A study of electrophoretic mobility of DNA in agarose and polyacrylamide gels.

The aim of this paper is to clarify the mechanism of gel electrophoresis of DNA under constant-field conditions. We have conducted a large number of experiments on double-stranded DNA varying in length between approximately 10 and approximately 50,000 base-pairs, in both agarose and polyacrylamide gels ranging from 0.5% to 12% concentration, and with electric field strengths ranging from 0.5 to 8 V/cm. We have made (logarithmic) plots of velocity against length of DNA for all of the various test conditions. At the left-hand side of these plots, all of the empirical curves have a unique, standard shape. When the curves are normalized so that their left-hand parts coincide, a second feature emerges in that, while for any given test the curve follows the "master curve" up to a certain point, it then "breaks away" and becomes horizontal. We describe these two patterns of behaviour as "regions 1 and 2", respectively. We find simple yet comprehensive empirical formulae that fit the observations in the two regions of behaviour: these express the velocity in terms of length of DNA, electric field strength and gel concentration. We then construct two separate theories for the two regions of behaviour. The first theory involves the statistics of motion of an object through a random array of gel obstacles, with the instantaneous speed depending on the number of obstacles with which the object is currently in contact. The second theory is based on the mechanical hypothesis (for which there is other, independent support) that the DNA moves through the gel by piling up against a barrier, which eventually breaks or deforms under the resulting force, thereby allowing the DNA to move on to the next barrier. The statistical theory is an adaptation of existing work, while the mechanical one is new. We also describe experiments on the migration of repeated-sequence, curved DNA with length up to 1500 base-pairs, and we discuss its behaviour in terms of our two theories. Our studies by electron microscopy are consistent with the view that this repeated-sequence DNA adopts a superhelical configuration. Finally, we show that a very wide range of observations may be understood clearly by means of our two theoretical schemes.

Improved functional recovery by ischaemic preconditioning is not mediated by adenosine in the globally ischaemic isolated rat heart.

OBJECTIVE: A brief period of ischaemia (5 min) and reperfusion (5 min), prior to a longer period of ischaemia and reperfusion, has been shown to reduce the extent of injury (necrosis, arrhythmias, or postischaemic contractile malfunction) caused by a subsequent longer period of ischaemia and reperfusion. Adenosine has been identified as a factor in the protection afforded against regional tissue necrosis by such preconditioning. The aim of this study was to assess the role of adenosine in preconditioning induced protection of postischaemic function in the globally ischaemic isolated rat heart. METHODS: The ability of global ischaemia to precondition against postischaemic contractile malfunction was first confirmed in the isolated ejecting rat heart preparation. Hearts (n = 6 per group) were perfused aerobically (37 degrees C, paced at 350 beats.min-1) for 20 min, at the end of which contractile function was measured. This was followed by 10 min of Langendorff perfusion (control group) or 5 min of global ischaemia plus 5 min of Langendorff reperfusion (preconditioned group). The hearts were then subjected to 20 min of global ischaemia (37 degrees C) and 35 min of reperfusion (15 min Langendorff and 20 min ejecting); function was then reassessed. RESULTS: Postischaemic recovery of aortic flow was 26(SEM 8)% in the control group v 57(4)% in the preconditioned group (p < 0.05). To assess whether exogenous adenosine could mimic this protection, the experiments were repeated with the 5 min period of ischaemic preconditioning replaced by 5 min of aerobic Langendorff perfusion with adenosine-containing buffer (100, 50, or 10 mumol.litre-1). No protection of postischaemic function was observed in any of the adenosine treated groups. In further experiments, we assessed whether ischaemic preconditioning persisted in the presence of the A1/A2 adenosine antagonist, 8 (p-sulphophenyl) theophylline (8-SPT). Since pacing was not used in these studies, the ability of ischaemia to precondition the myocardium was again confirmed; the protocol was then repeated with 8-SPT (10 mumol.litre-1) present in the perfusate throughout. Although 8-SPT depressed recovery in both control and preconditioned hearts it failed to abolish the protective effects of ischaemic preconditioning. CONCLUSIONS: There is no evidence from these results to support the involvement of adenosine to any major extent in preconditioning induced protection of postischaemic contractile function in the isolated rat heart.

