The Bactericidal Action of Peroxides; An E.P.R. Spin-Trapping Study

E.P.R. spin trapping has been employed to study radical production during the bactericidal action of three peroxide compounds (peracetic acid, 4-percarboxy-N-isobutyltrimellitimide and magnesium monoperoxyphthalate) upon both Gram negative (Escherichia Coll) and Gram positive (Staphylococcus Aureus) bacteria. Use of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has allowed direct detection of both carbon-centred and hydroxyl radicals, which are produced at varying rates for the different bacteria/peracid systems studied. The inhibition of bactericidal action, by DMPO and two antioxidants, Vitamin C and Trolox C, indicates that radicals are the lethal species and evidence is presented which suggests that radical production is internal to the bacterial cell. Hydroxyl radicals are believed to be the lethal species. The effect of added iron chelators and haem protein inhibitors indicates that iron species and haem proteins in particular are involved. A marked variation is found in observed hydroxyl-radical adduct signals with both the nature and concentration of peracid. A strong inverse correlation is found between the concentration of the observed radical adduct signal and the relative strength of the peroxide as a bactericide; use of a stable nitroxide as a radical scavenger confirms that strong bactericides produce radicals at a much faster rate than weak bactericides. Plots of radical generation versus time are correlated with % bacterial kill, offering further evidence that hydroxyl radicals are the lethal species.     

Permanent, nonleaching antibacterial surfaces. 1. Synthesis by atom transfer radical polymerization

We have grown an antimicrobial polymer directly on the surfaces of glass and paper using atom transfer radical polymerization (ATRP). The method described here results in potentially permanent nonleaching antibacterial surfaces without the need to chemically graft the antimicrobial material to the substratum. The tertiary amine 2-(dimethylamino)ethyl methacrylate was polymerized directly onto Whatman #1 filter paper or glass slides via atom transfer radical polymerization. Following the polymerization, the tertiary amino groups were quaternized using an alkyl halide to produce a large concentration of quaternary ammonium groups on the polymer-modified surfaces. Incubating the modified materials with either Escherichia coli or Bacillus subtilis demonstrated that the modified surfaces had substantial antimicrobial capacity. The permanence of the antimicrobial activity was demonstrated through repeated use of a modified glass without significant loss of activity. Quaternary amines are believed to cause cell death by disrupting cell membranes allowing release of the intracellular contents. Atomic force microscopic imaging of cells on modified glass surfaces supports this hypothesis.     

Metal use in ribonucleotide reductase R2, di-iron, di-manganese and heterodinuclear--an intricate bioinorganic workaround to use different metals for the same reaction

The ferritin-like superfamily comprises of several protein groups that utilize dinuclear metal sites for various functions, from iron storage to challenging oxidations of substrates. Ribonucleotide reductase R2 proteins use the metal site for the generation of a free radical required for the reduction of ribonucleotides to deoxyriboinucleotides, the building blocks of DNA. This ubiquitous and essential reaction has been studied for over four decades and the R2 proteins were, until recently, generally believed to employ the same cofactor and mechanism for radical generation. In this reaction, a stable tyrosyl radical is produced following activation and cleavage of molecular oxygen at a dinuclear iron site in the protein. Discoveries in the last few years have now firmly established that the radical generating reaction is not conserved among the R2 proteins but that different subgroups, that are structurally very similar, instead employ di-manganese or heterodinuclear Mn-Fe cofactors as radical generators. This is remarkable considering that the protein must exercise a strict control over oxygen activation, reactive metal-oxygen intermediate species and the resulting redox potential of the produced radical equivalent. Given the differences in redox properties between Mn and Fe, use of a different metal for this reaction requires associated adaptations of the R2 protein scaffold and the activation mechanism. Further analysis of the differences in protein sequence between R2 subgroups have also led to the discovery of new groups of R2-like proteins with completely different functions, expanding the chemical repertoire of the ferritin-like superfamily. This review describes the discoveries leading up to the identification of the different Mn-containing R2 protein groups and our current understanding of them. Hypotheses regarding the biochemical rationale to develop these chemically complex alternative solutions are also discussed.     

