The carbonate radical: its reactivity with oxygen, ammonia, amino acids, and melanins.

The carbonate radical (CO 3 (*-)) is of importance in biology and chemistry. We used pulse radiolysis to generate the CO 3 (*-) radical and show there is no reaction with oxygen. However, in the presence of ammonia the CO 3 (*-) radical is removed by NO (*), which itself arises from the scavenging of NH 2 (*) by oxygen, and the mechanism of this process is reported. The CO 3 (*-) radical shows complex decay patterns in the presence of ammonia, which can be understood as a balance between the radical-radical reaction CO 3 (*-) + CO 3 (*-) and CO 3 (*-) + NH 2 (*) (the amino radical). Also, we report reactivity with glycine and alanine and with melanin models. The CO 3 (*-) reacts with both dopa-melanin (DM, a model of black eumelanin) and with cysteinyl-dopa-melanin (CDM, a model of red/blond phaeomelanin). However, the reaction rate constant is much higher with CDM than with DM.

Chain scission of hyaluronan by carbonate and dichloride radical anions: potential reactive oxidative species in inflammation?

The reactions of the carbonate and dichloride radical anions, CO3- and Cl2-, with the extracellular matrix glycosaminoglycan hyaluronan (HA) have been studied using the kinetic technique of pulse radiolysis and also by steady-state irradiation combined with gel permeation chromatography/multiangle laser light scattering(gpc/MALLS) to measure the rates of reaction with HA and the yield of HA chain scission, respectively. For comparison, the same measurements were made for the reactions of the free radicals *OH, Br2*-, and N3*. The carbonate and dichloride radical anions were found to react relatively quickly with HA (7.0 x 10(5) and 6.9 x 10(6) dm3 mol(-1) s(-1), respectively) although they are much less reactive than the hydroxyl radical, *OH. Significant yields (20 and 38%, respectively) of chain scission of HA by these radical anions were also determined from the gpc/MALLS experiments, providing some support for their potential participation in the depolymerization of HA in vivo. These results are compared with data obtained for the other free radicals (hydroxyl, azide radicals, and dibromide radical anions) investigated in this study in order to gain an insight into their mechanism of reaction with HA. Earlier chain scission yields of HA by hydroxyl radicals determined by the authors have also been revised using the gpc/MALLS technique employed in the current study. The yields of 52% (absence of air) and 44% (in air) are much lower than the previous values. In the current study, the effect of oxygen on the yields of HA chain breaks is discussed in terms of the reactivity of HA peroxyl radicals in the presence of superoxide radical anions. The relevance of the results of this study to mechanisms of inflammation is discussed.

Triplet-state and singlet oxygen formation in fluorene-based alternating copolymers.

Data are reported on the triplet states of a series of fluorene-based A-alt-B type alternating copolymers based on pulse radiolysis-energy transfer and flash photolysis experiments. From the pulse radiolysis experiments, spectra are given for eight copolymers involving phenylene, thiophene, benzothiadiazole, and oligothienylenevinylene groups. Quantum yields for triplet-state formation (PhiT) have been obtained by flash photolysis following laser excitation and in one case by photoacoustic calorimetry. In addition, yields of sensitized formation of singlet oxygen have been determined by time-resolved phosphorescence and are, in general, in excellent agreement with the PhiT values. In all cases, the presence of thiophene units is seen to increase intersystem-crossing quantum yields, probably because of the presence of the heavy sulfur atom. However, with the poly[2,7-(9,9-bis(2'-ethylhexyl)fluorene)-alt-1,4-phenylene] (PFP), thiophene S,S-dioxide (PFTSO2) and benzothiadiazole (F8BT) copolymers, low yields of triplet formation are observed. With three of the copolymers, the energies of the triplet states have been determined. With PFP, the triplet energy is virtually identical to that of poly[2,7-(9,9-bis(2'-ethylhexyl)fluorene)]. In contrast, with fluorene-thiophene copolymers PFaT and PF3T, the triplet energies are closer to those of thiophene oligomers, indicating that there is significant conjugation between fluorene and thiophene units but also that there is a more localized triplet state than with the homopolymers.

Iron environment in soybean lipoxygenase-1.

