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Biomedical subjects

K R Fox

Publications and source records attributed to K R Fox.

At least 19 recordsLinked to original sources
PubMed1999

5-(1-propargylamino)-2'-deoxyuridine (UP): a novel thymidine analogue for generating DNA triplexes with increased stability.

We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (UP). The intra-molecular triplex A6-L-T6-L-(UP)5T (L = two octanediol residues) shows a single UV-melting transition which is >20 degrees higher than that of the parent triplex A6-L-T6-L-T6at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular triplex with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by UP, in a pH dependent fashion. Triplex stability increases by approximately 12 K for each T to UP substitution. Quantitative footprinting studies have examined the interaction of three UP-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5'-AGGAAGAGAAAAAAGAA. At pH 5.0, the UP-containing oligo-nucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (UP)6CUPT binds approximately 1000-fold more tightly than the unmodified oligonucleotide T6CTT. Oligonucleotides containing fewer UP residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.

DNA↗
PubMed1999

Towards mixed sequence recognition by triple helix formation.

The formation of intermolecular DNA triple helices offers the possibility of designing compounds with extensive sequence recognition properties which may be useful as antigene agents or tools in molecular biology. One major limitation of this approach is that these structures are generally restricted to homo-purine. homopyrimidine target sites. This review describes the strategies that have been employed to overcome this drawback and outlines the potential for triplex formation at mixed sequence DNA targets.

Binding Sites↗
PubMed1999

DNA triple-helix formation on nucleosome core particles. Effect of length of the oligopurine tract.

We have used DNase I footprinting to examine the formation of intermolecular triplexes on DNA fragments which have been complexed with nucleosome core particles. We have prepared five DNA fragments, based on the 160-bp tyrT sequence, which contain different length oligopurine tracts (up to 25 bp) at two different positions along the fragment, and have examined their availability for triple-helix formation after reconstituting onto nucleosome core particles. These results are compared with the formation of shorter triplexes in the same regions. In general we find that increasing the length of the complex does not facilitate nucleosomal triplex formation and that the most important factor affecting triplex formation is the position of the target site within the nucleosome-bound fragment. In some instances we find that longer oligonucleotides inhibit triplex formation. Although successful triplex formation was achieved on the longest nucleosome-bound oligopurine tracts, this was accompanied by changes in cleavage pattern that suggest oligonucleotide-induced changes in nucleosome structure.

Animals↗
PubMed1998

Triple-helix formation at different positions on nucleosomal DNA.

We have prepared a series of seven DNA fragments, based on the 160 base-pair tyrT sequence, which contain 12-14 base-pair oligopurine tracts at different positions, and have examined their availability for triple-helix formation after reconstituting onto nucleosome core particles. By using DNase I footprinting we find that in general, triplexes can only be formed at sites located toward the ends of nucleosomal DNA fragments. For the native fragment, bases 1-145 are in contact with the protein surface. Stable triplexes can be formed on these nucleosome-bound fragments for sites located before position 33 and beyond position 94. These are formed with both CT-containing oligonucleotides, generating parallel triplexes at pH 5.5, and GT-containing oligonucleotides forming antiparallel triplexes at pH 7.5. No antiparallel triplexes were formed at sites located between these positions. Parallel triplexes were also not formed at sites between positions 39-50 and 43-54 with oligonucleotide concentrations as high as 30 microM. However parallel triplex formation was evident at a site between positions 48 and 59, albeit with a reduced affinity compared to free DNA, suggesting that this oligopurine tract is less tightly associated with the nucleosome surface or that it has an altered translational position. The introduction of an oligopurine tract in the vicinity of the nucleosome dyad caused the fragment to adopt a different nucleosomal position, which could be targeted with parallel, but not antiparallel triplexes.

Base Sequence↗
PubMed1998

Triple helix formation at (AT)n adjacent to an oligopurine tract.

