Achalasia cardia: A five-year review in Hospital Tuanku Ja'afar, Seremban.

INTRODUCTION: Achalasia cardia is an oesophageal motility disorder that affects various age groups. This study focused on the epidemiological features of achalasia, its risk factors, treatment modalities offered and the clinical outcomes in a tertiary hospital. MATERIALS AND METHODS: A retrospective search was carried out on all patients with a diagnosis of achalasia cardia in Hospital Tuanku Ja'afar (HTJ), Seremban, Malaysia between 2014 and 2018. Demographic data, patient symptomatology, and definitive management options were determined from the records. Telephone interviews were conducted to evaluate patient satisfaction with the outcome of treatment. RESULTS: There were 30 patients with a newly diagnosed achalasia cardia in that 5-year period, with an equal incidence among men and women. The mean age of presentation was 44.63 ± 18.21 years. Malays formed the largest group. The mean weight and body mass index were 46.8 ± 10.4 kg and 18.0 ± 4.4 kg/m2 respectively. There was a wide range of duration of symptoms at presentation with a mean of 30.11 ± 35.29 months. Almost all patients presented with dysphagia (96.7%) while 70% also noted loss of weight. All patients underwent oesophagogastroduodenoscopy (OGDS) and 26 patients (86.7%) had barium swallow as part of diagnostic workup. A total of 18 patients underwent a laparoscopic Heller myotomy with or without Dor Fundoplication and/or cruroplasty while two patients (6.7%) underwent pneumatic dilatation as first treatment. Iatrogenic mucosal perforations were detected in 8 patients who underwent myotomy and fundoplication and were repaired intraoperatively. Of the patients who underwent myotomy and fundoplication, the mean weight increase was 15.6kg, increasing from 43.0 ± 8.4 kg to 58.6 ± 13.7 kg. All the patients who underwent treatment were satisfied with their treatment outcomes. CONCLUSION: Most patients with achalasia cardia deemed suitable for surgery and counselled accordingly accept surgery resulting in high levels of satisfaction and weight gain in almost all these patients. A small minority who opt for pneumatic dilatation may also achieve satisfactory outcomes comparable to surgery in the short term. Although rare, clinicians should be able to recognise this disease early as early intervention often leads to satisfactory longterm outcomes.

Electrocatalytic cyclization of dithiothreitol on a chemically modified electrode by analogy with protein action.

Electrocatalytic oxidative cyclization of dithiothreitol (DTT(SH)2) to a disulfide product was demonstrated on a Nafion/lead-ruthenium oxide pyrochlore chemically modified electrode (NPyCME). The process at the NPyCME with DTT(SH)2 is similar to the behaviour of protein in a disulfide linkage, which can be demonstrated by product analysis using HPLC coupled with UV spectroscopy. A possible electrocatalytic mechanism for DTT(SH)2 oxidation to dihydroxydithiane [i.e. cyclized DTT(S-S)] on the NPyCME was proposed in terms of Py-Ru(IV)/Py-Ru(VI) redox active sites. This physical aspect was further utilized for high precision analytical assays using flow injection analysis (FIA), with a linearity up to 50 microM and a detection limit (S/N = 3) of 28 nM (8.64 pg) in a 20 microL sample loop. This is the most sensitive method ever reported for DTT(SH)2 detection assays. The interference from dissolved oxygen, disulfide and glucose is almost negligible. The present method offers an easy route for extension to redox-related protein studies.

Structural events during the refolding of an all beta-sheet protein.

The refolding kinetics of the 140-residue, all beta-sheet, human fibroblast growth factor (hFGF-1) is studied using a variety of biophysical techniques such as stopped-flow fluorescence, stopped-flow circular dichroism, and quenched-flow hydrogen exchange in conjunction with multidimensional NMR spectroscopy. Urea-induced unfolding of hFGF-1 under equilibrium conditions reveals that the protein folds via a two-state (native <--> unfolded) mechanism without the accumulation of stable intermediates. However, measurement of the unfolding and refolding rates in various concentrations of urea shows that the refolding of hFGF-1 proceeds through accumulation of kinetic intermediates. Results of the quenched-flow hydrogen exchange experiments reveal that the hydrogen bonds linking the N- and C-terminal ends are the first to form during the refolding of hFGF-1. The basic beta-trefoil framework is provided by the simultaneous formation of beta-strands I, IV, IX, and X. The other beta-strands comprising the beta-barrel structure of hFGF-1 are formed relatively slowly with time constants ranging from 4 to 13 s.