The iron coordination in native, Fe(II), lipoxygenase has been studied by Extended X-Ray Absorption Fine Structure (EXAFS). The ligands are 6 +/- 1 nitrogen and/or oxygen ligands at 2.05-2.09 A, with a maximum variance of 0.09 A. The number of imidazole ligands is estimated at 4 +/- 1 using multiple scattering simulations. The remaining ligands are proposed to be carboxylate oxygens.

A pulse radiolysis study of the complexing of furocoumarins with DNA and proteins.

The complexing of furocoumarins with DNA in aqueous solution has been investigated using the fast-reaction technique of pulse radiolysis. Association constants are given for 8-methoxypsoralen, psoralen, khellin, angelicin, 5,7-dimethoxycoumarin, 5-methoxypsoralen and 4'-aminomethyl-4,5',8-trimethylpsoralen. In addition, the complexing of some furocoumarins to the protein, bovine serum albumin, has also been studied.

The reactivities of furocoumarin excited states with DNA in solution. A laser flash photolysis and fluorescence study.

The effect of DNA on both the fluorescence emission spectra and yields and lifetimes of the triplet stae of psoralen and 8-methoxypsoralen in aqueous solution has been determined. The changes in the fluorescence spectra are similar in nature for both of these furocoumarins and are attributed to binding of the drug to DNA. The yield of the 8-methoxypsoralen triplet state when bound to DNA was found to be similar, if not identical, to that measured in the absence of DNA. This contrasts sharply with data obtained for psoralen from which it is concluded that either the yield of bound psoralen triplet states is very low, if not zero, or that the lifetime of such species is less than 50 ns. The relevance of this data to the molecular basis of skin photosensitisation by furocoumarins is discussed.

Intramolecular electron transfer in the dipeptide, histidyltyrosine: a pulse radiolysis study.

The technique of pulse radiolysis has been used to investigate the possibility of intramolecular charge transfer in the dipeptide histidyltyrosine, following one-electron oxidation of one of its amino acid residues. The radical anion, Br2.- was found to react with the dipeptide at pH 6.0 with a bimolecular rate constant of 2.3+/-0.2 x 10(7) dm3 mol(-1)s(-1) suggesting that it reacts very selectively with the histidine moiety. Spectral observations at, or close to the end of this reaction show only the presence of a tyrosinyl free radical (TyrO.), however, indicating that fast (>10(6) s(-1) intramolecular charge transfer has taken place between histidine radicals (His+.) and tyrosine (TyrOH). This finding was supported by the direct observation of the rate of formation of TyrO. in experiments with the free amino acids, histidine and tyrosine, under conditions where Br2.- reacted selectively with histidine. The bimolecular rate constant for the reaction between His+. and TyrOH was found to be 2.4+/-0.5 x 10(6) dm3 mol(-1)s(-1). Taken together, the results of the study indicate that His+. is a relatively strong oxidising agent where (E (His+./His) > 770 mV at pH 6.0.

Singlet oxygen quenching and the redox properties of hydroxycinnamic acids.

The singlet oxygen quenching rate constants (kq) for a range of hydroxycinnamic acids in acetonitrile and D2O solutions were measured using time resolved near infrared phosphorescence in order to establish their antioxidant activity. The magnitude of kq observed depends on both the nature of the substituent groups and solvent polarity. The variations in kq depend on the energy of the hydroxycinnamic acid/molecular oxygen charge transfer states, (O2delta- ...HCAdelta+). In D2O the values of kq range from 4x10(7) M(-1) s(-1) to 4x10(6) M(-1) s(-1) for caffeic acid and o-coumaric acid respectively. In acetonitrile, the charge transfer energy levels are raised and this is reflected in lower singlet oxygen quenching rate constants with a kq value of 5x10(6) M(-1) s(-1) for caffeic acid. The phenoxyl radical spectra derived from the hydroxycinnamic acids were determined using pulse radiolysis of aqueous solutions and the reduction potentials were found to range from 534 to 596 mV. A linear correlation is observed between reduction potential, and hence free energy for electron transfer, and log kq. These correlations suggest a charge transfer mechanism for the quenching of singlet oxygen by the hydroxycinnamic acids.

The determination of the heparin neutralising capacity of protamine using acridine orange fluorescence.

A new method to estimate the heparin neutralising capacity of protamine has been investigated. The technique has been applied to two commercial heparin preparations tested against the W.H.O. 1st International Reference preparation of protamine. The technique is based on the fluorescence of acridine orange dye and the selective binding of heparin with protamine. A titration procedure has been devised and the two variations of the assay are compared and evaluated. The titration is rapid, accurate and convenient.

