche's syndrome are described. Angiographic documentation preceding coronary artery bypass grafting prevented an acutely ischemic leg in two of the cases.

Leriche's syndrome with total perfusion of the left lower extremity by way of the left internal mammary artery.

A case of collateral perfusion of a lower extremity by way of an internal mammary artery in the presence of Leriche's syndrome is described. The importance of recognizing this condition before coronary artery bypass grafting is emphasized.

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.

Evaluation of home blood pressure monitors in children and adolescents.

OBJECTIVE: This study was designed to adapt commercially available home blood pressure monitors for use in children ages 4 to 18 years and to compare the recordings obtained from the adapted devices to those obtained using a standard mercury sphygmomanometer. METHODS: Sequential same-arm blood pressures were measured by trained observers in 106 children, ages 4 to 18 years, using a calibrated mercury-gravity manometer (reference device) as the standard method, and 3 test devices (an aneroid manometer and two semiautomated oscillometric devices). For each patient, mid-arm circumference was measured and appropriate blood pressure cuff size was selected. Systolic and diastolic pressures were measured by trained observers using the reference device and the aneroid manometer in accordance with criteria established by the Second Task Force on Blood Pressure Control in Children. Other than variation in cuff size, all manufacturers' recommendations were followed for each test device. RESULTS: Outcome was assessed using criteria established by the British Hypertension Society (BHS) and the Association for the Advancement of Medical Instrumentation (AAMI). The aneroid manometer consistently received a grade of A using BHS criteria and also passed using AAMI criteria. Neither of the two semiautomated monitors achieved a passing grade, although the Labtron monitor performed slightly better than the Marshall 85 monitor. CONCLUSIONS: Home blood pressure monitors must be validated for use in children prior to widespread use. Given appropriate training and verification of observer accuracy, the aneroid manometer can be recommended for home use in children ages 4 to 18 years.

Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme.

The crystal structure of an unmodified hammerhead RNA in the absence of divalent metal ions has been solved, and it was shown that this ribozyme can cleave itself in the crystal when divalent metal ions are added. This biologically active RNA fold is the same as that found previously for two modified hammerhead ribozymes. Addition of divalent cations at low pH makes it possible to capture the uncleaved RNA in metal-bound form. A conformational intermediate, having an additional Mg(II) bound to the cleavage-site phosphate, was captured by freeze-trapping the RNA at an active pH prior to cleavage. The most significant conformational changes were limited to the active site of the ribozyme, and the changed conformation requires only small additional movements to reach a proposed transition-state.

Antibiotic interactions with the hammerhead ribozyme:tetracyclines as a new class of hammerhead inhibitor.

A screening of a range of common laboratory antibiotics for inhibition of the hammerhead ribozyme has shown that in addition to certain aminoglycosides (most notably neomycin B) the tetracyclines are also effective inhibitors, with chlorotetracycline being more effective than tetracycline. Inhibition by chlorotetracycline is not as strong as that by neomycin B but is more complicated, with at least two binding sites apparent. As with hammerhead inhibition by neomycin B, chlorotetracycline inhibition can be overcome by raising the concentration of the Mg2+ ion cofactor. We find that around six Mg2+ ions will displace neomycin B, compared with twelve for chlorotetracycline. Inhibition observed in the presence of mixtures of neomycin B and chlorotetracycline is consistent with separate binding sites on the hammerhead for these two classes of antibiotic. Under certain conditions of the mixing order and low concentration of chlorotetracycline, enhancement of single-turnover hammerhead cleavage by up to 20% is observed, with higher concentrations of antibiotic being inhibitory. We have also found that the presence of 2.5% (v/v) DMSO causes a 30% enhancement of the single-turnover cleavage. These results thus extend the range of known inhibitors of hammerhead cleavage, and also demonstrate how the cleavage can be accelerated.

The roles of the conserved pyrimidine bases in hammerhead ribozyme catalysis: evidence for a magnesium ion-binding site.

We report details of the synthesis and characterization of oligoribonucleotides containing 4-thiouridine or 2-pyrimidinone ribonucleoside (4HC). We have used these probes to examine the roles of the conserved pyrimidines in the central core of the hammerhead ribozyme. The effects on catalysis of singly-substituted hammerhead ribozyme and substrate strands were quantified in multiple-turnover reactions. Various effects were observed on kcat. and Km, with up to a 7-fold decrease and a 3-fold increase respectively. For substitutions with 4HC at positions 3 or 17, catalytic activity in single turnover reactions can be increased up to 8-fold equivalent to 40% of wild-type activity, by increasing the concentration of the Mg2+ cofactor, implying that these substitutions had a deleterious effect on Mg2+ binding. Calculations of the change in the apparent free energy of binding for variants at positions 3, 4 or 17 are each consistent with deletion of a single hydrogen-bond to an uncharged group in the ribozyme. The cytidine 5' to the scissile phosphate had not previously been thought to play a direct role in catalysis, however, removal of the exocyclic amino group decreased kcat. 4-fold. Recently, the crystal structures of a hammerhead ribozyme bound to either a non-cleavable 2'-deoxy substrate strand or a ribo-substrate strand have been reported. The kinetic properties of the variants described here are consistent with several key interactions seen in the crystals, in particular they provide experimental support for the assignment of the proposed catalytically active magnesium ion-binding site.

Effects of substitution of Thr63 by alanine on the structure and function of Lactobacillus casei dihydrofolate reductase.

A mutant of Lactobacillus casei dihydrofolate reductase has been constructed in which Thr63, a residue which interacts with the 2'-phosphate group of the bound coenzyme, is replaced by alanine. This substitution does not affect kcat, but produces an 800-fold increase in the Km for NADPH, which reflects dissociation of NADPH from the enzyme-NADPH-tetrahydrofolate complex, and a 625-fold increase (corresponding to 3.8 kcal/mol) in the dissociation constant for the enzyme-NADPH complex. The difference in magnitude of these effects indicates a small effect of the substitution on the negative cooperativity between NADPH and tetrahydrofolate. Stopped-flow studies of the kinetics of NADPH binding show that the weaker binding arises predominantly from a decrease in the association rate constant. NMR spectroscopy was used to compare the structures of the mutant and wild-type enzymes in solution, in their complexes with methotrexate and with methotrexate and NADPH. This showed that only minimal structural changes result from the mutation; a total of 47 residues were monitored from their resolved 1H resonances, and of these nine in the binary complex and six in the ternary differed in chemical shift between mutant and wild-type enzyme. These affected residues are confined to the immediate vicinity of residue 63. There is a substantial difference in the 31P chemical shift of the 2'-phosphate of the bound coenzyme, reflecting the loss of the interaction with the side chain of Thr63. The only changes in nuclear Overhauser effects (NOEs) observed were decreases in the intensity of NOEs between protons of the adenine ring of the bound coenzyme and the nearby residues Leu62 and Ile102, showing that the substitution of Thr63 does cause a change in the position or orientation of the adenine ring in its binding site.