Release of the neocarzinostatin chromophore from the holoprotein does not require major conformational change of the tertiary and secondary structures induced by trifluoroethanol.

Neocarzinostatin is a potent enediyne antitumor antibiotic complex in which a chromophore is noncovalently bound to a carrier protein. The protein regulates availability of the drug by proper release of the biologically active chromophore. To understand the physiological mechanism of the drug delivery system, we have examined the trifluoroethanol (TFE)-induced conformational changes of the protein with special emphasis on their relation to the release of the chromophore from holoneocarzinostatin. The effect of the alpha helix-inducing agent, TFE, on all the beta-sheet neocarzinostatin proteins was studied by circular dichroism, fluorescence, and (1)H NMR studies. By using binding of anilinonaphthalene sulfonic acid as a probe, we observed that the protein exists in a stable, partially structured intermediate state around 45-50% TFE, which is consistent with the results from tryptophan fluorescence and circular dichroism studies. The native state is stable until 20% TFE and is half-converted into the intermediate state at 30% TFE, which starts to collapse beyond 50%. High pressure liquid chromatographic analysis of the release of the chromophore caused by TFE treatment at 0 degrees C suggests that the release process, which occurs below 20% TFE, does not result from an observable conformational change in the protein. Kinetic measurements of the release of chromophore at 25 degrees C reveal that TFE does stimulate the rate of release, which increases sharply at 15% and reaches a maximum at 20% TFE, although no major secondary or tertiary structural change of the carrier protein is observed under these same conditions. Our data suggest that chromophore release results from a fluctuation of the protein structure that is stimulated by TFE. Complete release of the chromophore occurs at TFE concentrations where no overall observable unfolding of the apoprotein is seen. Thus, the results suggest that denaturation of the protein by TFE is not a necessary step for release of the tightly bound chromophore.

Giant neurofibromatosis of the chest wall: two patient reports.

Two patients with severely deforming giant neurofibromatosis of the chest wall secondary to von Recklinghausen's disease are presented. Pain, respiratory compromise, recurrent ulcerations, cosmetic considerations, and the malignant potential of these lesions indicated wide excision and reconstruction. It is impossible to completely eradicate all neurofibromas, which may affect virtually every nerve within the chest wall including the mediastinum. However, excision of the primary mass may reduce the possibility of malignant degeneration into neurofibrosarcoma or malignant schwannoma. This type of major, full-thickness chest wall resection is now possible using musculocutaneous flaps to achieve satisfactory closure with a single-stage procedure with minimal morbidity and a short hospital stay. At the 10-year follow-up, neither patient exhibited evidence of recurrence.

Bleeding ileal conduit stomal varices: diagnosis and management using transjugular transhepatic angiography and embolization.

An uncommon complication of ileal conduit urinary diversion is bleeding varices at the stoma site. Variceal formation is a complication of portal hypertension, which is most commonly due to intrinsic liver disease. Problematic recurrent bleeding is usually managed locally or by portosystemic shunt. We report a case of recurrent, massive ileal conduit variceal hemorrhage in a patient without a significantly elevated portosystemic gradient. Therefore, this patient was not a candidate for a shunt procedure. Using a transjugular transhepatic approach to the portal vein, the varices were embolized to stasis without any complications. The patient has subsequently experienced no further bleeding episodes.

Chromatographic and spectroscopic assignment of thiol induced cycloaromatizations of enediyne in neocarzinostatin.

Neocarzinostatin is an enediyne antitumor antibiotic. Upon attack by a thiol, the enediyne nucleus is cycloaromatized into two stable 1:1 thiol adducts. After analyzing products from various thiols, the chromatographic and spectroscopic characters that associate only with the cyclized aromatic moiety from enediyne nucleus were assigned. Based on HPLC analysis we have derived, products from picomole ranges of the drug sample can be detected. Confirming the type of cycloaromatization at nanomole ranges can be achieved by photodiode array UV spectroscopy. Three-dimensional fluorogram presents ten times more sensitive identification. The method provides a sensitive tool for massive screening study in microscale.

Stent-graft in the management of superior vena cava syndrome.

We report the use of a Dacron-covered Gianturco-Rösch Z (GRZ)-stent to treat malignant obstruction of the superior vena cava (SVC). Initial treatment with an uncovered GRZ-stent was suboptimal due to protrusion of tumor-thrombus through the stent struts into the SVC lumen. Placement of a coaxial Dacron-covered stent graft relieved the residual obstruction due to tumor within the SVC.