Positive inotropic effect of porcine left ventricular extract on canine ventricular muscle.

1. We previously isolated an extract from porcine left ventricle that possessed digitalis-like properties such as inhibition of cardiac and kidney Na+, K(+)-ATPase, displacement of [3H]-ouabain from its binding sites and cross reactivity with digoxin antibodies. The extract also had a positive inotropic effect on the guinea-pig heart. 2. In the present study the positive inotropic response of the extract was characterized in canine right ventricular trabeculae. Maximum inotropic response (501 +/- 20%) was produced by 300 microliters and the half maximal increase occurred with 125 microliters of the extract. 3. Ouabagenin produced aftercontractions in rapidly paced trabeculae. Equipotent and even greater amounts of the extract did not produce aftercontractions. 4. The extract increased the amplitude of the delayed component (P2) of biphasic contractions produced by replacing about 92-96% of the external Ca with Sr. A smaller increase in the size of the early component (P1) was also seen. 5. The extract decreased post-rest potentiation after rest for 30s and 2 min. After 8 min of rest, post-rest potentiation was converted to post-rest depression. 6. The extract (20 microliters) produced a decrease in the amplitude of the post-rest rapid cooling contracture (RCC) at all rest intervals. The steady state RCC, although greater than that in the control muscle, was increased to a lesser extent than the size of the steady state electrically driven contractions. 7. It is suggested that the extract from porcine left ventricle produces a positive inotropic response by increasing the trans-sarcolemmal influx of Ca. It also has additional effect(s) on the sarcoplasmic reticulum in that it may facilitate the loss of Ca from the sarcoplasmic reticulum and/or inhibit the uptake of Ca by the organelle.

Increased [3H]nitrendipine binding sites in rat heart during adult maturation and aging.

To study the effects of maturation and aging on calcium channels, we investigated the characteristics of binding of a radioligand, [3H]nitrendipine, to relatively pure sarcolemmal membranes from 2-, 12- and 24-month-old Sprague-Dawley rat hearts. Specific binding of [3H]nitrendipine was saturable, and the Scatchard analysis of the binding revealed a single class of binding sites. Binding of [3H]nitrendipine to the membrane of 12-month-old-rats was 50-75% greater than to the membrane of 2-month-old young adult rats with no further changes in binding during aging from 12 to 24 months. The maximum number of dihydropyridine binding sites (Bmax) was 70% higher in 12- and 24-month-old rat hearts (0.45 and 0.43 pmol/mg protein) than in 2-month-old rats (0.27 pmol/mg protein). The affinity for [3H]nitrendipine binding, on the other hand, was similar in all three age groups (KD values of 0.27, 0.31 and 0.29 nM in 2-, 12- and 24-month-old rats, respectively, at 25 degrees). Membranes of all three age groups showed a similar degree of enrichment in sarcolemmal marker enzymes, indicating that the difference in membrane purity was not a contributing factor to the observed increase in density. Furthermore, increased binding of [3H]nitrendipine to the membranes of older rat hearts was observed throughout the purification scheme. Since [3H]nitrendipine binding sites are considered to be specific sites for voltage-gated Ca2+ channels of the sarcolemma, it is concluded that the density of these channels in the myocardium increases during adult maturation and is maintained through senescence.

Mechanisms of reduced digitalis tolerance with advancing age in the guinea pig.

In the present study, arrhythmogenic toxicity of cardiac glycoside ouabain was investigated in guinea pigs after intravenous infusion (5 micrograms/kg/min). Guinea pigs of 18-24 months of age required significantly (P less than 0.05) lower doses of ouabain that 3-month-old animals (72 +/- 3 vs 100 +/- 3 micrograms/kg) for the initiation of cardiac arrhythmias. Investigation of Na(+)-Ca2+ exchange in the isolated sarcolemmal vesicles revealed a marked reduction in the Na(+)-dependent Ca2+ uptake. Kinetic analysis of these data has demonstrated a 70% reduction in Vmax and reduced affinity for Ca2+ in vesicles from 18-month-old as compared to 3-month-old guinea pigs. The rate of Na(+)-dependent Ca2+ efflux was also markedly lower in the vesicles of older animals, and the vesicles retained more Ca2+ after 3 min of Na(+)-dependent Ca2+ extrusion than did those from 3-month-old animals. The results suggest that the sensitivity to cardiac glycocide increases with age and may be associated with altered sarcolemmal Na(+)-Ca2+ exchange activity.

