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Biomedical subjects

J F Gibson

Publications and source records attributed to J F Gibson.

At least 19 recordsLinked to original sources
PubMed1993

Iron speciation at physiological pH in media containing ascorbate and oxygen.

The stability of iron ascorbate solutions was investigated, under both anaerobic and aerobic conditions, with the Fe2+ and Fe3+ indicators, respectively ferrozine and mimosine, at different pH values. The species present under the differing conditions were investigated by electron paramagnetic resonance (EPR) and Mössbauer spectroscopy and by gel-filtration chromatography. At physiological pH (6.8-7.4) iron ascorbate solutions rapidly form mononuclear chelatable Fe3+ species as reflected by indicator studies and EPR. Mössbauer spectroscopy fails to detect any Fe2+ species. EPR studies show a time-dependent decrease in rhombic Fe3+, particularly in oxygenated buffers, consistent with a conversion to polynuclear Fe. O2 uptake studies show that the conversion of Fe2+ to Fe3+ in Fe-ascorbate solutions at pH > 7.0 was accompanied by rapid O2 consumption but preceded depletion of ascorbate. Addition of high concentrations of mannitol (50-200 mM) reduces the O2 consumption and partly stabilizes the rapidly chelatable Fe form. Gel filtration studies show that the oxidation of Fe-ascorbate solutions at pH 7.4 is accompanied by an increase in the apparent relative molecular mass of the Fe, presumably due to Fe polymer formation. These studies indicate that inherent instability of Fe-ascorbate solutions above neutral pH and clearly have important implications in the use of ascorbate in studies of Fe physiology.

Animals↗
PubMed1987

Electron paramagnetic resonance studies of type 1 copper in type 2 depleted fungal laccase A.

The electron paramagnetic resonance (EPR) spectra of type 1 copper(II) in 63Cu-enriched Coriolus versicolor laccase A (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) have been studied. The X-band EPR spectrum in type 2 copper-depleted [63Cu]laccase A exhibited well-resolved ligand superhyperfine structure in the g perpendicular region. This structure was assigned to an interaction with two nitrogens and two protons, an assignment which is consistent with a model in which the two nitrogens belong to two histidine ligands and the two protons are the methylene protons of a coordinating cysteine. It also requires the delocalization of a substantial amount of the type 1 copper(II) unpaired electron density onto the cysteine sulphur.

Copper↗
PubMed1986

Relaxometry of ferritin solutions and the influence of the Fe3+ core ions.

The magnetic field dependence of 1/T1 over the range 0.01 to 50 MHz proton Larmor frequency (NMRD profile) is reported for water protons in solutions of horse spleen apoferritin, and of ferritin reconstituted at both low and high iron levels. The apoferritin results are in every way typical of diamagnetic spherical proteins of their size (K. Hallenga and S. H. Koenig, Biochemistry 15, 4255 (1976)). Titration of up to 24 ferrous ions per protein molecule, with subsequent oxidation to ferric, shows a nonlinear saturating contribution to the NMRD profile which is interpreted as arising from a small number of ferric ions (six to eight) bound close to the outside of each ferritin molecule, and a comparable number of interior sites. The latter become multiply occupied as the core grows and do not contribute measurably to 1/T1 in this state. The former sites are never more than singly occupied, and their contribution to the solvent proton relaxation rates is independent of the loading of the core. Measurements of 1/T2 at 20 MHz are quite in accord with theoretical expectations for apoferritin and ferritin with up to 24 ferric ions per molecule. However, a marked increase in 1/T2 is observed at higher iron loadings that we are unable to account for within the framework of the theory of outer sphere relaxation, even when the effects arising from inhomogeneities in the local magnetic field are included. A sample of human spleen hemosiderin was found to have the same 1/T1 NMRD profile as a comparable sample of ferritin.

Animals↗
PubMed1985

Electron spin resonance studies of splenic ferritin and haemosiderin.