Major injury leads to predominance of the T helper-2 lymphocyte phenotype and diminished interleukin-12 production associated with decreased resistance to infection.

OBJECTIVE: Patients with serious traumatic injury and major burns and an animal model of burn injury were studied to determine the effect of injury on the production of cytokines typical of the T helper-2 lymphocyte phenotype as opposed to the T helper-1 phenotype and on the production of interleukin-12. SUMMARY BACKGROUND DATA: Perturbations of natural and adoptive immunity are related to the increased susceptibility to infection manifested by seriously injured and burn patients. Earlier work has shown that impaired adoptive immunity after injury is characterized by diminished production of interleukin-2 (IL-2), a product of Th lymphocytes. Exposure of naive Th cells to certain antigens and cytokines causes conversion to either the Th-1 or the Th-2 phenotype. Th-1 cells produce IL-2 and interferon-gamma (IFN-tau) and initiate cellular immunity. Th-2 cells secrete interleukin-4 (IL-4) and interleukin-10 (IL-10) and stimulate production of certain antibodies. Conversion to the Th-1 phenotype is facilitated by IL-12, and conversion to the Th-2 phenotype is promoted by IL-4. The authors believed that serious injury might cause conversion of Th cells to the Th-2 as opposed to the Th-1 phenotype rather than generalized Th suppression. METHODS: The authors studied circulating peripheral blood mononuclear cells (PBMC) from 16 major burn and 8 trauma patients on 32 occasions early after injury and from 13 age- and sex-matched healthy individuals for cytokine production after phytohemagglutinin stimulation. Also studied was a mouse model of 20% burn injury known to mimic the immune abnormalities seen in humans with burns. Splenocytes from burn mice, 10 to 12 per group, were studied after activation by concanavalin A or by the bacterial antigen Staphylococcus aureus Cowan strain I for cytokine production and cytokine messenger RNA expression as determined by reverse transcriptase polymerase chain reaction. Burn mice were compared with sham-burn controls and attention was focused on day 10 after burn injury, a time when IL-2 production and resistance to infection are highly suppressed. Finally, burn and sham-burn animals, 20 per group, were treated in vivo with IL-12 (25 ng daily for 5 days) and observed for mortality after septic challenge (cecal ligation and puncture [CLP]) performed on day 10 after injury. RESULTS: Peripheral blood mononuclear cells from burn and trauma patients produced less IFN-tau, the index cytokine of Th-1 cells, than PBMCs from healthy individuals 1 to 14 days after burn injury (SE = 77.6 +/- 16 pg/mL patients vs. 141.3 +/- 35 pg/mL controls, p < 0.05). However, production of IL-4, the index cytokine of Th-2 cells, by patient PBMCs was increased (51.0 +/- 13.0 pg/mL patients vs. 26.9 +/- 2.5 controls, p < 0.05). Splenocytes from mice 10 days after burn injury, when compared with sham-burn controls, showed diminished production of IL-2 (1.04 +/- 0.91 units/mL burns vs. 5.8 +/- 0.55 units/mL controls, p < 0.05) and IFN-tau (1.05 +/- 0.7 units/mL burns vs. 12.0 +/- 8.9 units/mL controls, p < 0.05). However, burn splenocytes produced more IL-4 (2492 +/- 157.0 pg/mL burns vs. 672.0 +/- 22.7 pg/mL controls, p < 0.01) and IL-10 (695.2 +/- 20.8 pg/mL burns vs. 567.0 +/- 16.7 pg/mL controls, p < 0.05). Splenocyte production of IL-12 was also reduced after burn (0.20 +/- 0.035 units/mL) as compared with sham burn (0.46 +/- 0.08 units/mL, p < 0.05). The reduction in IL-2, IFN-tau, and IL-12 production by burn splenocytes was reflected by a tenfold decrease in expression of their respective cytokine mRNAs. In vivo IL-12 treatment of burn animals decreased mortality from CLP on day 10 after injury from 85% to 15% (sham-burn mortality after CLP, 15%, p < 0.05) and increased splenocyte IFN-tau production to supranormal levels. CONCLUSIONS: Serious injury induced diminished production of IL-1 2 and a shift to the Th-2 phenotype with increased production of IL-4 and IL-10, cytokines known to inhibit Th-1 function. The ability of exogenous IL-12 to restore Th-1 cytokine production and resistance to infection suggests a therapeutic role for IL-12 in the immune dysfunction seen after major injury.

The role of cyclic adenosine monophosphate in the suppression of cellular immunity after thermal injury.

