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Biomedical subjects

A Maxwell

Publications and source records attributed to A Maxwell.

At least 55 records · Page 3Linked to original sources
PubMed1998

Insulin-like growth factor axis of gluten-sensitive dogs during a gluten challenge.

Eight gluten-sensitive Irish setters underwent a gluten challenge to investigate changes in the insulin-like growth factor (IGF) axis. In the first study, they were challenged with an acute intraduodenal administration of tryptic-peptic gluten digest and then maintained on dietary gluten for three months. In the second study, the challenge came solely from dietary gluten fed for three months. After the acute intraduodenal administration of gluten, serum IGF-I levels decreased significantly by 21.8 per cent, (P = 0.01) on day 3 after challenge and then returned to normal. There was also a decrease (52.5 per cent, P < 0.03) in the levels of serum IGF-binding protein-3 (IGFBP-3) until day 14 after challenge but they had returned to normal by day 28. In two dogs IGFBP-3 levels decreased through specific serum protease activity. There were no changes in serum IGF-I or IGFBP levels during the second study after the dietary gluten challenge alone, or in four non-gluten-sensitive beagles studied as controls during the acute intraduodenal/dietary gluten challenge.

Animals↗
PubMed1998

Identification of a residue involved in transition-state stabilization in the ATPase reaction of DNA gyrase.

Examination of the X-ray crystal structure of the 43 kDa N-terminal domain of the DNA gyrase B protein (GyrB) shows that the majority of the interactions with bound ATP are made with subdomain 1 (residues 2-220). However, two residues from subdomain 2, Gln335 and Lys337, interact with the gamma-phosphate of ATP. The proposed roles for these residues include nucleotide binding, transition-state stabilization, and triggering protein conformational changes. We have used site-directed mutagenesis to convert Gln335 to Asn and Ala and Lys337 to Gln and Ala in the N-terminal domain of GyrB. Two of the resultant mutant proteins, GyrB43(Q335A) and GyrB43(K337Q), were shown to be correctly folded, and their interactions with ATP have been analyzed in detail. The Q335A protein is apparently unchanged with regard to nucleotide binding and hydrolysis, whereas the K337Q protein shows a modest decrease in nucleotide binding and a drastic reduction in ATPase activity. This is manifested by a approximately 10(3)-fold decrease in kcat. When the two mutations were moved into full-length GyrB, the Q335A mutation again showed little or no effect on activity, whereas the K337Q mutation had undetectable supercoiling and ATPase activities. We conclude that Gln335 is dispensable for ATP binding and hydrolysis by the gyrase B protein, whereas Lys337 has a critical role in the ATPase reaction and is likely to be a key residue in transition-state stabilization.

Adenosine Triphosphatases↗
PubMed1998

A method to measure clinical erosion: the effect of orange juice consumption on erosion of enamel.

OBJECTIVE: Acidic soft drinks are frequently implicated in dental erosion, but there are limited supporting data. Research is problematic due to the insidious nature of erosion and accuracy in assessing tissue loss. The aim of this study was to develop and validate, using a negative control, a model to accurately measure erosion in situ due to a single aetiological agent over a relatively short time period. METHODS: An intra-oral appliance capable of retaining an enamel sample was designed in order to assess the effect of orange juice consumption on enamel. The study was a single centre, randomized, placebo controlled, blind, crossover design. RESULTS: Ten subjects, each consuming 11 of orange juice per day for 15 days, showed significantly more erosion on the enamel specimens than the same subjects consuming 11 of water per day over the same time period, measurements undertaken with surfometry. The same investigation was performed in vitro. Again, orange juice was significantly more erosive; indeed, it was in the order of 10 times that produced in situ. Surface microhardness testing in situ and in vitro demonstrated statistically significant differences between exposed and unexposed areas after orange juice treatment. CONCLUSIONS: Changes produced by water either in situ or in vitro were always well within the baseline measurement parameters (+/- 0.3 micron) set down for the method and hence validated the clinical model in terms of reproducibility and accuracy in measurement. It is concluded that this method has confirmed the erosive potential of orange juice in situ. The method could have many applications to study dental erosion under highly controlled conditions and over realistic time periods.