Preparations of haemosiderin and ferritin isolated from iron-loaded human spleens were studied by electron spin resonance (ESR) spectroscopy at X-band (approx. 9.2 GHz). The spectra were mainly composed of two overlapping, broad features, one extremely anisotropic with its major component occurring at 0.1-0.2 T (feature A), the other nearly isotropic and occurring at around g = 2 (feature B). There is relatively more feature A and less feature B in ferritin than in haemosiderin. Both features originate from the iron oxyhydroxide crystallites of these iron proteins which, due to their small size, are superparamagnetic. Feature B is maximal in small cores or at high temperatures, where superparamagnetic fluctuations average out anisotropic magnetic interactions; feature A is greatest at low temperatures or in large cores, for which such fluctuations are blocked and an ESR spectrum characteristic of a magnetically ordered system is observed. It is concluded that there is no evidence in the ESR spectra for 'loose' protein-bound Fe3+ in ferritin or haemosiderin, and that haemosiderin cores are on average smaller than those of ferritin. The relationship of the ESR spectra between these two proteins supports the view that haemosiderin is derived from ferritin.

Animals↗
PubMed1985

The metabolic activation of 4,4'-methylene-bis-(2-chlorobenzeneamine) to a bacterial mutagen by hepatic postmitochondrial supernatant from human and other species.

4,4'-Methylene-bis-(2-chlorobenzeneamine) (MbOCA) is a commercially important industrial chemical that is carcinogenic in three animal species and mutagenic in the Ames test. The ability of hepatic postmitochondrial supernatant from humans, dogs, mice, and rats to activate MbOCA to a bacterial mutagen has been investigated using the Ames plate incorporation test and a bacterial fluctuation test. In the Ames plate test, hepatic S9 preparations from mice and Aroclor 1254-induced rats only were sufficiently active to produce a significant mutagenic response. Preincubation of MbOCA with S9 from human liver produced a slight increase in the number of revertants but not a doubling as compared to controls. However, using the more sensitive bacterial fluctuation test, liver S9 from all species activated MbOCA to a bacterial mutagen. The responses produced were dose-related and, for at least part of the dose range, were double the background levels observed in controls. The increases in the mutagenicity of MbOCA produced by liver S9 from humans, dogs, and rats were significant at the 0.1% level of probability. Liver S9 preparations from all species in which MbOCA is carcinogenic have now been demonstrated to be capable of activating this compound to a bacterial mutagen. The finding that S9 from human liver can also activate MbOCA to a mutagen increases the concern that it may be a human carcinogen.

Animals↗
PubMed1985

Filamentation of Escherichia coli K12 elicited by some monomeric, dimeric and trimeric complexes of ruthenium in various oxidation states.

A number of ruthenium complexes were tested for their ability to induce filamentation in Escherichia coli. These included monomeric and dimeric complexes with ruthenium in the II or III oxidation states, as well as mixed-valence complexes with ruthenium in the (II,III) oxidation states. In general, dimeric mixed-valence Ru(II,III) complexes were the most active class of compound, although some complexes of this type were relatively inactive. These were pyrazine- or bipyridyl-bridged complexes which are known to involve strong metal-ligand interaction, which stabilizes the Ru(II) oxidation state. Some Ru(III) complexes were also significantly active in induction of filamentous growth in E. coli. One of these was [Ru(NH3)5Cl]Cl2, which did not inhibit electron transport, Mg2+-ATPase activity or DNA synthesis in E. coli, but like [Ru2(NH3)6Br3]Br2 X H2O was a potent inhibitor of respiration-driven calcium transport in the organism. Filament-inducing activity of the complex was reduced in the presence of NaCl, but not in the presence of added Ca2+, ethanol, calcium pantothenate, or E. coli 'division promoting extract'. This behaviour is also similar to that of [Ru2(NH3)6Br3]Br2 X H2O. It is suggested that both complexes may induce filamentation in E. coli by a common mechanism, which may involve interference with calcium metabolism, or a wall or membrane target, rather than interaction with DNA.