We have used DNase I footprinting to investigate the recognition of (AT) n tracts in duplex DNA using GT-containing oligonucleotides designed to form alternating G.TA and T.AT triplets. Previous studies have shown that the formation of these complexes is facilitated by anchoring the triplex with a block of adjacent T.AT triplets, i.e. using T11(TG)6to recognize the target A11(AT)6. (AT)6T11. In the present study we have examined how the stability of these complexes is affected by the length of either the T.AT tract or the region of alternating G.TA and T.AT triplets, using oligonucleotides of type T x (TG) y to recognize the sequence A11(AT)11. We find that successful triplex formation at (AT)n (n = 3, 6 or 11) can be achieved with a stabilizing tail of 11xT.AT triplets. The affinity of the third strand increases with the length of the (GT) n tract, suggesting that the alternating G.TA and T.AT triplets are making a positive contribution to stability. These complexes are stabilized by the presence of manganese or a triplex-specific binding ligand. Shorter oligo-nucleotides, such as T7(TG)5, bind less tightly and require the addition of a triplex-binding ligand. T4(GT)5showed no binding under any conditions. Oligo-nucleotides forming a 3'-terminal T.AT are marginally more stable that those with a terminal G.TA. The stability of these complexes was further increased by replacing two of the T.AT triplets in the T n tail region with two C+.GC triplets.

Base Sequence↗
PubMed1998

DNA triple-helix formation on nucleosome-bound poly(dA).poly(dT) tracts.

We have used DNase I and hydroxyl-radical footprinting to examine the formation of intermolecular DNA triple helices on nucleosome-bound DNA fragments containing An.Tn tracts. We found that it is possible to form triplexes on these nucleosome-bound DNAs, but the stability of the complexes depends on the orientation of the A tract with respect to the protein surface. Hydroxyl-radical cleavage of these complexes suggests that the DNA fragments are still associated with the nucleosome. However, the phased cleavage pattern is lost in the vicinity of the triplex, suggesting that the DNA has locally moved away from the protein surface.

Base Sequence↗
PubMed1998

Is body composition important in young people's weight management decision-making?

AIM: Young people are often seen as an important target for prevention of overweight, but we know little about the factors which are important for their weight management decisions. This study aimed to evaluate the extent to which elements of body composition and dimensions were implicated in their decisions to change their weight. PARTICIPANTS: Participants were 116 male and 126 female volunteers from a tertiary college in the south-west of England with a mean age of 17.90 (s.d. = 1.90) y. MEASURES: Body composition measures included height (m), weight (kg), waist and hip circumferences (mm), shoulder and hip girths (mm) and skinfold thicknesses (triceps, calf and subscapular, mm). Students also provided self-reported information on dietary practices they had undertaken in the last year. RESULTS: More females reported attempting weight loss in the last year compared to males (51.5 vs 17.6%), although more males reported attempting weight gain (19.3 vs 2.3%). Reported dietary strategies for both males and females centred around eating less fatty foods, eating less than usual and exercising more. However, a significant proportion of females also reported excessive strategies, such as self-induced vomiting and regular crash dieting. Results indicated that females who desired to lose weight were significantly (P < 0.05) heavier, and bigger in terms of circumferences and skeletal breadths, compared to females who did not wish to change their weight. For males, the 'weight loss' group were significantly (P < 0.05) bigger, heavier and fatter compared to the 'no change' and 'weight gain' group. For females only, the previously significant main effect for body dimensions across weight management groups (Pillais F(14,188) = 3.81, P < 0.001) was extinguished when controlling for bone dimensions (Pillais F(12,198) = 1.68, P = 0.074). CONCLUSION: These results indicate that frame size, particularly in the lower trunk, is a more important predictor of female weight management decision-making, than their levels of fatness. For males, fatness appears to drive their weight management decision-making processes to a greater extent.

Adolescent↗
PubMed1998

Hodgkin's disease diagnosed by endoscopic ultrasound-guided fine needle aspiration of a periduodenal lymph node.