Adult↗
PubMed1998

Use of a rapid throughput in vivo screen to investigate inhibitors of eukaryotic topoisomerase II enzymes.

Topoisomerase II catalyzes the passage of one DNA helix through another via a transient double-stranded break. The essential nature of this enzyme in cell proliferation and its mechanism of action make it an ideal target for cytotoxic agents. Saccharomyces cerevisiae topoisomerase II has been frequently used as a model for testing potential inhibitors of eukaryotic topoisomerase II as antitumor agents. The standard in vivo method of estimating the sensitivity of S. cerevisiae to the antitopoisomerase drugs is via inhibition or kill curves which rely on viable-cell counts and is labor intensive. We present an alternative to this, a high-throughput in vivo screen. This method makes use of a drug-permeable S. cerevisiae strain lacking endogenous topoisomerase II, which is modified to express either human topoisomerase IIalpha or IIbeta or S. cerevisiae topoisomerase II carried on plasmids. Each modified strain expresses a full-length topoisomerase II enzyme, as opposed to the more commonly used temperature-sensitive S. cerevisiae mutant expressing yeast or yeast/human hybrid enzymes. A comparison of this new method with a plating-and-counting method gave similar drug sensitivity results, with increased accuracy and reduced manual input for the new method. The information generated has highlighted the sensitivities of different topoisomerase II enzymes and isoenzymes to several different classes of topoisomerase II inhibitor.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Nutritional modulation of canine insulin-like growth factors and their binding proteins.

The response of canine insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) to moderate nutritional restriction followed by refeeding has not previously been studied in detail. The purpose of these studies was to examine the effects of nutritional restriction on the IGF system of adult dogs. Normal serum IGF values were established after validation of heterologous RIAs for measuring canine IGFs-I and -II. Canine serum IGFBP profiles were examined by Western ligand blotting (WLB), using radiolabelled recombinant human (rh) IGF-I as the ligand, and were found to be similar to those of other species. IGF-I and IGFBP-3 concentrations correlated with body weight, thus reflecting breed size as previously shown, whereas IGF-II concentrations did not. IGFBP-2 serum concentrations and band intensity on WLB were increased compared with normal human serum IGFBP-2. Overnight fasting had no effect on IGF or IGFBP concentrations, including IGFBP-1, nor did refeeding. Prolonged restriction to 56% and then 42.5% of maintenance energy requirements for 2 weeks decreased IGF-I concentrations by 20.4% and 32.7% respectively. Feeding of the same diet ad libitum for 2 weeks normalised IGF-I concentrations. There were no changes in IGF-II or insulin levels. Serum IGFBP-2 concentrations increased with 56% restriction of maintenance energy (P = 0.03). We conclude that serum IGF-I is potentially a useful marker of short-term change in nutritional status in the adult dog.

Animals↗
PubMed1997

The DNA dependence of the ATPase activity of human DNA topoisomerase IIalpha.

We have purified human topoisomerase IIalpha from HeLa cells and studied its ATPase reaction. The ATPase activity is stimulated by DNA and shows apparent Michaelis-Menten kinetics. Although the ATPase activity of human topoisomerase IIalpha is lower than that of Saccharomyces cerevisiae, it is more active in decatenation, implying more efficient coupling of the ATPase to DNA strand passage under these conditions. Using plasmid pBR322 as the DNA cofactor, the reaction shows hyperstimulation by DNA at a base pair to enzyme dimer ratio of 100-200:1. When DNA fragments are used as the cofactor, the reaction requires > approximately 100 base pairs to stimulate the activity and fragments of approximately 300 base pairs show hyperstimulation. This behavior can be rationalized in terms of the enzyme requiring fragments that can bind to both the DNA gate and the ATP-operated clamp in order for the ATPase reaction to be stimulated. Hyperstimulation is a consequence of the saturation of DNA with enzyme. The mechanistic implications of these results are discussed.

