The development of the UK radon programme.

The natural radioactive gas, radon, is responsible for the largest component of the radiation dose received by the average UK citizen. The risks of exposure to radon have been demonstrated and quantified in epidemiological studies of those exposed at work and in the home. In the UK, measures are in place to identify and help control the exposures in those houses where levels are highest, to limit levels in new buildings and to control exposures in the workplace. This paper outlines the development of the programme, with special reference to the identification and remediation of homes with high radon levels.

Binding specificity of the Porphyromonas gingivalis heme and hemoglobin receptor HmuR, gingipain K, and gingipain R1 for heme, porphyrins, and metalloporphyrins.

Previous genetic and biochemical studies have confirmed that hemoglobin and hemin utilization in Porphyromonas gingivalis is mediated by the outer membrane hemoglobin and heme receptor HmuR, as well as gingipain K (Kgp), a lysine-specific cysteine protease, and gingipain R1 (HRgpA), one of two arginine-specific cysteine proteases. In this study we report on the binding specificity of the recombinant P. gingivalis HmuR protein and native gingipains for hemoglobin, hemin, various porphyrins, and metalloporphyrins as assessed by spectrophotometric assays, by affinity chromatography, and by enzyme-linked immunosorbent assay. Protoporphyrin, mesoporphyrin, deuteroporphyrin, hematoporphyrin, and some of their iron, copper, and zinc derivatives were examined to evaluate the role of both the central metal ion and the peripheral substituents on binding to recombinant HmuR and soluble gingipains. Scatchard analysis of hemin binding to Escherichia coli cells expressing recombinant membrane-associated six-His-tagged HmuR yielded a linear plot with a binding affinity of 2.4 x 10(-5) M. Recombinant E. coli cells bound the iron, copper, and zinc derivatives of protoporphyrin IX (PPIX) with similar affinities, and approximately four times more tightly than PPIX itself, which suggests that the active site of HmuR contains a histidine that binds the metal ion in the porphyrin ring. Furthermore, we found that recombinant HmuR prefers the ethyl and vinyl side chains of the PPIX molecule to either the larger hydroxyethyl or smaller hydrogen side chains. Kgp and HRgpA were demonstrated to bind various porphyrins and metalloporphyrins with affinities similar to those for hemin, indicating that the binding of Kgp and HRgpA to these porphyrins does not require a metal within the porphyrin ring. We did not detect the binding of RgpB, the arginine-specific cysteine protease that lacks a C-terminal hemagglutinin domain, to hemoglobin, porphyrins, or metalloporphyrins. Kgp and HRgpA, but not RgpB, were demonstrated to bind directly to soluble recombinant six-His-tagged HmuR. Several possible mechanisms for the cooperation between outer membrane receptor HmuR and proteases Kgp and HRgpA in hemin and hemoglobin binding and utilization are discussed.

Emerging strategies in microbial haem capture.

Gram-negative pathogenic bacteria have evolved novel strategies to obtain iron from host haem-sequestering proteins. These include the production of specific outer membrane receptors that bind directly to host haem-sequestering proteins, secreted haem-binding proteins (haemophores) that bind haem/haemoglobin/haemopexin and deliver the complex to a bacterial cell surface receptor and bacterial proteases that degrade haem-sequestering proteins. Once removed from haem-sequestering proteins, haem may be transported via the bacterial outer membrane receptor into the cell. Recent studies have begun to define the steps by which haem is removed from bacterial haem proteins and transported into the cell. This review describes recent work on the discovery and characterization of these systems. Reference is also made to the transport of haem in serum (via haemoglobin, haemoglobin/haptoglobin, haemopexin, albumin and lipoproteins) and to mechanisms of iron removal from the haem itself (probably via a haem oxygenase pathway in which the protoporphyrin ring is degraded). Haem protein-receptor interactions are discussed in terms of the criteria that govern protein-protein interactions in general, and connections between haem transport and the emerging field of metal transport via metallochaperones are outlined.

Radon exposures from the use of natural gas in buildings.

Low levels of natural radioactivity in the ground produce radon-222 and its decay products which can be entrained with gas streams and become distributed with gas supplies to commercial and domestic users. Levels of radon in blended gas received by most users are comparable with the levels that are present naturally in buildings as a result of ingress from the ground and this is further diluted during the combustion process. For typical rates of gas usage with an average radon level of about 200 Bq x m(-3), the estimated dose from the use of natural gas is estimated at 4 microSv, less than 1% of the dose from radon exposure at the average level in UK homes. Commercial users may receive somewhat higher doses, and the estimate for a critical group is a few tens of microsievert. The total radon emission to the environment is estimated at about 10(13) Bq x y(-1) which represents less than 10(-4) of the natural emission rate from the ground. There is some variability of radon levels in gas from different sources and it would be prudent to keep this source of exposure under review. A standard sampling and measurement protocol has been developed in conjunction with a technical group representing the industry.