Hodgkin's disease rarely presents as obstructive jaundice. We report a case of Hodgkin's disease arising in periduodenallymph nodes, presenting with biliary obstruction, definitively diagnosed on cytologic material obtained by endosonographically-guided real-time fine needle aspiration biopsy and confirmed at laparotomy. The medical literature pertaining to the use of endosonography and fine needle aspiration biopsy for pancreatic lesions and abdominal lymphoma is reviewed. Currently available data support the use of fine needle aspiration biopsy in establishing the diagnosis of lymphoma. This case highlights the utility of endoscopic ultrasonography with endosonographically guided real-time fine needle aspiration biopsy in diagnosing and managing patients with extrahepatic biliary obstruction or suspected abdominal lymphoma. Pairing endosonographically guided real-time fine needle aspiration biopsy with on-site cytologic assessment and immediate specimen triage can lead to definitive diagnosis of abdominal lymphoma, avoiding surgical intervention in many cases.

Adult↗
PubMed1998

First-cycle blood counts and subsequent neutropenia, dose reduction, or delay in early-stage breast cancer therapy.

PURPOSE: If patients could be ranked according to their projected need for supportive care therapy, then more efficient and less costly treatment algorithms might be developed. This work reports on the construction of a model of neutropenia, dose reduction, or delay that rank-orders patients according to their need for costly supportive care such as granulocyte growth factors. PATIENTS AND METHODS: A case series and consecutive sample of patients treated for breast cancer were studied. Patients had received standard-dose adjuvant chemotherapy for early-stage nonmetastatic breast cancer and were treated by four medical oncologists. Using 95 patients and validated with 80 additional patients, development models were constructed to predict one or more of the following events: neutropenia (absolute neutrophil count [ANC] < or = 250/microL), dose reduction > or = 15% of that scheduled, or treatment delay > or = 7 days. Two approaches to modeling were attempted. The pretreatment approach used only pretreatment predictors such as chemotherapy regimen and radiation history; the conditional approach included, in addition, blood count information obtained in the first cycle of treatment. RESULTS: The pretreatment model was unsuccessful at predicting neutropenia, dose reduction, or delay (c-statistic = 0.63). Conditional models were good predictors of subsequent events after cycle 1 (c-statistic = 0.87 and 0.78 for development and validation samples, respectively). The depth of the first-cycle ANC was an excellent predictor of events in subsequent cycles (P = .0001 to .004). Chemotherapy plus radiation also increased the risk of subsequent events (P = .0011 to .0901). Decline in hemoglobin (HGB) level during the first cycle of therapy was a significant predictor of events in the development study (P = .0074 and .0015), and although the trend was similar in the validation study, HGB decline failed to reach statistical significance. CONCLUSION: It is possible to rank patients according to their need of supportive care based on blood counts observed in the first cycle of therapy. Such rankings may aid in the choice of appropriate supportive care for patients with early-stage breast cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1998

Modeling the cost-effectiveness of granulocyte colony-stimulating factor use in early-stage breast cancer.

PURPOSE: To model the cost-effectiveness (CE) of granulocyte colony-stimulating factor (G-CSF) in early-stage breast cancer when its use is directed to those most in need of the medication. METHODS: A conditional CE model was developed for the use of G-CSF based on a ranking of patient need as determined by patient blood counts during the first cycle of chemotherapy. In the base case, no G-CSF was used. In the alternative case, G-CSF was used in the following manner. If the risk of a neutropenic event (as defined by a predictive model based on nadir absolute neutrophil count [ANC] and hemoglobin decrease in cycle 1) was equal to or exceeded a predetermined critical value "T," then patients would receive G-CSF in cycles 2 through 6 of chemotherapy. If the risk of an event was less than T, patients would not use G-CSF unless an event occurred, at which time G-CSF would be administered with every subsequent cycle. RESULTS: A decision rule (T) that would allow the most needy 50% of early-stage breast cancer patients to receive G-CSF after the first cycle of chemotherapy resulted in a CE ratio of $34,297 dollars per life-year saved (LYS). If only the most needy 10% of patients received G-CSF, then the associated CE ratio was $23,748/LYS; if 90% of patients could receive the medication, the CE ratio would be $76,487/LYS. These estimates were relatively insensitive to inpatient hospital cost estimates (inpatient costs for fever and neutropenia of $3,090 to $7,726 per admission produced dollar per LYS figures of $34,297 to $32,415, respectively). However, the model was sensitive to assumptions about the shape of the relationship between dose reduction and disease-free survival (DFS) at 3 years. CONCLUSION: Providing G-CSF to the neediest 50% of early-stage breast cancer patients (as defined by first-cycle blood counts) starting after the first cycle of chemotherapy is associated with a CE ratio of $34,297/LYS, which is well in the range of CE ratios for treatment of other common medical conditions. Furthermore, conditional CE studies, based on predictive models that incorporate individual patient risk, allow one to define populations for which therapy is, or is not, cost-effective. Limitations of our present understanding of the shape of the chemotherapy dose-response curve, especially at low levels of dose reductions, affect these results. Further work is required to define the shape of the dose-response curve in early-stage breast cancer.

