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G D Jones

Publications and source records attributed to G D Jones.

At least 55 records · Page 3Linked to original sources
PubMed1993

Yield of strand breaks as a function of scavenger concentration and LET for SV40 irradiated with 4He ions.

We have measured by gel electrophoresis the yields of single- and double-strand breaks (SSBs and DSBs) induced in aqueous solutions of SV40 DNA and the SV40 minichromosome by 137Cs gamma rays (mean LET 0.3 keV micron-1) and 4He ions (mean LETs 85, 102, and 152 keV microns-1). DNA SSBs are caused mainly by the hydroxyl radicals under these conditions and are reduced in yield as either the hydroxyl radical scavenger concentration or the LET is increased (over the range studied). The G(SSB) for 4He ion irradiation is less by a factor of up to 10 than the G(SSB) for gamma irradiation, depending upon the scavenger concentration. The difference in the yields of SSBs agrees well with the difference in the yields of hydroxyl radicals for the radiations in question. In contrast, the yields of DSBs are similar for gamma and 4He ion irradiation over much of the range of scavenging capacity studied. However, at the highest scavenger concentrations the yields of DSBs are greater for 4He ion irradiation. In addition, the yields of DSBs remain almost constant with increasing LET (over the range studied). Therefore the relative yield of DSBs per SSB increases with increasing LET, supporting the hypothesis that increasing LET leads to an increased clustering of damage in DNA.

DNA↗
PubMed1993

Changes in cytoplasmic pH in platelets activated through the high- and moderate-affinity receptor pathways by highly purified human alpha-thrombin.

Platelets in plasma were loaded with the probe BCECF/AM, and changes in cytoplasmic pH levels induced by highly purified human alpha-thrombin (2900 NIH U/mg) were studied in washed platelets having high- and moderate-affinity receptors and in platelets from which the high-affinity alpha-thrombin receptor had been removed by treatment with Serratia marcescens protease. In intact platelets, cytoplasmic acidification reached a maximum within 2 minutes of -0.072 +/- 0.009 pH units at 0.3 nmol/L alpha-thrombin concentration (0.03 U/ml). Cytoplasmic pH values were higher at both lower and higher alpha-thrombin concentrations and were significantly (p = 0.018) higher at 2 nmol/L alpha-thrombin, which induced -0.037 +/- 0.013 pH units of acidification. Five nanomoles of alpha-thrombin, however, induced cytoplasmic alkalinization of +0.027 +/- 0.033 pH units. In platelets lacking the high-affinity receptor where there is a 10 to 20-fold reduction in sensitivity to alpha-thrombin, acidification reached a maximum of -0.175 +/- 0.033 pH units at 2 nmol/L alpha-thrombin, but alkalinization was observed at 5 nmol/L (+0.038 +/- 0.025) and 10 nmol/L (+0.042 +/- 0.007) alpha-thrombin. These results show that the transition from acidification to alkalinization occurs in the same range of alpha-thrombin concentrations (2 to 5 nmol/L) in both preparations, despite the rightward shift in sensitivity caused by the absence of the high-affinity receptor. However, the maximum acidification reached in control platelets (-0.037 pH units at 2 nmol/L) was much less than the value obtained in platelets lacking the high-affinity receptor (-0.175 pH units at 2 nmol/L alpha-thrombin).(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1992

Spectroscopic identification of the haem ligands of cellobiose oxidase.

A spectroscopic study of the flavocytochrome b enzyme, cellobiose oxidase, employing optical, NMR, EPR and near infra-red MCD techniques, has identified the axial ligands of the b-type haem. These are a histidine and a methionine, and this ligation set is discussed in relation to the functional role of the haem group.

Carbohydrate Dehydrogenases↗
PubMed1992

Phencyclidine binds to blood platelets with high affinity and specifically inhibits their activation by adrenaline.

The ion channel probe phencyclidine [1-(1-phenylcyclohexyl)piperidine; PCP] selectively inhibited aggregation, secretion and ultrastructural changes in platelets induced by adrenaline, but did not affect activation induced by other common platelet agonists such as alpha-thrombin, ADP, collagen or ionophore A23187. [3H]PCP bound to platelets with high affinity (Kd 134 +/- 33 nM; 3600 +/- 1020 sites/platelet), as did the thienyl analogue [3H]TCP (1-[1-(2-thienyl)cyclohexyl]piperidine). PCP binding to platelets was increased 3-4-fold in N-methylglucamine buffer in the absence of Na+ ions. Binding was unaffected by haloperidol and was only weakly inhibited (EC50 10-20 microM), without significant stereoselectivity by the two sets of stereoselective ligands, dexoxadrol/levoxadrol and (+)MK801/(-)MK801. Binding of PCP was not competed for by adrenaline or yohimbine. Only the high-affinity binding of [3H]PCP to platelets was blocked by prior treatment of the platelets with the covalent affinity probe Metaphit, and these platelets no longer aggregated in response to adrenaline although they responded normally to alpha-thrombin, ADP and collagen. These results suggest that platelets contain high-affinity receptors for PCP that can modulate adrenaline-induced platelet activation.