Adenosine Triphosphatases↗
PubMed1997

DNA topology: topoisomerases keep it simple.

The ability of type II DNA topoisomerases to perturb the equilibrium distributions of DNA topoisomers is a consequence of their ability to hydrolyse ATP. A sliding mechanism of topoisomerase action has been proposed to account for this phenomenon.

Adenosine Triphosphate↗
PubMed1997

The interaction of the F plasmid killer protein, CcdB, with DNA gyrase: induction of DNA cleavage and blocking of transcription.

We have studied the interaction of the F plasmid killer protein CcdB with its intracellular target DNA gyrase. We confirm that CcdB can induce DNA cleavage by gyrase and show that this cleavage reaction requires ATP hydrolysis when the substrate is linear DNA, but is independent of hydrolysis when negatively supercoiled DNA is used. The 64 kDa domain of the gyrase A protein, which can catalyse DNA cleavage in the presence of the B protein and quinolone drugs, is unable to cleave DNA in the presence of CcdB unless the C-terminal 33 kDa domain of the gyrase A protein is also present. CcdB-induced DNA cleavage by gyrase requires a minimum length of DNA (> approximately 160 bp), whereas in the presence of quinolone drugs gyrase can cleave much shorter DNA molecules. We show that CcdB, like quinolones, can form a complex with gyrase which can block transcription by RNA polymerase. A model for the interaction of CcdB with gyrase involving the trapping of a post-strand-passage intermediate is suggested. We conclude that CcdB can stabilise a cleavage complex between DNA gyrase and DNA in a manner distinct from quinolones but, like the quinolone-induced cleavage complex, the CcdB-stabilised complex can also form a barrier to the passage of polymerases.

Bacterial Proteins↗
PubMed1997

Crystal structure of the breakage-reunion domain of DNA gyrase.

DNA gyrase is a type II DNA topoisomerase from bacteria that introduces supercoils into DNA. It catalyses the breakage of a DNA duplex (the G segment), the passage of another segment (the T segment) through the break, and then the reunification of the break. This activity involves the opening and dosing of a series of molecular 'gates' which is coupled to ATP hydrolysis. Here we present the crystal structure of the 'breakage-reunion' domain of the gyrase at 2.8 A resolution. Comparison of the structure of this 59K (relative molecular mass, 59,000) domain with that of a 92K fragment of yeast topoisomerase II reveals a very different quaternary organization, and we propose that the two structures represent two principal conformations that participate in the enzymatic pathway. The gyrase structure reveals a new dimer contact with a grooved concave surface for binding the G segment and a cluster of conserved charged residues surrounding the active-site tyrosines. It also shows how breakage of the G segment can occur and, together with the topoisomerase II structure, suggests a pathway by which the T segment can be released through the second gate of the enzyme. Mutations that confer resistance to the quinolone antibacterial agents cluster at the new dimer interface, indicating how these drugs might interact with the gyrase-DNA complex.

Amino Acid Sequence↗
PubMed1997

DNA gyrase can cleave short DNA fragments in the presence of quinolone drugs.

We have analysed the DNA cleavage reaction of DNA gyrase using oligonucleotides annealed to a single-stranded M13 derivative containing a preferred gyrase cleavage site. We find that gyrase can cleave duplexes down to approximately 20 bp in size in the presence of the quinolone drugs ciprofloxacin and oxolinic acid. Ciprofloxacin shows a variation in its site specificity with an apparent preference for G bases adjacent to the cleavage sites, whereas oxolinic acid stimulates cleavage predominantly at the previously determined site. With either drug, cleavage will not occur within 6 bases from the end of a DNA duplex or a nick. We suggest that cleavage site specificity with short DNA duplexes is determined by drug-DNA interactions whereas with longer fragments the positioning effect of the DNA wrap around gyrase prescribes the site of cleavage.