Enhancement in myocardial inotropic response to BAY K 8644 with advancing age.

In the present study, age-related alterations in cardiovascular response to Ca2+ agonist BAY K 8644 were investigated in rats. Dose response of BAY K 8644 (1-30 micrograms/kg) was studied in open chest rats by intravenous bolus administration. Maximum elevation of mean arterial pressure and (+)dp/dt of left ventricular pressure were significantly higher and the dose of BAY K 8644 required to produce half maximal response was substantially lower in 12 months old (4 micrograms/kg) than in 2 months old (10 micrograms/kg) rats. Larger doses of BAY K 8644 produced arrhythmias only in 12 months old rats, which was not totally abolished by nitroglycerine pretreatment. Perfusion of isolated rat hearts with 10(-6) M BAY K 8644 produced positive inotropic response, which was on the average 50% greater and developed much faster in 12 months old than in 2 months old rats. It is therefore concluded that the myocardial sensitivity to BAY K 8644 increases during adult maturation.

Expression of p53 predicts treatment failure in aggressive non-Hodgkin's lymphomas.

Approximately 50% of patients with aggressive non-Hodgkin's lymphomas (NHL) achieve a complete remission (CR) and cure with combination chemotherapy. The International Index is a useful clinical measure that predicts the patients' tolerance of therapy and likelihood of achieving CR, but it is not a direct measure of chemosensitivity. In this study we have investigated the predictive value of the tumor suppressor gene, p53, as a biological marker for response to treatment in the aggressive NHL. A retrospective study was carried out on 50 patients with aggressive NHL who were treated with CHOP chemotherapy. Treatment outcome was correlated with the expression of p53 in the lymphoma, as measured by routine immunohistochemistry using the monoclonal antibody Do-7. Forty percent of the lymphomas had >5% of the cells staining positively for p53 and this finding correlated significantly with response to treatment. Fifty percent of patients with p53 positive tumors achieved a CR versus 77% of patients with p53 negative tumors. In addition, the relapse rate and time to relapse were significantly different in the two groups. In the p53 positive group, 60% of patients relapsed in a median time of 6 months, whereas 26% of the p53 negative group relapsed with the time to relapse being >22 months. The overall survival of the p53 positive group (17 months) was significantly shorter than that of p53 negative group (>24 months). These results suggest that the immunohistochemical assessment of p53 is a simple and important prognostic measure for patients with aggressive NHL who are treated with CHOP chemotherapy.

Primary photophysical properties of ofloxacin.

Steady-state fluorescence has been used to study the excited singlet state of ofloxacin (OFLX) in aqueous solutions. Fluorescence emission was found to be pH dependent, with a maximum quantum yield of 0.17 at pH 7. Two pKa*s of around 2 and 8.5 were obtained for the excited singlet state. Laser flash photolysis and pulse radiolysis have been used to study the excited states and free radicals of OFLX in aqueous solutions. OFLX undergoes monophotonic photoionization from the excited singlet state with a quantum yield of 0.2. The cation radical so produced absorbs maximally at 770 nm with an extinction coefficient of 5000 +/- 500 dm3 mol-1 cm-1. This is confirmed by one-electron oxidation in the pulse radiolysis experiments. The hydrated electron produced in the photoionization process reacts with ground state OFLX with a rate constant of 2.0 +/- 0.2 x 10(10) dm3 mol-1 s-1, and the anion thus produced has two absorption bands at 410 nm (extinction coefficient = 3000 +/- 300 dm3 mol-1 cm-1) and at 530 nm. Triplet-triplet absorption has a maximum at 610 nm with an extinction coefficient of 11,000 +/- 1500 dm3 mol-1 cm-1. The quantum yield of triplet formation has been determined to be 0.33 +/- 0.05. In the presence of oxygen, the triplet reacts to form both excited singlet oxygen and superoxide anion with quantum yields of 0.13 and < or = 0.2, respectively. Moreover, superoxide anion is also formed by the reaction of oxygen with the hydrated electron from photoionization. Hence the photosensitivity due to OFLX could be initiated by the oxygen radicals and/or by OFLX radicals acting as haptens.

