O-2Preliminary results of the ARTISTIC study: a randomised trial in screening to improve cytology

Annually in the UK around 250 000 cervical smears show low-grade abnormalities. Alternative management policies following a low-grade smear are cytological surveillance or referral for colposcopy. Their effectiveness and cost-effectiveness, and the potential for human papillomavirus (HPV) testing to triage women to either management, has been debated. Trial of management of borderline and other low-grade abnormal smears (TOMBOLA) (a large RCT) addresses these uncertainties, considering clinical, psychosocial and economic outcomes. 4439 women aged 20–59, resident in Grampian, Tayside or Nottingham with a low-grade smear were randomised to cytological surveillance (six-monthly smears in primary care) or hospital-based colposcopy. At colposcopy, women with visible abnormality were randomised to immediate treatment or biopsy and recall for treatment if necessary. Recruitment HPV status was assessed using PCR techniques. Women were followed for three years to an exit colposcopy. Cumulative incidence of CIN2 or more severe disease (CIN2+) in the colposcopy arm was 7.9% per year, higher than in cytological surveillance (5.8%; OR = 1.43, 95% CI 1.23–1.67). This difference was less marked for CIN3+ (OR = 1.27, 1.04–1.55), suggesting spontaneous regression of some CIN2, and that initial colposcopy can lead to over-treatment. There was little difference in psychosocial outcomes between arms. In comparison of biopsy and recall versus immediate treatment, there was no difference in cumulative incidence of CIN2+ or psychosocial outcomes. There was over-treatment and increased frequency/duration of bleeding with immediate treatment. There was no compelling economic reason to favour any one management method. Testing for HPV does not appear to be effective in triage. Based on these findings, we make management recommendations for women with low-grade smears.     

Leishmanial phosphatase blocks neutrophil O-2 production

Leishmania donovani, the causative agent in kala-azar or visceral leishmaniasis, infects cells of the macrophage system. We show that a purified preparation of the tartrate-resistant acid phosphatase, isolated from the external surface of L. donovani promastigotes, inhibits superoxide anion production by human neutrophils. Preincubation of neutrophils for 15-30 min at 37 degrees C with 240 units (1 unit equals 1 nmol of 4-methylumbelliferyl phosphate cleaved per h) of the acid phosphatase decreases both the rate and extent of superoxide generation by 90% upon stimulation with the chemoattractant peptide fMet-Leu-Phe. The ability of the phosphatase to suppress superoxide anion production is abolished by heat inactivation of the enzyme or by incorporation of an acid phosphatase inhibitor in the preincubation medium, indicating that the effect is dependent on the catalytic activity of the enzyme. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.     

Biosynthesis of isochorismate in Klebsiella pneumoniae: origin of O-2

The shikimate metabolites are key precursors to a large number of natural products, including aromatic amino acids. Chorismic acid is an important branch point in the biosynthetic pathway to aromatic amino acids. Chorismic acid is also unique among natural products since it is the only compound known to undergo an enzymatic Claisen rearrangement. A metabolite of chorismic acid, isochorismic acid, first observed in Aerobacter aerogenes differs in its chemical structure by the location of the hydroxyl group and the double bonds. Isochorismic acid is a precursor to a growing number of shikimate-derived metabolites. Isochorismic acid has also been postulated to be an intermediate of m-carboxyaromatic amino acids, implying another enzymatic Claisen rearrangement. In this publication, we have isolated isochorismate synthase and found that on lyophilization the enzyme is stable for at least 6 months at -20 degrees C. Incubation of chorismate with this preparation in water enriched with 18O led to incorporation of one atom of 18O as proven from the fast atom bombardment mass spectra of the HPLC purified derived isochorismate.     

A quantitative method to determine the points of O-(2-hydroxypropyl) substitution in O-(2-hydroxypropyl)-guar

A method is described for locating the O-(2-hydroxypropyl) groups in O-(2-hydroxypropyl)-substituted guar. Per-O-methylation of the O-(2-hydroxypropyl)guar yielded guar that was partially O-methylated and partially O-(2-methoxypropyl)ated. This polymer was hydrolyzed, to afford a mixture of partially O-methylated monosaccharides and partially O-(2-methoxypropylated), partially O-methylated monosaccharides. These monosaccharide derivatives were reduced, and the alditols acetylated, to give a mixture of partially O-acetylated, partially O-methylated alditols with partially O-acetylated, partially O-(2-methoxypropyl)ated, partially O-methylated alditols. These alditol derivatives were identified by gas-liquid chromatography-mass spectrometry, and quantitated by gas-liquid chromatography.     