Effects of N-methyl hexanoylhydroxamic acid (NMHH) and myoglobin on endothelial damage by hydrogen peroxide.

OBJECTIVE: The aim was to investigate the interaction of the novel antioxidant N-methyl hexanoylhydroxamic acid (NMHH) with myoglobin in protecting endothelial cells against H2O2 mediated damage. METHODS: Cultured bovine aortic endothelial cells were exposed to 50-100 microM H2O2 for 10-60 min with and without NMHH and/or myoglobin, and immediate or delayed damage was assessed by lactate dehydrogenase release, 3H adenine uptake, a tetrazolium reduction assay, and microscopy. RESULTS: Brief exposure to low concentrations of H2O2 caused cell damage, for which the tetrazolium reduction assay was the most sensitive assay, and inhibited subsequent cell division. NMHH in concentrations from 50 to 200 microM protected against damage provided it was present at the time of adding H2O2, and the effect was markedly potentiated by 10 microM oxymyoglobin, which had little protective effect alone. CONCLUSIONS: NMHH is an effective antioxidant which is markedly potentiated by low concentrations of oxymyoglobin. Oxymyoglobin may potentiate NMHH by scavenging H2O2 through the rapid formation of ferrylmyoglobin, which is then reduced by NMHH. This synergism may be particularly relevant to the protection of myoglobin-rich cells such as myocytes.

Novel monohydroxamate drugs attenuate myocardial reperfusion-induced arrhythmias.

The novel monohydroxamates N-methyl hexanoylhydroxamic acid, N-methyl acetohydroxamic acid, and N-methyl butyrohydroxamic acid have antioxidant and iron chelating properties. They attenuated reperfusion-induced contractile dysfunction following long periods of ischaemia (50 min) in the isolated rat heart. Here we compare their effects and that of the trihydroxamate desferrioxamine on reperfusion-induced arrhythmias following short duration ischaemia (10 min). Isolated rat hearts were perfused by the Langendorff method, subjected to regional ischaemia and reperfusion. Arrhythmias induced during the first 5 min of reperfusion were quantified. Drugs (all at 150 microM) were introduced during the last 2 min of ischaemia and remained throughout reperfusion. Although the monohydroxamate- and desferrioxamine-treated hearts showed a reduction in the incidence of ventricular tachycardia and fibrillation, only the reduction in the incidence of sustained fibrillation ( > 3 min duration) in N-methyl acetohydroxamic acid--(27%), N-methyl hexanoylhydroxamic acid--(27%) and desferrioxamine-treated hearts (20%) was statistically significant (p < 0.05 vs control 73%; n = 15). There was a reduction in the severity of the arrhythmias, manifest as a significant increase in the duration of sinus rhythm in all the monohydroxamate-treated hearts, and a significant reduction (vs control 218 +/- 29 s; mean +/- SEM) in the duration of ventricular fibrillation in hearts treated with N-methyl acetohydroxamic acid (101 +/- 31 s) and desferrioxamine (112 +/- 30 s). This improvement was offset by an increase in the duration of ventricular tachycardia, in hearts treated with N-methyl acetohydroxamic acid, N-methyl butyrohydroxamic acid and desferrioxamine. These results suggest that these novel monohydroxamates, particularly N-methyl acetohydroxamic acid, attenuate reperfusion-induced arrhythmias in this model when introduced during the ischaemic period.

Non-oxidative modification of low density lipoprotein by ruptured myocytes.

In this study, the interaction of ruptured cardiac myocytes with low density lipoprotein (LDL) has been investigated and the consequent extent of uptake by macrophages. The results show that lysate released from ruptured myocytes is capable of inducing LDL oxidation and that the resulting modified form is recognised and degraded by macrophages. Peroxyl radical scavengers inhibit the LDL oxidation but not the macrophage uptake suggesting that LDL can be modified by mechanisms that are independent of oxidative processes by intracellular constituents of cardiac myocytes.

Lung damage in mice from cyclophosphamide and thoracic irradiation: the effect of timing.