CO2.- radical induced cleavage of disulfide bonds in proteins. A gamma-ray and pulse radiolysis mechanistic investigation

Disulfide bond reduction by the CO2.- radical was investigated in aponeocarzinostatin, aporiboflavin-binding protein, and bovine immunoglobulin. Protein-bound cysteine free thiols were formed under gamma-ray irradiation in the course of a pH-dependent and protein concentration dependent chain reaction. The chain efficiency increased upon acidification of the medium, with an apparent pKa around 5, and decreased abruptly below pH 3.6. It decreased also at neutral pH as cysteine accumulated. From pulse radiolysis analysis, CO2.- proved able to induce rapid one-electron oxidation of thiols and of tyrosine phenolic groups in addition to one-electron donation to exposed disulfide bonds. The bulk rate constant of CO2.- uptake by the native proteins was 5- to 10-fold faster at pH 3 than at pH 8, and the protonated form of the disulfide radical anion, [symbol: see text], appeared to be the major protein radical species formed under acidic conditions. The main decay path of [symbol: see text] consisted of the rapid formation of a thiyl radical intermediate [symbol: see text] in equilibrium with the closed, cyclic form. The thiyl radical was subsequently reduced to the sulfhydryl level [symbol: see text] on reaction with formate, generating 1 mol of the CO2.- radical, thus propagating the chain reaction. The disulfide radical anion [symbol: see text] at pH 8 decayed through competing intramolecular and/or intermolecular routes including disproportionation, protein-protein cross-linking, electron transfer with tyrosine residues, and reaction with sulfhydryl groups in prereduced systems. Disproportionation and cross-linking were observed with the riboflavin-binding protein solely. Formation of the disulfide radical cation [symbol: see text], phenoxyl radical Tyr-O. disproportionation, and phenoxyl radical induced oxidation of preformed thiol groups should also be taken into consideration to explain the fate of the oxygen-centered phenoxyl radical.     

Breast cancer in northern Alberta: pilot study in computerized registration.

Analysis of data from 643 breast cancer patients seen between 1971 and 1973 in northern Alberta was undertaken as a preliminary study leading towards a comprehensive breast registry. Age at first treatment and menopausal status were found to be related significantly to the clinical stage of the disease. Other data reported included age at menarche, lymph node involvement and methods of primary treatment. A decline in use of the radical mastectomy was noted. The comprehensive breast registry, which will be used to identify high-risk groups, assess treatment modalities, test hypotheses and generate ideas, has a high probability of success because of compulsory registration of new cases of breast cancer in Alberta and collection of data by the same four individuals.     

Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation

The generation of oxygen radicals and the process of lipid peroxidation have become a focus of attention for investigators in the fields of central nervous system (CNS) trauma and stroke (e.g., ischemia). Considering our level of understanding of free radical and lipid peroxidation chemistry, absolute proof for their involvement in the pathophysiology of traumatic and ischemic damage to the CNS has been meager. While direct, unequivocal evidence for the participation of free radicals and lipid peroxidation as primary contributors to the death of neuronal tissue waits to be established, numerous recent studies have provided considerable support for the occurrence of free radical and lipid peroxidation reactions in the injured or ischemic CNS. In addition, the pharmacological use of antioxidants and free radical scavengers in the treatment of experimental CNS trauma and ischemia has provided convincing, although indirect evidence, for the involvement of oxygen radicals and lipid peroxidation in these conditions. The intent of this and its companion paper is to review: 1) the biochemical processes which may give rise to free radical reactions in the CNS, 2) the environment of the ischemic cell as it may affect the generation of oxygen radicals and the catalysis of lipid peroxidation reactions, 3) the evidence for the involvement of free radical mechanisms in CNS trauma and ischemia, and 4) the pathophysiological consequences of these phenomena.     

Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline-N-oxide and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide as spin traps for HO* and SO4*-.

To spin trap hydroxyl radical (HO*) with in vivo detection of the resultant radical adducts, the use of two spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) (10 mmol/kg) has been compared. In mice treatment with 5-aminolevulinic acid and Fe3+ resulted in detection of adducts of hydroxyl radicals (HO*), but only with use of DEPMPO. Similarly, 'HO* adducts' generated via nucleophilic substitution of SO4*- adducts formed in vivo could be observed only when using DEPMPO as the spin trap. The reasons for the differences observed between DEPMPO and DMPO are likely due to different in vivo lifetimes of their hydroxyl radical adducts. These results seem to be the first direct in vivo EPR detection of hydroxyl radical adducts.     