Kinetic study of O-2 DISMUTATION BY BOVINE SUPEROXIDE DISMUTASE. Evidence for saturation of the catalytic sites by O-2.

Dismutation of O₂^? by bovine copper-zinc superoxide $\text{di}$ smutase has been studied at different O₂^? concentrations with a polarographic method. Saturation of the enzyme by the substrate was observed and K_m and Vmax values were calculated. Inhibition by OH^? and CN^? was shown to be of the competitive type. The data support on inner sphere mechanism for the reaction between O₂^? and copper.     

The NADPH-dependent O-.2-generating oxidase from human neutrophils

A subcellular particulate fraction from normal neutrophils that was enriched in NADPH-dependent O-.2-generating activity (Gabig, T. G., Schervish, E. W., and Santinga, J. T. (1982) J. Biol. Chem. 257, 4114-4119) has been further characterized. This preparation contained 0.25 +/- 0.02 nmol of flavin adenine dinucleotide/mg of protein and 0.28 +/- 0.01 nmol of cytochrome b/mg of protein. Measurable amounts of riboflavin or flavin mononucleotide were not present. The flavoprotein was completely resolved from the cytochrome b by selective bile salt extraction of the particulate oxidase fraction. The identical subcellular particulate fraction was studied in the neutrophils from two male patients with chronic granulomatous disease. The neutrophil oxidase fraction from one of the chronic granulomatous disease patients had a cytochrome b component that was spectrally abnormal, but a normal content of flavin adenine dinucleotide. The fraction from this patient's neutrophils corresponding to the resolved flavoprotein from normal cells had fluorescence excitation and emission spectra that were identical to the normal flavoprotein. The neutrophil oxidase fraction from the second chronic granulomatous disease patient had a quantitatively and spectrally normal cytochrome b but less than 8% of the normal amount of flavin adenine dinucleotide. The fraction from the latter patient's neutrophils corresponding to the resolved flavoprotein from normal cells had no detectable flavoprotein by fluorescence excitation and emission spectroscopy. It is postulated that these two patients represent distinct mutants in two separate components of the neutrophil NADPH-dependent O-.2-generating oxidase system, flavoprotein and cytochrome b.     

Cooperation between PDGF and FGF converts slowly dividing O-2Aadult progenitor cells to rapidly dividing cells with characteristics of O- 2Aperinatal progenitor cells

We have shown previously that oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells isolated from adult rat optic nerves can be distinguished in vitro from their perinatal counterparts on the basis of their much slower rates of division, differentiation, and migration when grown in the presence of cortical astrocytes or PDGF. This behavior is consistent with in vivo observations that there is only a modest production of oligodendrocytes in the adult CNS. As such a behavior is inconsistent with the likely need for a rapid generation of oligodendrocytes following demyelinating damage to the mature CNS, we have been concerned with identifying in vitro conditions that allow O-2Aadult progenitor cells to generate rapidly large numbers of progeny cells. We now provide evidence that many slowly dividing O-2Aadult progenitor cells can be converted to rapidly dividing cells by exposing adult optic nerve cultures to both PDGF and bFGF. In addition, these O-2Aadult progenitor cells appear to acquire other properties of O-2Aperinatal progenitor cells, such as bipolar morphology and high rate of migration. Although many O-2Aadult progenitor cells in cultures exposed to bFGF alone also divide rapidly, these cells are multipolar and migrate little in vitro. Oligodendrocytic differentiation of O-2Aadult progenitor cells, which express receptors for bFGF in vitro, is almost completely inhibited in cultures exposed to bFGF or bFGF plus PDGF. As bFGF and PDGF appear to be upregulated and/or released after injury to the adult brain, this particular in vitro response of O-2Aadult progenitor cells to PDGF and bFGF may be of importance in the generation of large numbers of new oligodendrocytes in vivo following demyelination.     

Study protocol of the YOU CALL - WE CALL TRIAL: impact of a multimodal support intervention after a "mild" stroke

Background

More than 60% of new strokes each year are "mild" in severity and this proportion is expected to rise in the years to come. Within our current health care system those with "mild" stroke are typically discharged home within days, without further referral to health or rehabilitation services other than advice to see their family physician. Those with mild stroke often have limited access to support from health professionals with stroke-specific knowledge who would typically provide critical information on topics such as secondary stroke prevention, community reintegration, medication counselling and problem solving with regard to specific concerns that arise. Isolation and lack of knowledge may lead to a worsening of health problems including stroke recurrence and unnecessary and costly health care utilization. The purpose of this study is to assess the effectiveness, for individuals who experience a first "mild" stroke, of a sustainable, low cost, multimodal support intervention (comprising information, education and telephone support) - "WE CALL" compared to a passive intervention (providing the name and phone number of a resource person available if they feel the need to) - "YOU CALL", on two primary outcomes: unplanned-use of health services for negative events and quality of life.