Design, synthesis and DNA-cleavage of Gly-Gly-His-naphthalene diimide conjugates.

Naphthalene diimides with one and two metal-chelating Gly-Gly-His (GGH) motifs have been synthesized. Both conjugates induce single-stranded cleavage of plasmid pBR322 DNA in the presence of nickel and Oxone and are approximately 100-fold more efficient than Ni(II) x GH-CONH2 itself.

Cleavage of abasic sites in DNA by intercalator-amines.

Anthraquinone and naphthalene diimide intercalators with amine-containing side chains cleave plasmid DNA at abasic sites (apurinic or apyrimidinic (AP) sites). The intercalator-amine is substantially more effective than the amine itself; many intercalators with diamine side chains cleave most of the abasic sites at micromolar concentration (30 min at 37 degrees C). Intercalators with two amino moieties in the side chain are more efficient than those with one, arguing for a role for each of two amines in the cleavage mechanism. Side chains ending in tertiary amines are somewhat more effective than those ending in primary amines, indicating that imine formation is not required for cleavage at the abasic site. We also report a systematic study of abasic site cleavage by polyamines, including piperidine, spermine, spermidine and 12 other di-, tri- and tetra-amines. For polyamines as well as intercalator-amines, examples with three carbon atoms between neighboring nitrogens atoms cleave most efficiently. This may reflect a particularly favorable geometry for proton abstraction for these species. The effect of nitrogen-nitrogen spacing on the pKa values of the nitrogens may contribute as well. Overall, cleavage of plasmid DNA at adventitious abasic sites by intercalator-amines bearing two nitrogens in a single side chain occurs readily.

Dimerization of tetracationic porphyrins: ionic strength dependence.

Cationic porphyrins are under study in a number of contexts including their interaction with biological targets, as possible therapeutic agents and as building blocks for molecular devices such as molecular photodiodes and solar cells. Many cationic porphyrins dimerize readily in aqueous solution. Dimerization in turn can control the properties of the porphyrin as well as its binding to its target. The propensity of a porphyrin to dimerize in aqueous solution can be estimated by recording the optical spectrum of the solution as a function of the concentration of added salt. Analysis of the data in terms of the Debye-Hückel formalism gives an estimate of the extent of dimerization as a function of ionic strength. Data for TMPyP4 [meso-tetrakis(4-N-methylpyridinium)porphyrin] and its butyl and octyl homologs; TMAP [meso-tetrakis(4-N,N,N-trimethylanilinium)porphyrin]; T theta PP [meso-tetrakis[4-N-[(3-(trimethyl-ammonio)propyl)oxy]phenyl]porphyrin] and the ferrocenyl porphyrin P3Fc are discussed. Dimerization may affect binding of the cationic porphyrins to their targets, e.g., DNA.

Capillary electrophoretic separation of cationic porphyrins.

Cationic porphyrins have a wide variety of uses including those as nucleic acid binding and cleaving agents, as potential pharmacological agents, as electron donor/acceptors in intramolecular electron transfer processes and as analytical reagents. Herein, we report the separation of cationic porphyrins by capillary electrophoresis on fused silica in phosphate buffer at pH 2-5. The porphyrins studied in this work were synthesized from alkylation of the parent tetrapyridylporphyrin (TPyP) to give various pyridinium porphyrins. For example, methylation of TPyP gives a mixture of the mono-, cis-di-, trans-di-, tri- and tetramethylated porphyrins [e.g., 5,10,15,20-tetrakis(N-methyl-4-pyridiniumyl)-21H,23H-p orphyrin, TMPyP(4)]. Capillary electrophoresis on a synthetic mixture showed separation of four of these compounds. Mixtures after alkylation with iodopropionic acid and bromopropylamine were also separated. The cis-di- and trimethylated TMPyP derivatives were separated on a small preparative scale by centrifugal partition chromatography. Capillary electrophoresis was also used to separate metallo TMPyP(4) complexes including those of cobalt, copper, iron, manganese, palladium, tin, vanadium and zinc. The conformational isomers (atropisomers) of 5,10,15,20-tetrakis(N-methyl-2-pyridiniumyl)-21H,23H-p orphyrin, TMPyP(2), were also separated. Net charge, molecular mass and molecular shape all contribute to the differential retention of cationic porphyrins under capillary electrophoresis conditions. Additional factors affecting the separations, including aggregation and protonation of the porphyrins, were probed by evaluating the separation of TMPyP(4) and its butyl and octyl analogs as a function of solution conditions. Cationic porphyrins are difficult to separate using traditional chromatographic methods; capillary electrophoresis and centrifugal partition chromatography provide excellent new techniques for separation of this class of compounds.