Breast Neoplasms↗
PubMed1998

Motivation for physical activity and weight management.

The importance of exercise for health and long-term weight management is now well-established. However, the critical challenge is facilitating increased physical activity in the large sector of young to middle-aged adults who are sedentary and already overweight. The psychology of exercise initiation and adherence in the overweight is seriously under-researched. However, it is possible to infer findings from the general population and from other health behaviours to develop effective exercise promotion strategies. Motivation and barriers to exercise, exercise-related beliefs, attitudes, and self-efficacy, and the formulation of self-perceptions and identity towards exercise need to be considered in interventions and campaigns to capture the interests of the general public. Readiness to change and behaviour change strategies need to be considered and incorporated into social support structures to facilitate individual behaviour change. This could be delivered through community, workplace and primary health care settings.

Adult↗
PubMed1997

Recognition of GC base pairs by triplex forming oligonucleotides containing nucleosides derived from 2-aminopyridine.

We have attempted to alleviate the pH dependency of triplex recognition of guanine by using intermolecular triplexes containing 2-amino-5-(2-deoxy-d-ribofuranosyl)pyridine (AP) as an analogue of 2'-deoxycytidine (dC). We find that for the beta-anomer of AP, the complex between (AP)6T6and the target site G6A6*T6C6is stable, generating a clear DNase I footprint at oligonucleotide concentrations as low as 0.25 microM at pH 5.0, in contrast to 50 microM C6T6which has no effect on the cleavage pattern. This complex is still stable at pH 6.5 producing a footprint with 1 microM oligonucleotide. Oligonucleotides containing the alpha-anomer of AP are much less effective than the beta-anomer, though in some instances they are more stable than the unmodified oligonucleotides. The results of molecular dynamics studies on a range of AP-containing triplexes has rationalized the observed stability behaviour in terms of hydrogen-bonding behaviour.

5-Methylcytosine↗
PubMed1997

Interaction of minor groove binding ligands with long AT tracts.

We have used quantitative DNase I footprinting to examine the ability of distamycin and Hoechst 33258 to discriminate between different arrangements of AT residues, using synthetic DNA fragments containing multiple blocks of (A/T)6or (A/T)10in identical sequence environments. Previous studies have shown that these ligands bind less well to (A/T)4sites containing TpA steps. We find that in (A/T)6tracts distamycin shows little discrimination between the various sites, binding approximately 2-fold stronger to TAATTA than (TA)3, T3A3and GAATTC. In contrast, Hoechst 33258 binds approximately 20-fold more tightly to GAATTC and TAATTA than T3A3and (TA)3. Hydroxyl radical footprinting reveals that both ligands bind in similar locations at the centre of each AT tract. At (A/T)10sites distamycin binds with similar affinity to T5A5, (TA)5and AATT, though bands in the centre of (TA)5are protected at approximately 50-fold lower concentration than those towards the edges. Hoechst 33258 shows a similar pattern of preference, with strong binding to AATT, T5A5and the centre of (TA)5. Hydroxyl radical footprinting reveals that at low concentrations both ligands bind at the centre of (TA)5and A5T5, while at higher concentrations ligand molecules bind to each end of the (A/T)10tracts. At T5A5two ligand molecules bind at either end of the site, even at the lowest ligand concentration, consistent with the suggestion that these compounds avoid the TpA step. Similar DNase I footprinting experiments with a DNA fragment containing T n (n = 3-6) tracts reveals that both ligands bind in the order T3< T4 << T5 = T6.