Endogenous inotropic substance from heart tissue has digitalis-like properties.

In the past few years, we developed an extraction procedure which we successfully used to isolate a crude fraction containing digitalis-like substance (DLS) from porcine left ventricular tissue. In this study, the crude fraction was found to cross-react with digoxin antibodies and showed immunoreactivity of 4.25 +/- 0.6 ng digoxin equivalent/ml. On further purification of the crude fraction using silica gel G column chromatography, a fraction C was obtained, which was highly positive inotropic on canine trabeculae and it dose-dependently inhibited ouabain sensitive 86Rb+ uptake in rat heart slices. A 50% inhibition of uptake was obtained by 25 microliters of fraction C. Fraction C also inhibited canine kidney Na+, K(+)-ATPase (Sigma, U.S.A.) dose-dependently and a 50% inhibition of this enzyme required 17 microliters of fraction C. Ashing of the fraction C at 500 degrees C resulted in loss of inotropic and enzyme inhibitory activities, indicating an organic nature of the unknown digitalis-like substance.

Antiestrogen use and survival of women with non-small cell lung cancer in Manitoba, Canada.

Lung cancer is the leading cause of cancer death worldwide. Sex differences in lung cancer incidence and survival are known. Female sex is an independent good prognostic factor. Estrogens appear to play a key role in lung cancer outcomes. Accordingly, antiestrogen use may also influence survival in female non-small cell lung cancer (NSCLC) patients. In this study, we compared survival among antiestrogen users and nonusers. We performed a retrospective population-based study. Using the Manitoba Cancer Registry (MCR), we identified all women diagnosed with NSCLC from 2000 to 2007. The population-based Drug Program Information Network was accessed to establish which patients received antiestrogens. Demographic data (e.g., smoking patterns, stage, histology) were gathered from the MCR and by chart review. Survival differences between antiestrogen-exposed and not exposed groups were compared using multivariable Cox regression. Two thousand three hundred twenty women fit our patient criteria, of which 156 had received antiestrogens. Exposure to antiestrogens was associated with a significantly decreased mortality in those exposed both before and after the diagnosis of NSCLC (adjusted hazard ratio, 0.42, p = 0.0006). This association remained consistent across age and stage groups. Antiestrogen use before and after the diagnosis of NSCLC is associated with decreased mortality. This supports previous evidence that estrogens may play a key role in the biology and outcomes of NSCLC and suggests a potential therapeutic use for these agents in this disease.

Erratum: Corrigendum: Survival and treatment patterns in elderly patients with advanced non-small-cell lung cancer in Manitoba.

[This corrects the article on p. e238 in vol. 18, PMID: 21980255.].

Survival and treatment patterns in elderly patients with advanced non-small-cell lung cancer in Manitoba.

Lung cancer is the leading cause of cancer death worldwide. Non-small-cell lung cancer (nsclc) is the most common form of lung cancer, with a median age at diagnosis of 70 years. These elderly patients are often underrepresented in the randomized clinical trials upon which chemotherapy plans are based. The objective of the present study was to determine the patterns of treatment and survival in elderly patients with advanced nsclc in Manitoba.An eligible cohort of elderly patients over 70 years of age at diagnosis (n = 497) with advanced nsclc was identified from the provincial cancer registry database for the period 2001-2004. Of the 497 patients identified, only 147 had been evaluated by a medical oncologist, and 82 of the 147 had received chemotherapy treatment, which is 16.5% of the initial cohort.Patients who received chemotherapy were younger than those who did not receive chemotherapy. Most patients receiving chemotherapy (84%) received doublet chemotherapy, with an almost equal split between cisplatin and carboplatin treatment. The median survival times for patients in this cohort were 64 weeks (stage iii nsclc) and 56 weeks (stage iv) with chemotherapy treatment, and 46 weeks (stage iii) and 26 weeks (stage iv) without chemotherapy.Although 50% of patients with advanced nsclc are more than 70 years of age, few are evaluated by a medical oncologist and even fewer are treated with chemotherapy. However, it should be noted that, in the elderly patients who were treated, survival times are comparable to those experienced by younger patients, which is indicative of a benefit of chemotherapy treatment for those elderly patients.