Bacteriophage M13↗
PubMed1997

Exploiting nucleotide thiophosphates to probe mechanistic aspects of Escherichia coli DNA gyrase.

The interaction of DNA gyrase with ATP has been probed using a range of thiophosphate ATP analogs. ATP gammaS is not detectably hydrolyzed by gyrase but can support limited, probably catalytic, DNA supercoiling. ATP gammaS is a good inhibitor of both ATP hydrolysis and ATP-supported supercoiling. In contrast, both ATP alphaS(Rp) and ATP betaS(Rp) have been shown to be good substrates for the ATPase reaction of gyrase and to support catalytic DNA supercoiling. The corresponding Sp diastereoisomers do not support significant levels of supercoiling and are not readily hydrolyzed, but are shown to be reasonable inhibitors of gyrase. For ATP alphaS(Rp), the supercoiling and ATPase activities appear to be tightly coupled with the thionucleotide being apparently a better substrate than ATP in terms of both DNA supercoiling and nucleotide hydrolysis. In the case of ATP betaS(Rp), DNA supercoiling and nucleotide hydrolysis appear to be uncoupled in that ATP betaS(Rp) is almost as good a substrate as ATP for the ATPase reaction of both intact gyrase and the 43 kDa GyrB fragment, whereas it only supports slow DNA supercoiling; the mechanistic consequences of these observations are discussed in terms of a new model for energy coupling in gyrase. DNA gyrase has been shown to be capable of catalyzing DNA supercoiling in the presence of Mg2+, Ca2+, and Mn2+ but not Zn2+, Co2+, Ni2+, or Cd2+. The pronounced diastereoselectivity seen in both the DNA supercoiling and ATPase activity with ATP alphaS and ATP betaS together with evidence from the X-ray structure of the 43 kDa GyrB-ADPNP-Mg complex is consistent with metal ion coordination at both of these sites, and probably to the gamma-phosphoryl center during turnover. Thus, the absolute configuration of the catalytically active Mg2+-ATP complex is likely to involve coordination to the pro-S oxygens at both P alpha and P beta, leading to the alpha,beta,gamma-tridentate Mg-ATP complex with the lambda-exo configuration.

Adenosine Triphosphatases↗
PubMed1997

DNA gyrase as a drug target.

DNA gyrase is a remarkable enzyme, catalysing the seemingly complex reaction of DNA supercoiling. As gyrase is essential in prokaryotes, it is a good target for antibacterial agents. These agents have diverse chemical structures and interact with gyrase in a variety of ways.

Coumarins↗
PubMed1996

Probing the role of the ATP-operated clamp in the strand-passage reaction of DNA gyrase.

The high-resolution structure of the 43 kDa N-terminal fragment of the DNA gyrase B protein shows a large cavity within the protein dimer. The approximate size of this cavity is 20 A, suggesting it could accommodate a DNA helix. Computer-modelling studies of this cavity suggest that it contains a constriction, reducing the width to approximately 13 A, principally caused by the side chain of Arg286. We have used site-directed mutagenesis to alter this residue to Gln. Gyrase bearing this mutation shows virtually no supercoiling activity and near-normal relaxation and DNA cleavage activities. The mutated protein has ATPase activity which cannot be stimulated by DNA. These data support the proposed role of the 43 kDa domain as an ATP-operated clamp which binds DNA during the supercoiling cycle. The lack of DNA-dependent ATPase of the mutant may indicate that binding of DNA within the clamp is a prerequisite for stimulation of the ATPase activity.

Adenosine Triphosphatases↗
PubMed1996

Conversion of DNA gyrase into a conventional type II topoisomerase.