Binding, Competitive↗
PubMed1991

Kinetics of radiation-induced strand break formation in single-stranded pyrimidine polynucleotides in the presence and absence of oxygen; a time-resolved light-scattering study.

Time-resolved reductions in the light-scattering intensity (LSI) of aqueous oxic and anoxic solutions of poly-C and poly-U at pH 7.8, following pulse-irradiation, have been studied as indices of strand break formation. With doses of 3-24 Gy per pulse, a number of kinetically distinct strand breakage components have been detected. A comparison of the LSI responses obtained from irradiations conducted under N2O with those conducted under air or O2 show no marked difference in the overall extent of LSI change. However, the immediate and fast (t 1/2 less than or equal to 50 microseconds) reduction in LSI, accounting for about 18-19% of the pyrimidine polynucleotide's total LSI response in oxic solution, is reduced in the absence of oxygen, to about 12% of the total LSI response found with poly-C and to about 9% for poly-U. For poly-C there is a five-fold enhancement in the rate of major strand breakage under anoxia [k1(N2O) = 7.9s-1] whereas for poly-U a more modest enhancement (about two-fold) is observed. These enhanced rates are mirrored by those for the losses of the principal optical anoxic absorptions (observed pulse radiolytically) that are assigned to the pyrimidine 6-yl base radicals. Such findings support a proposal that the rate-limiting step of major strand breakage for pyrimidine polynucleotides is a base radical mediated hydrogen atom abstraction reaction (Lemaire et al. 1987, Hildenbrand and Schulte-Frohlinde 1989). Irradiation of poly-C and poly-U in N2O/O2 (4:1, v/v) saturated solutions yields LSI changes much larger than those noted under N2O and air (or O2), which are in turn approximately double the responses observed under N2. This indicates that the major strand breaking species of water radiolysis is the OH-radical and that there is an oxygen enhancement of single strand breakage of about 1.9 for poly-C and 1.6 for poly-U.

DNA Damage↗
PubMed1990

Reactions of reduced cellobiose oxidase with oxygen. Is cellobiose oxidase primarily an oxidase?

We report rapid-mixing experiments in which cellobiose oxidase, fully reduced with cellobiose, is allowed to react with excess molecular O2. Analysis of the progress curves and their comparison with computer simulations suggests that O2 reacts only with the cytochrome b-type haem and with a rate constant of approx. 0.5 s-1. In steady state the cytochrome b is partially oxidized, whereas the flavin remains largely reduced. This situation may be contrasted with that when dichloroindophenol is substituted for O2. Under these conditions the reactions are rapid (millisecond time range), and the redox centres in the enzyme appear to be oxidized simultaneously.

Anaerobiosis↗
PubMed1990

The kinetics of radiation-induced strand breakage in polynucleotides in the presence of oxygen: a time-resolved light-scattering study.

The time-resolved light-scattering changes of aqueous, aerated solutions of poly-C, poly-U and poly-A at pH 7.8, following pulse irradiation, have been studied as indices of strand break formation. With doses of 4-24 Gy/pulse a number of kinetically distinct components have been detected. For the poly-pyrimidines an immediate and fast reduction (tau 1/2 less than or equal to 50 microseconds) in light-scattering intensity (LSI), accounting for approximately 20% of the total LSI change, is followed by a much slower loss (k1 approximately 1.6 s-1) which constitutes their major LSI change. For poly-A a similar fast component is observed, present to an extent equivalent to the one noted with poly-C; it constitutes, however, over 50% of the purine polynucleotide's total response, with the remainder of the change being a slower loss (tau 1/2 approximately 0.09 s). Optical pulse radiolysis studies of poly-C and poly-U, in support of the LSI investigations, show that transient absorbances in a region assigned to base peroxyl radicals decay in a complex fashion, with some at a rate equivalent to that for the slow (major) component of LSI loss. These observations support a proposal that the rate-limiting step of major strand breakage for these polynucleotides, in the presence of oxygen, is a base peroxyl radical-mediated abstraction of a H-atom from an adjacent sugar moiety (Bothe et al. 1986), with the resulting sugar peroxyl radicals then leading to strand break formation at a rate equivalent to that for loss of the initial, fast LSI components. These latter processes are attributed to strand breaks arising from the direct interaction of .OH with the polynucleotide sugar phosphate backbone.