The development of lung damage in mice following thoracic irradiation is enhanced by cyclophosphamide (CY). CY was given at various times from 28 days before to 28 days after irradiation. Although increased damage was seen at all times, the extent showed marked variation according to the time interval. The most marked variations were seen when CY was given within 24 hours of irradiation; maximal enhancement was seen when CY was given before or with irradiation and minimal response when given 12 hrs before or 12 hrs after irradiation. It may therefore be prudent for the clinician to avoid such close time intervals between cytotoxic drugs and irradiation, unless a specific time-related therapeutic gain can be exploited.

The effects of anesthetics and misonidazole on the development of radiation-induced lung damage in mice.

The measurement of breathing frequency as a functional end-point of radiation-induced lung injury in mice allowed two phases of damage to be discerned; the first was manifest at 12-20 weeks after irradiation, the second beyond 28 weeks. Anesthesia by pentobarbitone sodium or steroids gave significant radioprotection of the lung during the early pneumonitic phase. Addition of the hypoxic cell sensitizer misonidazole removed the protective influence of the anesthetics but did not sensitize the lungs of unanesthetized mice. No anesthetic protection was detected for the late response, showing evidence for dissociation between early and late lung damage. The degree of epilation was measured on the dorsal thoracic region of the same mice. Protection by anesthetics and its reversal by misonidazole was also demonstrated. These results provide a warning of potential hazards in the laboratory evaluation of chemical radiosensitizers. The use of anesthetics at the time of irradiation could lead to an exaggerated enhancement of normal tissue damage.

Granisetron in the prevention of irradiation-induced emesis.

A double-blind double-dummy, comparative study was carried out in 30 patients receiving highly emetogenic fast dose rate, single fraction total body irradiation prior to bone marrow transplantation. Patients were randomised into one of two groups, receiving either granisetron, a specific 5-HT3 antagonist or a combination of metoclopramide, dexamethasone phosphate and lorazepam to assess the comparative efficacy of the two regimens in the control of irradiation-induced nausea and vomiting. After 24 h eight patient (53%) treated with granisetron showed a complete response compared with two patients (13%) in the comparator group. The control of vomiting by granisetron, over both 24 h and 7-day periods (P = 0.001 and P = 0.004), was significantly better than that seen with the comparator and required significantly fewer rescue doses. The safety profiles in the two groups appeared similar, with the exception that granisetron produced less drowsiness than the comparator.

Incidence of cataracts after single fraction total body irradiation: the role of steroids and graft versus host disease.

Eighty-eight patients who received single fraction total body irradiation (sfTBI) as part of their conditioning for allogeneic BMT have been evaluated for the risk of cataract formation. Thirty-eight (43%) have developed cataracts; 11 required surgery. With 9.5-13.6 years follow-up (median 10.7 years), all 12 recipients of unmanipulated marrow allografts have developed cataracts; the actuarial risk of needing surgery was 32 (+/- 18%, 95% confidence intervals (CI)). Ten of these 12 required high-dose steroids (prednisolone > 1 mg/kg/day) for the treatment of GVHD. Seventy-six patients received T cell-depleted allografts; 14 of 76 required post-transplant immunosuppression with high-dose steroids. With 1-9.4 years follow-up (median 5 years), the actuarial risk of cataract formation in T cell-depleted allograft recipients is 72% (+/- 52% CI), the actuarial risk for needing surgery is 20% (+/- 9% CI). Recipients of sfTBI and non-T cell-depleted allografts had a significantly greater risk of developing cataracts (p = 0.003, long rank test) and of needing surgery (p < 0.05, log rank test) than patients receiving T cell-depleted BM. Cataracts occurred more frequently in patients requiring post-transplant immunosuppression with steroids (relative risk 2.12, p < 0.01 log rank test).

Immunotherapy with interleukin 2 after ABMT in AML.