Adenosine Triphosphatases↗
PubMed1984

Biochemical studies on the isolation and characterization of human spleen haemosiderin.

Haemosiderin was isolated from thalassaemic human spleens by centrifugation through concentrated KI solutions. A method for solubilizing haemosiderin was developed which leaves the iron oxyhydroxide cores and constituent polypeptides intact, facilitating further purification and analysis. Purified haemosiderin contained no detectable haem, trace amounts of carbohydrate, and iron and phosphorus in a molar ration of 6:1; much of the phosphate may be present as core-adsorbed. Several lipids were present, but it is not certain whether these are contaminants or components of the haemosiderin granules. In all preparations examined, a characteristic group of six to seven peptides of apparent Mr 12 900-17 800 were found, with a major band at Mr 14 500 and, in addition, a minor component of Mr 42 000; these peptides co-chromatographed with the cores. Negatively stained electron micrographs suggest that these peptides form an incomplete shell about the cores, consistent with the view that haemosiderin is a proteolytic product of ferritin.

Amino Acids↗
PubMed1984

Mössbauer spectroscopic studies of human haemosiderin and ferritin.

Ferritin and haemosiderin isolated from iron-overloaded human spleens have been investigated by 57Fe Mössbauer spectroscopy at temperatures between 1.3 and 200 K and also in applied magnetic fields. Virtually identical spectra were obtained from both materials at the high and low-temperature ends of this range, and also at 4.2 K in an applied magnetic field of 10 T; this indicates that both must contain iron in a closely similar chemical form. The difference between the two materials lies in the temperature dependence of their Mössbauer spectra in the intermediate temperature range, between 10 and 100 K. The temperature dependence of the Mössbauer spectra is characteristic of superparamagnetic behaviour, which occurs when a magnetically ordered material is present in the form of small particles. The details of this temperature dependence are related to the distribution of particle sizes and the magnetic anisotropy constant of each substance. Electron microscopy shows the haemosiderin cores to be markedly smaller on average than those of ferritin. Combining the Mössbauer spectroscopy and electron microscopy data we have shown that the magnetic anistropy constant of haemosiderin is considerably larger than that of ferritin. This is thought to result from the smaller core size and less symmetrical protein shell of the former. These data are consistent with the proposal that haemosiderin is derived from ferritin.

Ferritins↗
PubMed1984

Haemosiderin and tissue damage.

High levels of haemosiderin occur in iron overload syndromes such as idiopathic haemochromatosis or secondary iron overload in thalassaemic patients; haemosiderin is the predominant iron-storage compound in such cases. It consists of a large aggregate of FeOOH cores, many of which have an incomplete shell of protein, and is probably derived from ferritin by lysosomal proteolysis. In addition, some chemical degradation of the ferritin cores appears to occur on conversion to haemosiderin. Other biochemical components are phosphate and magnesium, which may be adsorbed to the core surface, and perhaps certain lipids. Haemosiderin may have a central role, either directly or indirectly, in iron cytotoxicity and therefore the chemistry and biochemistry of this material warrants further study.

Electron Spin Resonance Spectroscopy↗
PubMed1984

Filamentous growth of Escherichia coli K12 elicited by dimeric, mixed-valence complexes of ruthenium.

Dimeric, mixed-valence [(Ru(II),Ru(III)] compounds of ruthenium caused filament formation in growing cultures of Escherichia coli K12. Three compounds with the general formula Ru2(NH3)6X5 X H2O (where X is a halide) were tested; in order of decreasing effectiveness (and with the concentration giving maximum effect), these were the bromo (10(-5) M), chloro (10(-4) to 10(-5) M), and iodo (10(-3) to 10(-4) M) analogues. Filamentation elicited by the bromo and chloro compounds was spontaneously reversible after 3-4 h, and tentatively attributed to oxidation of the active mixed-valence form to inactive Ru(III) complexes. Several compounds known to accelerate division of filaments formed under other conditions were ineffective in reversing the filamentation, but the presence of 0.43 M-dimethylsulphoxide totally inhibited filamentation caused by the bromo or chloro compounds and by cis-Pt(NH3)2Cl2 (cisplatin), an established filamenting and antitumour agent. The ruthenium complexes bound to mammalian DNA, but were without effect on the UV spectrum or cellular content of DNA in E. coli, despite showing marked mutagenic activity in reverse mutation tests with Salmonella typhimurium. Cells remained sensitive to inhibition of division by the ruthenium complexes until immediately prior to the division event. Possibilities for the (probably complex) mode of action and the potential of related compounds for therapeutic use are discussed.