Electrostatic and steric control of electron self-exchange in cytochromes c, c551, and b5.

The ionic strength dependence of the electron self-exchange rate constants of cytochromes c, c551, and b5 has been analyzed in terms of a monopole-dipole formalism (van Leeuwen, J.W. 1983. Biochim. Biophys. Acta. 743:408-421). The dipole moments of the reduced and oxidized forms of Ps. aeruginosa cytochrome c551 are 190 and 210 D, respectively (calculated from the crystal structure). The projections of these on the vector from the center of mass through the exposed heme edge are 120 and 150 D. For cytochrome b5, the dipole moments calculated from the crystal structure are 500 and 460 D for the reduced and oxidized protein; the projections of these dipole moments through the exposed heme edge are -330 and -280 D. A fit of the ionic strength dependence of the electron self-exchange rate constants gives -280 (reduced) and -250 (oxidized) D for the center of mass to heme edge vector. The self-exchange rate constants extrapolated to infinite ionic strength of cytochrome c, c551, and b5 are 5.1 x 10(5), 2 x 10(7), and 3.7 x 10(5) M-1 s-1, respectively. The extension of the monopole-dipole approach to other cytochrome-cytochrome electron transfer reactions is discussed. The control of electron transfer by the size and shape of the protein is investigated using a model which accounts for the distance of the heme from each of the surface atoms of the protein. These calculations indicate that the difference between the electrostatically corrected self-exchange rate constants of cytochromes c and c551 is due only in part to the different sizes and heme exposures of the two proteins.

NMR study of the alkaline isomerization of ferricytochrome c.

The pH-induced isomerization of horse heart cytochrome c has been studied by 1H NMR. We find that the transition occurring in D2O with a pKa measured as 9.5 +/- 0.1 is from the native species to a mixture of two basic forms which have very similar NMR spectra. The heme methyl peaks of these two forms have been assigned by 2D exchange NMR. The forward rate constant (native to alkaline cytochrome c) has a value of 4.0 +/- 0.6 s-1 at 27 degrees C and is independent of pH; the reverse rate constant is pH-dependent. The activation parameters are delta H not equal to = 12.8 +/- 0.8 kcal.mol1, delta S not equal to = -12.9 +/- 2.0 e.u. for the forward reaction and delta H not equal to = 6.0 +/- 0.3 kcal.mol-1, delta S not equal to = -35.1 +/- 1.3 e.u. for the reverse reaction (pH* = 9.28). delta H degree and delta S degree for the isomerization are 6.7 +/- 0.6 kcal.mol-1 and 21.9 +/- 1.0 e.u., respectively.

Electron self-exchange in Pseudomonas cytochromes.

Electron self-exchange has been measured by an NMR technique for cytochromes c551 from Pseudomonas aeruginosa and Pseudomonas stutzeri. The rate for P. aeruginosa cyt c551 is 1.2 x 10(7) M-1 s-1 at 40 degrees C in 50 mM phosphate at pH 7. For P. stutzeri, under the same conditions, the rate is 4 x 10(7) M-1 s-1. For both cytochromes, the rate was independent of ionic strength up to 0.5 M in added NaC1, the enthalpy of activation was 20 +/- 4 kcal mol-1, and the entropy of activation was 38 +/- 10 cal mol-1 deg-1.

Myocardial infarction with normal results of coronary angiography following diltiazem withdrawal.

Abrupt withdrawal of calcium channel blocking agents has been associated with symptoms of ischemic heart disease, but acute myocardial infarction has not been noted. Herein is described a severely uremic patient who had an acute myocardial infarction shortly after discontinuance of diltiazem, although results of subsequent coronary arteriography were normal. It is postulated that myocardial damage occurred because of increased intracellular calcium flux, augmented myocardial contractility, and/or drug withdrawal-related coronary spasm.

Occupational exposure to natural radiation.