BACKGROUND AND OBJECTIVE: Cyclic adenosine monophosphate (cAMP) is an intracellular second messenger that is known to convey inhibitory signals for T-cell proliferation and function. We investigated the association between this molecule and the profound immunosuppression that accompanies thermal injury. DESIGN: Mice were randomized into two groups: one group was subjected to a 20% full-thickness scald burn; the second to a sham burn (control). The mice were killed on days 4, 7, or 10 after the burn injury and splenocytes were pooled and cultured for 15 minutes in the presence or absence of prostaglandin E2 (PGE2). RESULTS: Levels of cAMP in splenocytes were significantly elevated on day 7 after burn in the burn group compared with the sham controls (P < .05, Wilcoxon Rank Sum Test). Incubation of splenocytes with PGE2 resulted in significantly greater levels of intracellular cAMP in cells from the burn group compared with controls on days 4, 7, and 10. Incubation of normal splenocytes with dibutyryl cAMP in the presence of concanavalin A significantly decreased cell proliferation and the production of interleukin-2. The decrease in interleukin-2 production was evident at the level of messenger RNA expression. Stimulation of splenocytes with a combination of phorbol ester and calcium ionophore, bypassing all membrane-associated events prior to protein kinase C activation, reversed the inhibitory effects of dibutyryl cAMP. Incubation of splenocytes from burned animals with H-8, a selective inhibitor of cAMP-dependent protein kinases, restored the proliferative response to that of sham controls on days 4, 7, and 10 after thermal injury. CONCLUSIONS: These data indicate that elevated levels of intracellular cAMP, combined with an increased production of cAMP in response to circulating PGE2, may play a fundamental role in suppression of the immune response following thermal injury and that cAMP exerts its immunomodulatory effects prior to protein kinase C activation.

Spontaneous generation of a biradical species of neocarzinostatin chromophore: role in DNA bulge-specific cleavage.

Detailed structure determination of the major and minor base-catalyzed degradation products of the chromophore of the enediyne anticancer antibiotic neocarzinostatin in the absence of DNA demonstrates that the enolate Michael addition reaction leading to a spirolactone cumulene intermediate is a spontaneous, stereoselective process. The implications of these findings for the mechanism of the thiol-independent, site-specific cleavage by the so-generated radical species of the drug at a DNA bulge are described.

Sources of hydrogen abstraction by activated neocarzinostatin chromophore.

Activation of the enediyne neocarzinostatin chromophore (NCS-Chrom) by thiol addition at C-12 generates a diradical species with radical centers at C-2 and C-6, which abstract hydrogens from deoxyribose in the minor groove of DNA. Since hydrogen abstraction from DNA accounts for only part of the hydrogen incorporated at these sites, it is important to determine the other possible sources. At low concentration of thiol, a condition resembling that during NCS-Chrom-induced DNA damage, the major non-DNA hydrogen donation source was found to be the carbon-bound hydrogen of the aqueous methanol solvent, rather than the expected sulfur-bound hydrogen of the thiol. Further, experiments with the gamma-L-glutamyl-DL-cysteinylglycine labeled with deuterium on the alpha- or beta-carbons to the sulfur showed small amounts of internal transfer of hydrogen into C-2 of the drug from the naturally occurring L,L diastereomer only. Quantitation of the hydrogen transfer was accomplished by separation of the L,DL diastereomeric mixtures of the thiol-NCS-Chrom adducts. In all, these various hydrogen donation sources can account for at least 70-80% of the hydrogen incorporated at C-2 of the drug under DNA damage conditions. Selective quenching of the radical at C-2 could account for the predominance of single-stranded over double-stranded DNA lesions.

Murine memory B cells are multi-isotype expressors.

Flow cytometric analyses of the surface immunoglobulins of murine memory B cells revealed the existence of populations expressing multiple isotypes, including an IgM+/IgG+ population that could be stimulated in vitro with antigen to secrete both IgM and IgG. Female BALB/c mice were immunized with R-phycoerythrin (RPE), a fluorescent photosynthetic accessory protein from red algae. Pooled splenocytes from these mice at different stages of immunization were stained with RPE as well as with allophycocyanin- and fluorescein-conjugated anti-isotype antibodies and analysed on a two-laser FACS. RPE-binding cell sub-populations were defined and selectively sorted to verify their phenotype and to demonstrate that the various subpopulations (IgM+/IgG+, IgM+/IgG-, IgM-/IgG+) had different isotype-secretion patterns when challenged with RPE in vitro. These results re-affirm the notion that a transcriptional processing mechanism may be responsible for the simultaneous expression of multiple isotypes in memory cells.