Binding Sites↗
PubMed1997

Relative stability of triplexes containing different numbers of T.AT and C+.GC triplets.

We have used DNase I footprinting to compare the stability of parallel triple helices containing different numbers of T.AT and C+. GC triplets. We have targeted a fragment containing the 17mer sequence 5'-AGGAAGAGAAAAAAGAA with the 9mer oligonucleotides 5'-TCCTTCTCT, 5'-TTCTCTTTT and 5'-TTTTTTCTT, which form triplexes at the 5'-end, centre and 3'-end of the target site respectively. Quantitative DNase I footprinting has shown that at pH 5.0 the dissociation constants of these oligonucleotides are 0.13, 4.7 and >30 microM respectively, revealing that increasing the proportion of C+.GC triplets increases triplex stability. The results suggest that the positive charge on the protonated cytosine contributes to triplex stability, either by a favourable interaction with the stacked pisystem or by screening the charge on the phosphate groups. In the presence of a naphthylquinoline triplex binding ligand all three oligonucleotides bind with similar affinities. At pH 6.0 these triplexes only form in the presence of the triplex binding ligand, while at pH 7.5 footprints are only seen with the oligonucleotide which generates the fewest number of C+.GC triplets (TTTTTTCTT) in the presence of the ligand.

Adenine↗
PubMed1997

DNA triple helix formation at oligopurine sites containing multiple contiguous pyrimidines.

We have used DNase I footprinting to assess the formation of triple helices at 15mer oligopurine target sites which are interrupted by several (up to four) adjacent central pyrimidine residues. Third strand oligonucleotides were designed to generate complexes containing central (X.TA)nor (X.CG)n triplets (X = each base in turn) surrounded by C+.GC and T.AT triplets. It has previously been shown that G.TA and T.CG are the most stable triplets for recognition of single TA and CG interruptions. We show that these triplets are the most useful for recognizing consecutive pyrimidine interruptions and find that addition of each pyrimidine residue leads to a 30-fold decrease in third strand affinity. The addition of 10 microM naphthylquinoline triplex-binding ligand stabilizes each complex so that all the oligonucleotides produce footprints at similar concentrations (0.3 microM). Targets containing two pyrimidines are only bound by oligonucleotides generating (G.TA)2 and (T.CG)2 with a further 30-fold decrease in affinity. (G.TA)2 is slightly more stable than (T.CG)2. In the presence of the triplex-binding ligand the order of stability is (G.TA)2 > (C.TA)2 > (T.TA)2 > (A.TA)2 and (T.CG)2 > (C.CG)2 > (G.CG)2 = (A.CG)2. No oligonucleotide footprints are generated at target sites containing three consecutive pyrimidines, though addition of 10 microM triplex-binding ligand produces stable complexes with oligonucleotides generating (G.TA)3, (T.CG)3 and (C.CG)3, with a further 30-fold reduction in affinity. No footprints are generated at targets containing four Ts, though the ligand induces a weak interaction with the oligonucleotide generating (T.CG)4.

Base Sequence↗
PubMed1997

Kinetic studies on the formation of intermolecular triple helices.

We have used DNase I footprinting to examine the association kinetics of GA-, GT- and CT-containing oligonucleotides with the target sequence (GGA)5GG. (CCT)5CC. These reactions are slow yielding bimolecular association rate constants between 50 and 2000 M-1s-1. We find that GT-containing oligonucleotides bind much faster than GA- or CT-containing third strands. In each case the observed rate constants are faster at the centre than at the edges of the target site. Although several explanations can be offered for this observation, it is consistent with a model in which triplex formation at this repetitive site is achieved via intermediate complexes in which the third strand is not properly aligned with its target and which subsequently migrate to the correct position.

DNA Footprinting↗