Cell Division↗
PubMed1983

Temperature dependence of the electronic spin-lattice relaxation time in a 2-iron-2-sulphur model complex.

The complex [Fe2S2(S2-o-xylyl)2]2- in DMF (dimethylformamide), DMSO (dimethylsulphoxide) or a 1:1 DMF/DMSO mixture, a model for the chromophore in the 2Fe-2S proteins (ferredoxins), has been reduced and studied by conventional EPR over a temperature range. The low-field feature of the spectrum, Hz, has been computer simulated in order to analyse the lineshape in terms of a convolution product of Lorentzian and Gaussian distributions. The Gaussian contribution to the linewidth and a fixed part of the Lorentzian contribution, which is a function of the solvent and the way it freezes, were measured at a low temperature (less than or equal to 100 K) and subtracted from the linewidths in the higher-temperature range (130-200 K). The variable Lorentzian contribution thus obtained was related to spin-lattice relaxation times. The spin-lattice relaxation times of the sample having 1:1 DMSO/DMF solvent were measured in the range 6 to 11 K by the saturating pulse technique and in the range 10 to 65 K by continuous saturation methods. Up to 65 K the results follow the law 1/T1 alpha T4.5, a relationship which is not readily interpreted in terms of a simple Debye model. At higher temperatures the results may be interpreted in terms either of a dominant Orbach mechanism involving excited states at approx. 900 +/- 50 cm-1 (DMSO, DMF) or 770 +/- 50 cm-1 (1:1 DMSO/DMF), or of a Raman process in which 1/T1 alpha T7.5. The former is compatible with the two-phonon process already described in some ferredoxins, especially those with little anisotropy (gy - gx approximately 0.0) which have characteristically high [J] values.

Dimethyl Sulfoxide↗
PubMed1983

Urinary mutagenicity assays: a problem arising from the presence of histidine associated growth factors in XAD-2 prepared urine concentrates, with particular relevance to assays carried out using the bacterial fluctuation test.

The appearance of mutagenic activity in urine samples from a group of nurses and from unexposed individuals has been investigated using the bacterial fluctuation test. Apparent mutagenic activity was seen in samples from all subjects and did not appear to be related to any specific occupational or environmental exposure. This activity seems to be related to the presence of histidine or histidine-related auxotrophic growth factors in the urine concentrates, not completely removed by the recommended XAD-2 column procedure. It is suggested that the reliance on XAD-2 columns for the removal of histidine may produce spurious results when using this assay to screen populations for exposure to mutagenic chemicals.

Animals↗
PubMed1982

A dimeric complex of ruthenium: a new inhibitor of respiration-driven calcium transport in Escherichia coli K12.

Succinate-driven calcium uptake by everted membrane vesicles from Escherichia coli was sensitive to 2 mM-KCN and a dimeric, mixed valence complex of ruthenium, Ru2 (NH3)6Br5(H2O) (100 micro M), but relatively insensitive to 100 micro M-Ruthenium Red. The sensitivity of uptake to KCN and the slight inhibition caused by Ruthenium Red could be attributed to the different potencies of these compounds as respiratory inhibitors. Respiration was completely insensitive to the dimeric ruthenium complex, however, suggesting that this compound is an inhibitor of respiration-driven calcium transport.