Natural sources of radiation can make an important contribution to the exposures of people at work. Two areas of interest are work with minerals having elevated concentrations of activity and work in buildings where radon daughter concentrations are elevated. The Euratom Directive on ionising radiation requires that the handling of radioactive substances be reported to national authorities. National authorities may waive this requirement where the activity per unit mass is below 100 Bq g-1, or for solid natural radioactive substances, 500 Bq g-1. An investigation was undertaken in five factories to determine whether work with minerals having levels of natural activity below these might lead to significant doses. Models based on the data collected were used to relate the activity in the minerals to the effective dose equivalent arising from gamma radiation, inhalation of radon daughters, and intake of long-lived activity. These assessments show that the activity concentration at which exposures to airborne dust could lead to doses equal to one-tenth of the dose limit for workers are 0.3 Bq g-1 for thorium-232 and 1 Bq g-1 for uranium-238. Above these values, radiological supervision may be necessary. In a separate study, measurements of radon daughter concentrations were made in seventy workplaces. Concentrations in some premises approached or exceeded the derived air concentration for occupational exposure. The highest concentrations were found in premises with low ventilation rates.

Use of the nuclear Overhauser effect to assign 1H NMR resonances in a low-spin paramagnetic hemin.

The nuclear Overhauser effect has been used to assign the 1H resonances of the paramagnetic low-spin biscyano complex of Fe(III) protoporphyrin. When the meso protons are irradiated, changes in integrated signal intensity are seen at neighboring methyl or methylene groups and vice versa. Although the changes ar small (less than 1% negative NOEs for Fe(III)protoporphyrin(CN)2 in Me2SO-d6 at 30 degrees C and 360 MHz), they can be seen clearly. This technique has been used to assign the 6-alpha-CH2 (6.21), 7-alpha-CH2 (5.82), beta-meso (0.50) and delta-meso (0.03 ppm) resonances of this species. The nuclear Overhauser effect will allow rapid assignment of 1H NMR resonances in a wide variety of low-spin paramagnetic hemins.

Comparative efficacy and safety of bumetanide and furosemide in long-term treatment of edema due to congestive heart failure.

Bumetanide and furosemide were compared for efficacy in reducing edema due to congestive heart failure in 28 patients (21 receiving bumetanide and seven receiving furosemide) in a long-term study for periods from one week to 18 months. In both groups the patients showed decreases in body weight, abdominal girth, edema, hepatomegaly, blood pressure, and heart rate. Commonly observed decreases frequently achieved statistical significance, more often with bumetanide, but the differences between treatments were rarely statistically significant. Both drugs were generally well tolerated. A breast nodule and gynecomastia were each reported once in the bumetanide group as was gynecomastia in one patient who had been on furosemide, all remotely related to test drugs. Soft stools, flatulence, mild constipation, and diminished vision each reported once in the bumetanide group were judged to be unrelated or remotely related to the drug therapy. Tendencies toward hypokalemia, hypochloremia, alkalosis, and hyperuricemia without clinical gout were deemed the result of the pharmacologic action of the diuretics. Others were attributable to the underlying disease state of these patients. Both diuretics proved to be effective in the treatment of cardiac edema and other manifestations of heart failure. Bumetanide treatment beyond six months in 11 patients indicated continued safety as well as efficacy.

Use of catecholamines in acute myocardial infarction.

We review the hemodynamic effects and clinical usefulness of five natural and synthetic catecholamines. Their actions are best understood by an appreciation of the relative ability of each catecholamine to activate alpha, beta 1 and beta 2 adrenergic receptors in the myocardium and peripheral vasculature. Epinephrine, the first catecholamine isolated, is shown to have little useful role in the therapy of acute myocardial infarction. L-norepinephrine has powerful alpha and moderate beta 1 effects. It is the catecholamine of choice in the initial treatment of cardiogenic shock associated with acute myocardial infarction. Isproterenol markedly increases myocardial contractility and heart rate by beta 1 stimulation, while simultaneously decreasing peripheral vascular resistance and, therefore, arterial pressure through its action on beta 2 receptors. It increases cardiac output, but its metabolic costs are high in the presence of ischemia. Its role in the therapy of acute myocardial infarction has largely been supplanted by more selective agents. Dopamine causes slightly less vasoconstriction than l-norepinephrine and slightly less myocardial stimulation than isoproterenol. In low doses, it increases renal and mesenteric blood flow by activation of a non-adrenergic receptor. Tachycardia and associated metabolic deterioration render it a second-line drug in the treatment of severe cardiogenic shock. Dobutamine, a new synthetic catecholamine, has primarily beta 1 activity. It increases myocardial contractility with little effect on heart rate or peripheral vascular resistance. It is ineffective in cardiogenic shock, but may eventually be shown to have a role in the treatment of left ventricular failure uncomplicated by severe hypotension.