DNA Damage↗
PubMed1989

Determination of 2-n-octadecylindole-5-carboxylic acid in human plasma by high-performance liquid chromatography.

A method is described for the determination of the novel hypocholesterolaemic drug, 2-n-octadecylindole-5-carboxylic acid (I) in plasma. A homologue of I is used as the internal standard. Methanol is added to the plasma sample in order to precipitate the plasma proteins, followed by centrifugation and removal of the supernatant. This is reduced to dryness by heating under oxygen-free nitrogen, prior to reconstitution in the chromatographic mobile phase. The solution is assayed by injection on to a 5 micron particle size ODS2 analytical column, protected by a disposable RP-18 packed guard column, using an isocratic mobile phase of acetonitrile-water-isopropyl alcohol-formic acid (75:275:150:2.5, v/v). Detection is by ultraviolet absorbance at 276 nm. At a flow-rate of 1.5 ml min-1 and ambient temperature, the retention time of the drug is 16 min, whilst that for the internal standard is 21 min. This method has been validated and successfully used to assay clinical trial plasma samples. Basic pharmacokinetic parameters are presented.

Chromatography, High Pressure Liquid↗
PubMed1989

Effects of separate proteolytic and high-affinity binding activities of human thrombin on rapid platelet activation. A quenched-flow study.

We have used a general quenched-flow approach to study platelet function as early as 0.3 s after stimulation with three types of human thrombin: alpha-thrombin, gamma-thrombin, which is proteolytically active but does not bind to the high-affinity sites, and di-isopropyl fluorophosphate-derivatized (DIP)-alpha-thrombin, an active site-inhibited analogue that does bind to the high-affinity site. Large doses of gamma-thrombin evoked moderate aggregation and serotonin release, but minimal phosphorylation of the 20 and 47 kDa proteins. The initial (1.5-3.0 s) increase in cystolic free calcium concentration ([Ca2+]i) indicated by Indo-1 was also diminished, but by 5 s was nearly as high (1.0 microM) as with alpha-thrombin. A large dose of DIP-alpha-thrombin, on the other hand, induced minimal aggregation, serotonin secretion and [Ca2+]i response within 6 s. There was, however, a transient dephosphorylation of the 20 kDa protein. When combined, gamma- and DIP-alpha-thrombin were approximately additive in their ability to induce aggregation and serotonin secretion, but strongly synergistic in phosphorylating the 20 and 47 kDa proteins. The [Ca2+]i increase was not, however, enhanced over that induced by gamma-thrombin alone. These results demonstrate that phosphorylation of either the 20 or 47 kDa proteins is not correlated with [Ca2+]i dynamics and is neither required nor directly involved in platelet aggregation and secretion induced by thrombin. The high-affinity binding activity of thrombin is not necessary for rapid platelet Ca2+ influx, aggregation and serotonin release within the first critical seconds of activation.

Blood Platelets↗
PubMed1989

Hypolipidemic 2-[4-(1,1-dimethylethyl)phenyl]-4H-3,1-benzoxazin-4-ones. Structure-activity relationships of a novel series of high-density lipoprotein elevators.

The preparation and plasma lipid altering characteristics of a series of 4H-3,1-benzoxazin-4-ones are described. Hypocholesterolemic, hypotriglyceridemic, and high-density-lipoprotein elevating properties are found for derivatives bearing a 4-(1,1-dimethylethyl)phenyl group at the 2-position, and this activity is displayed in both hypercholesterolemic and in normolipidemic rats when the ring system is substituted at position 6 with hydrogen, methyl, chloro, or iodo groups, and is optimal when the 6-position is substituted by a bromine atom. Evidence is presented suggesting that a metabolite or degradation product is responsible for the changes in lipoprotein concentration observed with active molecules of this type. Synthesis of anticipated degradation products of the active molecules gave products displaying the expected in vivo activity, but no improvement in the narrow therapeutic margin of the best compound, 6-bromo-2-[4-(1,1-dimethylethyl)phenyl]-4H-3,1-benzoxazin-4-one, was obtained.

Animals↗
PubMed1988

Rapid kinetic studies of the reduction of cellobiose oxidase from the white-rot fungus Sporotrichum pulverulentum by cellobiose.