Biological Transport, Active↗
PubMed1980

Effects of trialkyllead compounds on growth, respiration and ion transport in Escherichia coli K12.

Triethyllead and tripropyllead cations affected growth, energy metabolism and ion transport in Escherichia coli K12. The tripropyllead compound was more liposoluble than the triethyl analogue and was also more effective in inhibiting cell growth and the oxygen uptake of both intact cells and membrane particles. Triethyllead acetate (5 microM) inhibited growth on non-fermentable carbon sources, such as glycerol and succinate, more markedly than on glucose. At higher concentrations, triethyllead caused significant inhibition of respiration rates of intact cells; the concentration giving 50% inhibition was 60 microM for glycerol-grown cells and 150 microM for glucose-grown cells. Oxidation of succinate by membrane particles was less sensitive to inhibition by the tripropyl- or triethyllead compounds than were the oxidations of DL-lactate or NADH. Triethyllead acetate [1.9 mumol (mg membrane protein)-1] inhibited the reduction by NADH of cytochromes; evidence for more than one site of inhibition in the respiratory chain was obtained. Membrane-bound ATPase activity was strongly inhibited by triethyllead acetate in the absence or presence of Cl-. The concentration of inhibitor giving 50% inhibition [0.02 mumol (mg membrane protein)-1] was about two orders of magnitude lower than that required for 50% inhibition of substrate oxidation rates in membranes. Triethyllead acetate (1 microM) induced swelling of spheroplasts in iso-osmotic solutions of either NH4Cl or NH4Br, presumably as a result of the mediation by the organolead compound of Cl-/OH- and Br-/OH- antiports across the cytoplasmic membrane. Similar exchanges of OH- for F-, NO3- or SO4(2)- or the uniport of H+ could not be demonstrated. Comparisons are drawn between the effects of trialkyllead compounds and those of the more widely studied trialkyltin compounds.

Adenosine Triphosphatases↗
PubMed1980

Adenosine triphosphatase activity and its sensitivity to ruthenium red oscillate during the cell cycle of Escherichia coli K12.

A procedure has been developed for the large-scale fractionation into size and age classes of bacteria from exponentially growing cultures of Escherichia coli K12 by centrifugation through an equivolumetric gradient of sucrose in a zonal rotor. The resolution attained is superior to that in methods of this type that have been described previously. The activity of adenosine triphosphatase (ATPase) was assayed in extracts from bacteria separated into size classes by this method and from synchronous cultures prepared by size selection. Activity approximately doubled during a cell cycle, but the experimental data did not fit models of either continuously or exponentially increasing activity during the cycle. It is suggested that ATPase activity oscillates during the cell cycle with maxima at about 0.37 and 0.80 of a cycle. The fluctuations in activity greatly exceed the variations due to experimental error and, in the case of synchronous cultures, do not arise from perturbations in growth behaviour following zonal gradient selection. Sensitivity of ATPase activity to 75 micrometer-Ruthenium Red also fluctuates during the cell cycle, with maximum inhibition (60 to 80%) occurring near the middle of the cycle, a time that does not coincide with maximum enzyme activity.

Adenosine Triphosphatases↗
PubMed1979

Manganese as a calcium probe: electron paramagnetic resonance and nuclear magnetic resonance spectroscopy of intact cells.

When Lettree cells are exposed to Mn2+, the cation becomes associated with cells in two ways: in a relatively loose and mobile manner that gives a six-line EPR spectrum designated Mnb*, and in an immobile, relatively tight manner that gives no detectable EPR specrtum, designated Mnb. Mnb* is probably on the surface of cells; most Mnb is probably inside cells. NMR measurements of Lettree cell suspensions show two water proton relaxation rates and confirm the existence of cell-associated Mn. Human erythrocytes, on the other hand, bind no Mn2+ under these conditions, as judged by EPR and NMR measurements. Virally-treated Lettree cells show an increase in Mnb (but not in Mnb*). They also show a third water proton relaxation rate.

Animals↗