Myeloablative chemo (+/- radio) therapy and rescue with ABMT has been used as final consolidation therapy in 18 patients with AML in first remission. In seven (6 autologous, 1 syngeneic) marrow reinfusion was followed by iv IL-2. One patient, who commenced IL-2 4 days after BMT, died from pulmonary oedema due to the capillary leak syndrome. Thereafter, treatment with IL-2 was delayed until the platelet count reached 30 x 10(9)/l. All patients developed reversible hypotension (treated with infusion of colloid), but treatment was otherwise well tolerated. With 21-58 months (median 32 months) from the time of ABMT there has been one relapse (actuarial risk 17%, 95% confidence intervals (CI) 3-31%). The disease-free survival is 71% (95% CI 38-100%). Eleven patients with comparable remission induction and consolidation therapy, and an identical interval between diagnosis and ABMT (5-11 months, median 6 months) received an autograft without immunotherapy. With 24-45 months (median 29 months) follow-up the actuarial disease-free survival is 36% (95% CI 8-64%), the actuarial relapse risk is 54% (95% CI 18-90%). We conclude that immunotherapy given after ABMT to patients with AML in first remission when the platelet count exceeds 30 x 10(9)/l is safe and may induce an immunological environment which results in the elimination of residual leukaemia.

Granisetron, a selective 5-HT3 receptor antagonist, for the prevention of radiation induced emesis during total body irradiation.

The antiemetic efficacy of granisetron was tested in an open trial in patients undergoing highly emetogenic treatment by single fraction total body irradiation. Thirty-two consecutive patients were entered. Results were both patient- and observer-rated. Following a single intravenous dose of granisetron 18 patients (56.3%) experienced total protection and a further 13 (40.6%) had major antiemetic protection with four of these patients experiencing nausea only. One patient experienced an anaphylactic reaction on infusion of monoclonal antibody-treated donor marrow 5 h after administration of the trial drug and vomited on multiple occasions. The reaction was associated with hypotension. A further patient experienced transient hypotension secondary to septicaemia 8 h after receiving granisetron. Three patients required a second dose. Headache was the most frequent side-effect occurring in three patients, but in to of these patients the test drug was not thought to be implicated. In conclusion granisetron is a highly effective agent in controlling radiation induced emesis with a favourable toxicity profile.

Mobile gene cassettes and integrons in evolution.

Integrons and the site-specific recombination systems encoded by them provide a simple mechanism for the addition of new genes to bacterial chromosomes. Although there is substantial divergence among the four known integron-encoded integrases, they all recognize the recombination sites, known as 59-base elements, that are associated with genes that are packaged in gene cassettes. In contrast, the integron-associated recombination sites, attl sites, are preferentially recognized by the cognate integrase.

Effects of pulsed electromagnetic fields on Na+ fluxes across stripped rabbit colon epithelium.

The effect of pulsed electromagnetic fields on the electrical potential and two-way flux of Na+ across the epithelium of the rabbit colon in vitro was investigated. In control experiments the transepithelial mucosal-to-serosal and serosal-to-mucosal fluxes (Jm----s and Js----m) were constant over the experimental period. When the epithelium was at right angles to the applied electromagnetic field, the Jm----s flux of Na+ was reduced, whereas Js----m was enhanced. When the epithelium was rotated 180 degrees, Jm----s was enhanced, whereas Js----m was reduced. When the epithelium was mounted parallel to the magnetic field, both Jm----s and Js----m of Na+ were increased, the latter continuing to increase after the field was turned off. When the tissue was rotated 180 degrees, the same enhanced flux was observed, but now the Jm----s flux showed the greatest increase, which again occurred in the period after the field was turned off. The rate of decrease of transepithelial potential difference in all orientations was less than the control. Also, the conductance increased in orientations 2-4 and decreased in orientation 1 after the field had been applied. This suggests that pulsed electromagnetic fields can have a direct effect on the movement of Na+ across tissue and transepithelial potentials. The mechanism may depend on several factors, such as induced changes in certain ion pumps, the membrane potential, and the surface charge of cell wall proteins.

Lung damage from cytotoxic drugs.