DNA gyrase is unique among topoisomerases in its ability to introduce negative supercoils into closed-circular DNA. We have demonstrated that deletion of the C-terminal DNA-binding domain of the A subunit of gyrase gives rise to an enzyme that cannot supercoil DNA but relaxes DNA in an ATP-dependent manner. Novobiocin, a competitive inhibitor of ATP binding by gyrase, inhibits this reaction. The truncated enzyme, unlike gyrase, does not introduce a right-handed wrap when bound to DNA and stabilizes DNA crossovers; characteristics reminiscent of conventional type II topoisomerases. This new enzyme form can decatenate DNA circles with increased efficiency compared with intact gyrase and, as a result, can complement the temperature-sensitive phenotype of a parCts mutant. Thus these results suggest that the unique properties of DNA gyrase are attributable to the wrapping of DNA around the C-terminal DNA-binding domains of the A subunits and provide an insight into the mechanism of type II topoisomerases.

Animals↗
PubMed1996

DNA cleavage is not required for the binding of quinolone drugs to the DNA gyrase-DNA complex.

The primary target for the quinolone group of antibacterial agents is DNA gyrase. One model for the interaction of quinolone drugs with gyrase and DNA suggests that the drugs bind to the single-stranded regions revealed following DNA cleavage by the enzyme. We have tested this hypothesis by using mutants which have the active-site tyrosine in the gyrase A subunit altered to phenylalanine or serine. We have found that proteins bearing these mutations are still able to bind drug, suggesting that DNA cleavage is not a prerequisite for drug binding. We have also found that the blocking of transcription by RNA polymerase in vitro by the gyrase-quinolone complex on DNA does not occur when the active-site tyrosine is mutated to serine; i.e., polymerase blocking requires DNA cleavage.

Anti-Infective Agents↗
PubMed1996

The interaction of coumarin antibiotics with fragments of DNA gyrase B protein.

DNA gyrase is the target of the coumarin group of antibacterial agents. The drugs are known to inhibit the ATPase activity of gyrase and bind to the 24-kDa N-terminal subdomain of gyrase B protein. Supercoiling assays with intact DNA gyrase and ATPase assays with a 43-kDa N-terminal fragment of the B protein suggest that the drugs bind tightly, with Kd values <10(-7) M. In addition, the ATPase data suggest that 1 coumermycin molecule interacts with 2 molecules of the 43-kDa protein while the other coumarins form a 1:1 complex. This result is confirmed by cross-linking experiments. Rapid gel-filtration experiments show that the binding of ADPNP(5'-adneylyl beta,gamm-imidodiphosphate) and coumarins to the 43-kDa protein is mutally exclusive, consistent with a competitive mode of action for the drugs. Rapid gel-filtration binding experiments using both the 24-and 43-kDa proteins also show that the drugs bind with association rate constants of >10(5) M-1.s-1, and dissociation rate constants of approximately 3x10(-3)s-1 and approximately 4x10(-3)s-1 for the 43-and 24-kDa proteins, respectively. Titration calorimetry shows that the Kd values for coumarins binding to both proteins are approximately 10-8M and that binding is enthalpy driven.

Adenylyl Imidodiphosphate↗
PubMed1996

The nature of inhibition of DNA gyrase by the coumarins and the cyclothialidines revealed by X-ray crystallography.

This study describes the first crystal structures of a complex between a DNA topoisomerase and a drug. We present the structures of a 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein in complexes with two different inhibitors of the ATPase activity of DNA gyrase, namely the coumarin antibiotic, novobiocin, and GR122222X, a member of the cyclothialidine family. These structures are compared with the crystal structure of the complex with an ATP analogue, adenylyl-beta-gamma-imidodiphosphate (ADPNP). The likely mechanism, by which mutant gyrase B proteins become resistant to inhibition by novobiocin are discussed in light of these comparisons. The three ligands are quite dissimilar in chemical structure and bind to the protein in very different ways, but their binding is competitive because of a small degree of overlap of their binding sites. These crystal structures consequently describe a chemically well characterized ligand binding surface and provide useful information to assist in the design of novel ligands.

Adenylyl Imidodiphosphate↗