Spectroscopic studies of chemically modified synthetic melanins

The infrared and electron spin resonance spectra of synthetic 3,4-dihydroxyphenylalanine (DOPA) and tyrosine melanins and chemically modified melanin samples were determined, and it was shown that unmodified and reduced DOPA melanins exhibited similar ir spectra. Oxidized DOPA melanins showed a higher number of carboxy groups in the sample. A significant increase of free radical content in reduced DOPA melanin and a decrease of free radical content in oxidized DOPA melanin in comparison to unmodified samples were demonstrated by the use of ESR methodology. Methylation of tyrosine melanin with an excess of diazomethane gave very rich ir spectra as compared to melanins methylated with methanol saturated by gaseous HCl. In tyrosine melanin samples the esterification of carboxy groups with methanol caused a decrease in the free radical content. When diazomethane was used, the methylated melanin samples had free radical levels reduced to only about 4% of the total observed for unmodified tyrosine melanin.     

Electric potential at the entrance of the amphotericin channel. Channel specificity

The electric potential at the entrance of the amphotericin channel was varied by changing the membrane surface charge, modifying the charged groups of amphotericin molecule or adding of MgSO4. It has been shown that the zero current potential and channel selectivity depend on the potential at the entrance of the channel. It has been found that anion and cation current through amphotericin channel are coupled. Possible usage of CMF for studying radical stages in catalytic Fe2+-oxidation of liposomes heterogeneous processes has been shown.     

Studies on the metal-ion and lipoxygenase-catalysed breakdown of hydroperoxides using electron-spin-resonance spectroscopy.

The breakdown of cumene hydroperoxide and peroxidized fatty acids by iron is shown, by use of the spin trap 5,5-dimethyl-l-pyrroline-N-oxide, to be sensitive to (a) the oxidation state of the metal and (b) the nature of the chelating ligands. The initial step in the Fe2+-catalysed breakdown is the production of an alkoxyl radical by one-electron reduction, and this type of radical has been successfully trapped from each substrate. Subsequent reactions of this alkoxyl species produce both carbon-centred and peroxyl radicals, depending on the concentrations of the reagents present. The use of the same spin trap in microsomal systems undergoing either NADPH-supported or Fe2+-induced peroxidation led to the detection of low concentrations of radical adducts, among which are signals that are believed to be due to lipid alkoxyl radicals. Reaction of polyunsaturated fatty acid hydroperoxides with both Fe2+ and lipoxygenase under anaerobic conditions gives rise to signals not only from the alkoxy-radical adduct, but also from a further species which is tentatively identified as being due to an acyl [RC(O).]-radical adduct; chemical studies lend support to this assignment.     

Syntheses and radical scavenging activities of resveratrol derivatives

Nine new resveratrol derivatives, having bromo, iodo, and fluoroethyl groups, were designed and synthesized. All compounds having free phenol groups showed good free radical scavenging activity. Among them, 2-bromoresveratrol 19 has a similar free radical scavenging activity to (+)-catechin.     

Endogenous scavenger levels (vitamin E and A) of patients with chronic alcoholic liver disease under different environmental conditions.

Serum level of endogenous scavengers (E and A vitamin) was studied in groups of patients with various chronic alcoholic liver diseases and in a healthy control group on polluted and non-polluted areas. Vitamin levels in patients with chronic liver disease are diminished in comparison to the healthy in general, but mainly in the cirrhotic group. Diminution of vitamin E levels was observed in earlier phase of liver disease than that of vitamin A levels. Patients and healthy control on polluted area showed more expressed diminution of vitamin levels than the same groups on non-polluted area. Free radical parameter (RBC diene conjugate content) and characteristic alcoholic parameters (serum GOT, gamma-GT, cholesterol level and liver GOT, gamma-GT content in biopsy specimen) were used to explain the differences between the same investigated groups on polluted and non-polluted areas. As conclusion can be supposed that industrial pollution of environment has a worsening effect in diseases with free radical mechanism.     

The YF161D1 mutant of Synechocystis 6803 exhibits an EPR signal from a light-induced photosystem II radical.