The reactions between cellobiose and cellobiose oxidase were investigated by stopped-flow spectrophotometry. Under anaerobic conditions rapid reduction of the associated flavin is followed by slower reduction of cytochrome b. The kinetic difference spectra are reported. The rate of flavin reduction depends on the cellobiose concentration (with an apparent second-order rate constant of approx. 10(5) M-1.s-1) but reaches a rate limit of approx. 20 s-1. In contrast, the rate of cytochrome b reduction decreases at high cellobiose concentrations. Kinetic titrations of the flavin and cytochrome b moieties yield the stoichiometries of the separate reactions, i.e. the number of moles of cellobiose needed to fully reduce 1 mole of each redox component. The rate constant for cytochrome b reduction, unlike that for flavin reduction, increased with enzyme concentration, prompting the conclusion that any given cytochrome b centre is reduced preferentially by flavin groups in different molecules rather than by its partner flavin within the same monomer. These data are discussed in the context of a scheme that rationalizes them and accounts for the overall stoichiometry in which three two-electron donors (cellobiose molecules) reduce two three-electron acceptors (the flavin-cytochrome b of cellobiose oxidase).

Carbohydrate Dehydrogenases↗
PubMed1988

Effect of epinephrine on rapid ADP-induced aggregation, protein phosphorylation, and cytoplasmic calcium dynamics of platelets: a quenched-flow study.

We have used a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation with a low concentration of adenosine 5'-diphosphate (ADP) (0.5 mumol/L), epinephrine (15 mumol/L), or a combination of the two. Compared with ADP alone, the combination nearly doubled the rate of aggregation as measured by the loss of single particles. Our aim was to determine whether the aggregation, protein phosphorylation, and cytoplasmic calcium responses to this potentiating combination of ADP and epinephrine were analogous to those caused by a maximal concentration of ADP (10 mumol/L) and also to determine whether cyclooxygenase was involved in this potentiation. Phosphorylation of myosin light chain (MLC) and the 47 kd protein were analyzed during the first 5 seconds of platelet response and were related to the progress of aggregation. Unlike aggregation, ADP-induced phosphorylations of both MLC and the 47 kd protein were inhibited rather than potentiated by epinephrine. Pretreatment of the platelets with indomethacin did not affect aggregation or MLC phosphorylation, but increased 47 kd phosphorylation at 0.6 seconds and eliminated it at 3 seconds. Cytoplasmic free calcium ([Ca2+]i) of indo-1-loaded platelets were monitored with our continuous-flow fluorescence approach. Although epinephrine itself did not change [Ca2+]i, it potentiated the [Ca2+]i rise induced by a low dose of ADP. A comparison of these results with platelet activation caused by 10 mumol/L ADP indicates that MLC and 47 kd phosphorylations are not correlated with [Ca2+]i dynamics and are not required or directly involved in platelet aggregation.

Adenosine Diphosphate↗
PubMed1988

Subsecond calcium dynamics in ADP- and thrombin-stimulated platelets: a continuous-flow approach using indo-1.

The regulation and kinetics (less than 5 seconds) of cytosolic calcium changes ([Ca2+]i) in stimulated blood platelets have been investigated under physiological blood flow conditions. Using a newly-developed continuous-flow approach with indo-1-loaded human platelets, adenosine diphosphate (ADP, 10 mumol/L) and thrombin (5 U/mL) were equally effective in significantly increasing [Ca2+]i by 0.5 seconds. ADP induced a transient [Ca2+]i peak of 1 to 2 mumol/L near 2 seconds, whereas thrombin caused a sustained and larger response. The first phase (less than 2 seconds) was not influenced by a lack of extracellular Ca2+, in contrast to the subsequent [Ca2+]i increase that only reached about 0.7 mumol/L for either ADP or thrombin. The shear rates used in our continuous-flow apparatus were physiological (less than 1,258 sec-1) and only slightly increased the basal [Ca2+]i of 0.1 mumol/L. Platelet aggregation (less than 5 seconds), assessed by single-particle counting, was not altered in platelets loaded with indo-1/AM (2.5 mumol/L).

Adenosine Diphosphate↗
PubMed1986

Resolution, purification and some properties of the multiple forms of cellobiose quinone dehydrogenase from the white-rot fungus Sporotrichum pulverulentum.

Four forms of cellobiose quinone dehydrogenase have been purified from the white-rot fungus Sporotrichum pulverulentum. The Mr of the enzyme has been estimated by sedimentation equilibrium to be 57,800 and by SDS/polyacrylamide-gel to be 60,000. These enzymes are clearly monomers. Cellobiose quinone dehydrogenases contain FAD and variable amounts of a green chromophore which we suggest is 6-hydroxy-FAD. The superoxide anion and H2O2 are the products of its reaction with oxygen. All of the isoenzymes from any one preparation display similar kinetic parameters. However, these vary between preparations. The only apparent difference between the four separable isoenzymes is their neutral-sugar content.

Amino Acids↗