Method/Design

We will recruit 384 adults who meet inclusion criteria for a first mild stroke across six Canadian sites. Baseline measures will be taken within the first month after stroke onset. Participants will be stratified according to comorbidity level and randomised to one of two groups: YOU CALL or WE CALL. Both interventions will be offered over a six months period. Primary outcomes include unplanned use of heath services for negative event (frequency calendar) and quality of life (EQ-5D and Quality of Life Index). Secondary outcomes include participation level (LIFE-H), depression (Beck Depression Inventory II) and use of health services for health promotion or prevention (frequency calendar). Blind assessors will gather data at mid-intervention, end of intervention and one year follow up.

Discussion

If effective, this multimodal intervention could be delivered in both urban and rural environments. For example, existing infrastructure such as regional stroke centers and existing secondary stroke prevention clinics, make this intervention, if effective, deliverable and sustainable.

Trial Registration

ISRCTN95662526     

The enzymatic basis for O-.2 production by human neutrophils

O-.2 production is the first step in the generation of a group of powerful microbicidal oxidants by neutrophils. The production of O-.2 is catalyzed by a membrane-bound, NADPH-preferring flavoprotein oxidase, a conclusion supported by much evidence including the discovery of a new form of chronic granulomatous disease caused by a mutation affecting that oxidase directly. Also involved in the O-.2-forming reaction is a b-type cytochrome; the role of this cytochrome is as yet undefined, though it does not appear to be on the direct route of electron transfer between NADPH and oxygen. It has been postulated that quinones too participate in the O-.2-forming reaction, but further work is necessary to define their role more fully.     

Lucigenin: redox potential in aqueous media and redox cycling with O-(2) production

The use of lucigenin luminescence as a measure of ?O-(2) has been questioned because lucigenin has been shown to be capable of mediating the production of O-(2). This being the case, lucigenin can signal the presence of O-(2) even in systems not producing it in the absence of lucigenin. The reduction potential of lucigenin should be in accord with its ability to mediate O-(2) production; but it has not heretofore been measured in aqueous media. The problems facing such measurement are the insolubility of the divalently reduced form, which deposits on the electrode, and the slow conformational transition that follows the second electron transfer and which interferes with reversibility. We have now used rapid scan cyclic voltammetry to determine that the reduction potential for lucigenin is -0.14 +/- 0.02 V versus the normal hydrogen electrode. This value applies to both the first and the second electron transfers to lucigenin and it is in accord with the facile mediation of O-(2) production by this compound.     

Ion channel expression by white matter glia: the O-2A glial progenitor cell.

We describe electrophysiological properties of the O-2A glial progenitor cell in a new serum-free culture system. O-2A progenitors have many properties characteristic of neurons: they have glutamate-activated ion channels, express the neuronal form of the sodium channel, fire single regenerative potentials, and synthesize the neurotransmitter GABA by an alternative synthetic pathway. Nearly identical properties were observed in acutely isolated O-2A progenitors, indicating that this phenotype is not an artifact of culture. The O-2A did not express a simple subset of channel types found in its descendant cells, the type-2 astrocyte and oligodendrocyte, studied in the same culture system. During development, these electrophysiological properties may contribute to O-2A function in vivo.     

Radiation sensitization by oxygen of in vitro mammalian cells: is .O-2 involved?

Oxygen is a potent sensitizer of cells exposed to ionizing radiation, and, although the exact chemical mechanisms are not fully understood, some evidence suggests that this sensitization may involve the formation of superoxide anion radicals (.O-2) [F. Lavelle, A. M. Michelson, and L. Dimitrijevic, Biochem. Biophys. Res. Commun. 55, 350-357 (1973); A. Petkau and W. S. Chelack, Int. J. Radiat. Biol. 26, 421-426 (1974); L. W. Oberley, A. L. Lindgren, S. A. Baker, and R. H. Stevens, Radiat. Res. 68, 320-328 (1976)] To test this hypothesis, we compared the sensitivity of Chinese hamster V79 cells irradiated in O2/N2 and O2/N2O gas mixtures with and without the addition of other radical scavenging agents. In these tests, although oxygen was present, be blocked the radiation-induced reactions of O2 which produce .O-2. We found that the total amount of biological damage depends simply on the concentration of O2 that is present; the overall sensitivity is not reduced when .O-2 cannot be formed. Thus radiation sensitization by O2--at least of this cell line--does not require the formation of superoxide anion radicals.     