The currently accepted model for the location of the redox-active tyrosines, D and Z, in photosystem II suggests that they are symmetrically located on the D1 and D2 polypeptides, which are believed to form the heterodimer core of the reaction center. Z, the electron conduit from the manganese catalytic site to the primary chlorophyll donor, has been identified with tyrosine-161 of D1. The YF161D1 mutant of Synechocystis 6803 [Debus, R. J., Barry, B. A., Sithole, I., Babcock, G. T., & McIntosh, L. (1988b) Biochemistry 27, 9071-9074; Metz, J. G., Nixon, P. J., Rogner, M., Brudvig, G. W., & Diner, B. A. (1989) Biochemistry 28, 6960-6969], in which this tyrosine has been changed to a phenylalanine, should have no light-induced EPR (electron paramagnetic resonance) signal from a tyrosine radical. This negative result has indeed been obtained in analysis of one of two independently constructed mutants through the use of a non-oxygen-evolving core preparation (Metz et al., 1989). Here, we present an analysis of a YF161D1 mutant through the use of a photosystem II purification procedure that gives oxygen-evolving particles from wild-type Synechocystis cultures. In our mutant preparation, a light-induced EPR signal from a photosystem II radical is observed under conditions in which, in a wild-type preparation, we can accumulate an EPR signal from Z+. This EPR signal has a different lineshape from that of the Z+ tyrosine radical, and spin quantitation shows that this radical can be produced in up to 60% of the mutant reaction centers. The EPR lineshape of this radical suggests that photosystem II reaction centers of the YF161D1 mutant contain a redox-active amino acid.     

腹腔镜结肠癌根治术与传统开腹结肠癌根治术的临床对比分析

目的:探讨腹腔镜结肠癌根治术与传统开腹结肠癌根治术疗效的差别。方法:随机将86例结肠癌患者分为两组,各43例。腹腔镜组采用腹腔镜结肠癌根治术,传统开腹组采用开腹结肠癌根治术,观察两组患者手术情况及术后并发症情况。结果:腹腔镜组手术时间、淋巴结清扫数与传统开腹组比较无显著差异(P0.05);腹腔镜组术中出血量少于传统开腹组、住院时间短于传统开腹组(P0.05);腹腔镜组排气时间短于传统开腹组(P0.01),两组并发症发生率差异有统计学意义(P0.01)。结论:腹腔镜结肠癌根治术具有术中出血量少、创伤小、术后恢复快、住院时间明显缩短等特点,可达到传统开腹手术的效果,值得广泛应用。     

Cyclodecapeptides to mimic the radical site of tyrosyl-containing proteins.

Tyrosyl radicals are involved in many biologically important processes. The development of model compounds to mimic radical enzyme active sites, such as galactose oxidase (GO), has widely contributed to an enhanced understanding of their spectral properties, structural attributes and even reactivity. An emerging approach towards the synthesis of such active site mimetics is the use of peptidic ligands. The potential of cyclodecapeptides to bear phenoxyl radicals has been evaluated through three compounds. LH(4) (2+) is a cyclodecapetide containing two histidine residues (mimicking His(496) and His(581) of GO) and two tyrosine residues (mimicking Tyr(495) and the Tyr(272)* radical of GO). L(tBu)H(4) (2+) and L(OMe)H(4) (2+) incorporate 2,4,6-protected phenols in place of each tyrosine in LH(4) (2+). The deprotonation constants of each peptide determined by potentiometric titrations showed that there are some interactions between the acido-basic residues. Cyclic voltammetric studies revealed that only the peptides incorporating 2,4,6-protected phenolates exhibit reversible redox couples and are thus precursors of radicals stable enough to persist in solution. These studies also showed L(OMe2-) to possess the lower oxidation potential, indicating that this peptide, in its radical form, is the most stabilized. The electrochemically generated radical species have been characterized by EPR spectroscopy.     

Chemical modification of horseradish peroxidase. Preparation and characterization of tracer enzymes with different isoelectric points.

Horseradish peroxidase (HRP), a plant glycoprotein with a molecular weight of 40,000 D and a molecular radius (ae) of 30 A, has been modified chemically to prepare tracer molecules with different molecular charge. Modification of free carboxyl groups on the enzyme is achieved by carbodiimide activation and subsequent reaction of activated carboxyl groups with a nucleophile; uncharged groups or radicals containing additional positively charged moieties are introduced into the protein molecule resulting in an increased net positive charge of the tracer. Amino groups in the protein molecule are modified by acetylation or succinylation; this reaction will increase the net negative charge of the enzyme by either introducing an uncharged group or an additional carboxyl radical. The tracer molecules so obtained are then characterized in terms of molecular size and charge by column chromatography and isoelectric focusing respectively. The enzymatic activity as measured by 3,3'-diaminobenzidine reaction, the pH optimum and the absorption spectra for the modified enzymes remain virtually unchanged.     