Collagen shield heparin delivery for prevention of postoperative fibrin.

We studied collagen shield heparin delivery to the rabbit eye utilizing radiolabeled heparin as well as a fibrin inhibition assay. Radiolabeled heparin studies revealed significant tritium delivery to the cornea, aqueous, and iris, with only trace levels detectable for the lens, vitreous, and sclera. An aqueous fibrin inhibition assay revealed that a single collagen shield soaked in heparin achieved anterior chamber anticoagulant levels that paralleled the time course of the radiolabeled heparin delivery and resulted in fibrin inhibition during the 6-hour study period. Subconjunctival heparin injection did not alter baseline aqueous anticoagulant activity. No complications related to collagen shield heparin delivery were encountered. These studies suggest that a heparin-hydrated collagen shield may prevent postoperative fibrin formation in eyes at risk for this complication, including eyes undergoing surgery for the complications of proliferative diabetic retinopathy proliferative vitreoretinopathy, and glaucoma filtration surgery.

Sites in the diyne-ene bicyclic core of neocarzinostatin chromophore responsible for hydrogen abstraction from DNA.

The antitumor antibiotic neocarzinostatin exhibits its main drug action by abstracting hydrogen from DNA deoxyribose with consequent strand breakage or related lesions. All biological activities of the drug derive solely from a nonprotein chromophoric substance (NCS-chrom) consisting of a novel epoxy-bicyclo-diyne-ene system. Thiol or sodium borohydride activates NCS-chrom into a labile, reactive species that induces DNA damage but causes inactivation of the drug in the absence of the target DNA. Mass spectrometric studies indicate that the isolated thiol-activated NCS-chrom product in the presence of DNA has the same molecular weight as the thiol-inactivated NCS-chrom product in the absence of DNA. No deuterium is incorporated into the chromophore from the deuterium-labeled sulfhydryl group. Since three deuterium atoms can be incorporated into the drug by treatment with sodium borodeuteride without DNA, adding an unlabeled DNA under parallel conditions permitted the ready identification of the activated NCS-chrom product that abstracted hydrogen from the DNA. Not only does the activated NCS-chrom product have the same structure as the inactivated drug without DNA, but two of the incorporated deuterium atoms have been substituted by hydrogen. With the aid of NMR spectrometry, the two replaced hydrogen atoms are found to be incorporated into the C-2 and C-6 positions of the bicyclo-diyne-ene ring of NCS-chrom and are derived neither from borodeuteride nor from the hydroxyl functions of the solvents. In accord with current proposals, the two hydrogens incorporated into the drug may come from closely opposed sites on the complementary strands of the DNA at which the drug is bound.(ABSTRACT TRUNCATED AT 250 WORDS)

3'-Formyl phosphate-ended DNA: high-energy intermediate in antibiotic-induced DNA sugar damage.

Under anaerobic conditions where the nitroaromatic radiation-sensitizer misonidazole substitutes for dioxygen, DNA strand breakage (gaps with phosphate residues at each end) by the nonprotein chromophore of the antitumor antibiotic neocarzinostatin (NCS-Chrom) is associated with the generation of a reactive form of formate from the C-5' of deoxyribose of thymidylate residues. Such lesions account for a minority (10-15%) of the strand breakage found in the aerobic reaction without misonidazole. Amino-containing nucleophiles such as tris(hydroxymethyl)aminomethane (Tris) and hydroxylamine act as acceptors for the activated formate. The amount of [3H]formyl hydroxamate produced from DNA labeled with [5'-3H]thymidine is comparable to the spontaneously released thymine. During the course of the reaction, misonidazole undergoes a DNA-dependent reduction and subsequent conjugation with glutathione used to activate NCS-Chrom. From these and earlier results, we propose a possible mechanism in which the carbon-centered radical formed at C-5' by hydrogen atom abstraction by thiol-activated NCS-Chrom reacts anaerobically with misonidazole to form a nitroxyl-radical-adduct intermediate, which fragments to produce an oxy radical at C-5'. beta-Fragmentation results in cleavage between C-5' and C-4' with the generation of 3'-formyl phosphate-ended DNA, a high-energy form of formate, which spontaneously hydrolyzes, releasing formate and creating a 3'-phosphate end, or transfers the formyl moiety to available nucleophiles. A similar mechanism, involving dioxygen addition, is probably responsible for the 10-15% DNA gap formation in the aerobic reaction.