TRIAL: a tool for finding distant structural similarities

Finding structural similarities in distantly related proteins can reveal functional relationships that can not be identified using sequence comparison. Given two proteins A and B and threshold ε ?, we develop an algorithm, TRiplet-based Iterative ALignment (TRIAL) for computing the transformation of B that maximizes the number of aligned residues such that the root mean square deviation (RMSD) of the alignment is at most ε ?. Our algorithm is designed with the specific goal of effectively handling proteins with low similarity in primary structure, where existing algorithms perform particularly poorly. Experiments show that our method outperforms existing methods. TRIAL alignment brings the secondary structures of distantly related proteins to similar orientations. It also finds larger number of secondary structure matches at lower RMSD values and increased overall alignment lengths. Its classification accuracy is up to 63 percent better than other methods, including CE and DALI. TRIAL successfully aligns 83 percent of the residues from the smaller protein in reasonable time while other methods align only 29 to 65 percent of the residues for the same set of proteins.     

Inactivation of the human CuZn superoxide dismutase during exposure to O-2 and H2O2

Human copper-zinc superoxide dismutase undergoes inactivation when exposed to O₂^? and H₂O₂ generated during the oxidation of acetaldehyde by xanthine oxidase at pH 7.4 and 37° C. In contrast, human manganese superoxide dismutase is not inactivated under the same conditions. Catalase and Mn-superoxide dismutase protect CuZn superoxide dismutase from inactivation. Similar protection is observed with hydroxyl radical (OH.) scavengers, such as formate and mannitol. In contrast, other OH. scavengers such as ethanol and tert-butyl alcohol, have no protective action. The latter results indicate that “free OH.” is not responsible for the inactivation. Furthermore, H₂O₂ generated during the oxidation of glucose by glucose oxidase, i.e., without production of O₂^?, does not induce CuZn superoxide dismutase inactivation. A mechanism accounting for this $\text{O}{}_2{}^{\mathrm{?}}\text{H}{}_2\text{O}{}_2$-dependent inactivation of CuZn superoxide dismutase is proposed.     

Synthesis and properties of diastereoisomers of adenosine 5'-(O-1-thiotriphosphate) and adenosine 5'-(O-2-thiotriphosphate).

The chemical synthesis of adenosine 5'-(O-1-thiotriphosphate) (ATPalphaS) and adenosine 5'-(O-2-thiotriphosphate) (ATPbetaS) is described. Both exist as a pair of diastereomers, A and B. The isomers of ATPalphaS can be distinguished on the basis of their different reaction rates with myokinase as well as nucleoside diphosphate kinase. With both enzymes, isomer A reacts fast whereas isomer B reacts considerably more slowly. Phosphorylation of a mixture of isomers of ADPalphaS with pyruvate or acetate kinase yields ATPalphaS, isomer A, whereas the phosphoryl transfer with creatine or arginine kinase yields isomer B. The isomers of ATPbetaS differ in their reactivity with myosin. Isomer A is readily hydrolyzed, whereas isomer B is not. However, isomer B reacts faster with nucleoside diphosphate kinase and ADP than isomer A. Phosphoryl transfer with pyruvate kinase onto ADPbetaS yields ATPbetaS, isomer A, with acetate kinase, isomer B.     

Substance P Receptors on O-2A Progenitor Cells and Type-2 Astrocytes In Vitro

Abstract: Bradykinin- and substance P (SP)-stimulated second messenger studies in isolated subsets of neuroglia showed bradykinin-stimulated synthesis of phospho- inositides (PI) in type-1 astrocytes and oligodendrocytes. SP-stimulated PI accumulation was restricted to oligoden- drocyte/type-2 astrocyte progenitor cells and type-2 astrocytes. These data were confirmed by analysis of calcium transients in single cells. In a regional study, SP-stimulated PI accumulation in primary astrocyte cultures was restricted to white matter. We conclude that regional heterogeneity in the expression of peptide receptors in cultures of primary astrocytes arises from a restricted distribution on subsets of macroglia. SP receptors restricted on cells of the oligodendrocyte/type-2 astrocyte type-2 lineage in vitro, coupled with in vivo observations by others, suggests that SP receptor expression is conserved on subsets of macroglia in vitro and possibly reactive astrocytes in vivo.