Beyondism: Raymond B. Cattell and the new eugenics

A significant confusion has arisen out of the mass of work done on the history of eugenics in the last two decades. Early scholars of the subject treated eugenics as a marginalized or obsolete movement of the radical right. Subsequent research has shown that eugenic ideas were adopted in diverse national settings by very different groups, including – among others – liberals, communists and Catholics, as well as radical rightists. This complexity is sometimes taken to mean that eugenics has no special ideological associations, that it is historically and potentially a beast of a thousand heads. It is not. Although people of varied ideological commitments have been attracted to eugenics, ideologues of the radical right, and above all interwar fascists, have been uniquely and centrally involved in its development. Fascism and the radical right are also complex entities, but for all the heterogeneity of both eugenics and fascism, the special historical relationship between the two cannot be ignored. This relationship is exemplified in the work of the influential psychologist, Raymond B. Cattell. Cattell was an early supporter of German national socialism and his work should be understood in the context of interwar fascism. The new religious movement that he founded, ‘Beyondism’, is a neo-fascist contrivance. Cattell now promulgates ideas that he formulated within a demimonde of radical eugenists and neo-fascists that includes such associates as Revilo Oliver, Roger Pearson, Wilmot Robertson and Robert K. Graham. These ideas and Cattell's role in the history of eugenics deserve deeper analysis than they have hitherto received. Far from being of merely antiquarian interest, his work currently encourages the propagation of radical eugenist ideology. It is unconscionable for scholars to permit these ideas to go unchallenged, and indeed honored and emulated by a new generation of ideologues and academicians whose work helps to dignify the most destructive political ideas of the twentieth century. This revised version was published online in August 2006 with corrections to the Cover Date.     

Restoration of antioxidants in liver by methionine feeding in experimental rat urolithiasis.

The effect of methionine or citrate on antioxidant defense system has been studied in urolithic rat. Liver weight and its protein concentration did not change in the rats fed with calculi producing diet (CPD) when compared to normal diet fed rats. Feeding rats along with citrate (c-CPD) or methionine (m-CPD) improved their body weight gain. Liver microsomes and mitochondria fractions of CPD and c-CPD fed groups showed increased susceptibility for lipid peroxidation in presence of ascorbate and t-butyl hydroperoxide when compared to either control or m-CPD fed groups. Increased superoxide dismutase and xanthine oxidase activities, decreased catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase activities, decreased concentrations of reduced glutathione, total thiols, ascorbic acid and vitamin-E and increased formation of hydroxyl radical, hydroperoxides and diene conjugates were observed in the liver of both CPD fed group as well as c-CPD fed group. Except SOD and xanthine oxidase, all other parameters were normalized in m-CPD fed group. This suggested that feeding methionine reduced the susceptibility for lipid peroxidation by restoration of the level of free radical scavengers.     

Scavenger and antioxidant properties of ten synthetic flavones.

To study the effect of the hydroxyl groups on biological activities of flavones, we synthesized 10 polyhydroxyflavones with varied substitution patterns. The abilities of the 10 compounds to act as radical scavengers were investigated using chemiluminescence in two biological models: the xanthine/xanthine oxidase system and the oxidative burst of rat alveolar macrophages. Stable radical formation was observed by electron spin resonance (ESR) spectroscopy. We found that the presence of the pyrogallol moiety in the B component of flavones gave rise to radical scavenger activity and that C-6 substituted hydroxyl group may also provide the basis for biological activity. Furthermore, compounds with a hydroxyl at C-7 position appeared to be xanthine oxidase inhibitors. One particular compound exhibited radical scavenger activity and xanthine oxidase inhibition. This type of compound should prove to be useful in the treatment of ischemia, for which both properties were required.     

乳腺癌保乳改良根治术与传统改良根治术临床疗效的比较

目的:对比分析保乳改良根治术与传统改良根治术治疗乳腺癌的临床疗效。方法:将行手术治疗的415例女性乳腺癌患者按手术方式分为保乳改良根治术组76例与传统改良根治术组339例,两组患者术后均接受辅助治疗。比较两组患者术后满意度,术后并发症,复发、转移及死亡率,评价保乳改良根治术的临床疗效。结果:两组患者的发病年龄,术后病理类型,失随访情况,转移、死亡及复发率比较无统计学意义(P0.05)。保乳改良根治术组皮下积液发生率明显低于传统改良根治术组(P0.05),术后满意度明显优于传统改良根治术组(P0.05),其他并发症比较无显著差异(P0.05)。结论:保乳改良根治术能得到和传统改良根治术同样的疗效,且具有损伤小、并发症少、外形美观、术后满意度高等优势。