Generation of superoxide free radical by neocarzinostatin and its possible role in DNA damage.

Spectroscopic analysis of the reduction of both nitro blue tetrazolium and ferricytochrome c induced by neocarzinostatin shows that superoxide free radical is produced during the spontaneous degradation of the antibiotic. The amount of superoxide free radical produced from neocarzinostatin is not affected by the presence of thiol, although earlier work has shown that DNA damage is stimulated at least 1000-fold by thiol. Transition metals are not involved in this reaction. Although superoxide dismutase inhibits the reduction of nitro blue tetrazolium and cytochrome c induced by neocarzinostatin, neither it nor catalase interferes with the action of neocarzinostatin on DNA, whether or not drug has been activated by thiol. The pH profiles for spontaneous base release and alkali-labile base release (a measure of nucleoside 5'-aldehyde formation at a strand break) do not correspond with that for the generation of superoxide free radical from neocarzinostatin. The same holds for supercoiled DNA cutting by neocarzinostatin chromophore in the absence of a thiol, which is an acid-favored reaction. These results indicate that the generation of superoxide free radical by the drug does not correlate with DNA damage activity, whether or not thiol is present. Furthermore, the failure of hydroxyl free-radical scavengers to inhibit drug-induced single-strand breaks in supercoiled DNA in the absence of thiol also indicates that a diffusible hydroxyl free radical is most probably not involved in this reaction.

Molecular mechanism of novel DNA sugar damage by an antitumour protein antibiotic.

Neocarzinostatin (NCS) belongs to a family of antitumour protein antibiotics that selectively inhibit DNA synthesis. Replicon initiation in mammalian cells is selectively inhibited by NCS, and cells defective in DNA repair, such as ataxia telangiectasia fibroblasts, are especially sensitive to NCS as they are to X-ray. The holoantibiotic consists of a nonprotein chromophore (Mr = 659), tightly and specifically bound to an apoprotein (Mr = 10,700). The apoprotein protects the highly labile chromophore from degradation in aqueous solution; all the activity resides in the nonprotein chromophore. The latter binds specifically to DNA, especially to regions rich in T and A residues, with a tight binding site consisting of four base pairs. NCS chromophore consists of three main structural subunits: a naphthoic acid derivative, an amino-sugar and a connecting highly unsaturated middle component (C12H5) with a strained ether (probably epoxide) and cyclic carbonate. The authors have proposed that the naphthoic acid subunit intercalates DNA and the positively charged amino sugar binds electrostatically to the negatively charged sugar phosphate backbone of DNA; these two anchors serve to juxtapose the middle piece with the deoxyribose of mainly thymidylate residues in DNA. Upon activation of the drug by a thiol (which forms an adduct with the middle piece) and in the presence of O2, there is a selective oxidation of the 5'-C of deoxyribose to produce a DNA strand break with a phosphate at the 3'-end and a nucleoside 5'-aldehyde at the other. Kinetic analysis shows that one molecule of thiol adds to DNA-bound NCS chromophore even in the absence of oxygen; this is rapidly followed by the consumption of 1 mol of O2 and then another mol of thiol. The oxygen of the 5'-aldehyde is derived from O2, not H2O. Even in the absence of O2 the NCS chromophore abstracts a hydrogen from C-5' of deoxyribose in DNA, presumably generating a carbon-centred radical intermediate in the DNA (other mechanisms have not been eliminated) which can add O2 to form a peroxy derivative. The second molecule of thiol may be involved in the cleavage of this complex to form the 5'-aldehyde at the strand break. There is no evidence for the involvement of metals or a diffusible form of reduced oxygen.(ABSTRACT TRUNCATED AT 400 WORDS)

Incorporation of 18O2 into thymidine 5'-aldehyde in neocarzinostatin chromophore-damaged DNA.

Strand scission of DNA by the chromophore of neocarzinostatin converts the 5'-hydroxyl of deoxyribose to a 5'-aldehyde. The origin of the aldehydic oxygen has now been elucidated by mass spectrometry. DNA-associated thymidine 5'-aldehyde produced by treatment of DNA with neocarzinostatin chromophore in 2H218O/16O2 or in 2H216O/18O2 was reduced, liberated by nuclease treatment, permethylated, and analyzed by gas chromatography-mass spectrometry. The data clearly show that molecular oxygen is the only source of the 5'-aldehydic oxygen. The addition of molecular oxygen at C-5', possibly via a reactive form of neocarzinostatin chromophore, must be involved; a carbonium ion intermediate at C-5' is ruled out.