Bleomycin, busulphan, and methotrexate are by far the commonest cytotoxic drugs to cause interstitial pneumonitis. However, many other cytotoxic drugs have been reported to produce similar lung damage. Combined effects of these drugs, and of the drugs with other agents that cause lung damage, such as oxygen and radiation, may result in enhancement of lung damage. Early diagnosis, made possible by awareness of this complication and its correct investigation, may reduce severe morbidity and mortality. In some instances, factors that predispose to lung damage are known, and these have been studied in experimental animals.

The effects of vasoactive intestinal polypeptide on the electrical activity of guinea-pig intestinal smooth muscle.

The effect of vasoactive intestinal polypeptide (VIP) on the electrical activity of the smooth muscle of the guinea-pig taenia coli has been examined using the single sucrose gap apparatus. VIP usually caused a slowly developing, long-lasting membrane hyperpolarisation, although sometimes it reduced the frequency of spontaneous spike discharge without hyperpolarising the membrane. In contrast, non-adrenergic, non-cholinergic nerve stimulation initiated rapid membrane hyperpolarisation followed by post inhibitory excitation. In the presence of apamin, the VIP response was little affected, whereas the responses to non-adrenergic, non-cholinergic nerve stimulation were either markedly antagonised or reversed to membrane depolarisation. Consideration is given to the possible role of either VIP or ATP as the neurotransmitter responsible for the inhibitory junction potential elicited in the guinea-pig taenia coli in response to non-adrenergic inhibitory nerve stimulation.

Effects of co-supplementation of iron with ascorbic acid on antioxidant--pro-oxidant balance in the guinea pig.

The relationship between intake of iron with ascorbic acid and their uptake into the plasma and liver of guinea pigs was studied. The influence on the antioxidant/pro-oxidant balance of liver microsomes was also determined. Animals were fed a standard pelleted diet low in iron and ascorbic acid for 35 days. The pellet diet was supplemented by oral dosing with a solution containing either maintenance dietary levels of ascorbic acid and iron, or one of three regimens that increased the dosage of these substances ten fold. There were no significant differences in animal growth rate or food intake between these regimens. Liver and plasma total ascorbate levels were significantly increased (p < 0.05) in animals receiving either ascorbic acid alone (liver 126 +/- 36 micrograms/g tissue wet wt. and plasma 51.7 +/- 17.0 microM; n = 9) or ascorbic acid and iron (105 +/- 18 micrograms/g and 40.3 +/- 15.3.0 microM; n = 8) compared to controls (84 +/- 36 micrograms/g and 15.3 +/- 8.5 microM; n = 11). Total iron levels in the liver (76.7 +/- 7.3 micrograms/g; control; n = 6) and plasma (2.4 +/- 0.03 mg/l; control) were not significantly raised in animals under these conditions of iron or ascorbate intake. Liver microsomes isolated from animals receiving iron had a greater susceptibility to oxidative stress in terms of malondialdehyde production during auto-oxidation compared to those from control animals under the same conditions. This effect was eliminated on combining ascorbic acid with the iron supplementation, suggesting that oral administration of vitamin C has a protective rather than a pro-oxidant effect under these circumstances.

The effects of ascorbic acid and iron co-supplementation on the proliferation of 3T3 fibroblasts.

Exposure of 3T3 fibroblasts to FeII reveals a concentration-dependent inhibition of cell proliferation compared to control cells, the apparent threshold for this iron-mediated effect being 5 microM FeII. The inhibition of cell proliferation was accompanied by an enhancement of total malondialdehyde (MDA) levels (as detected directly by hplc) in the cells at higher iron concentrations. The co-supplementation of FeII with varying concentrations of ascorbic acid over the range 5 microM to 240 microM had no significant effect on the threshold for iron toxicity or lipid peroxidation. These results show that there is neither a significant exacerbation of the pro-oxidant effect of FeII nor any protective effect of ascorbate when cultures of 3T3 mouse fibroblasts are exposed to co-supplementation regimes of iron with ascorbic acid.

Do iron and vitamin C co-supplementation influence platelet function or LDL oxidizability in healthy volunteers?

OBJECTIVE: To examine the effect of co-supplementation with iron and vitamin C on antioxidant status, platelet function and low density lipoprotein oxidation in normal healthy volunteers. DESIGN: The study was carried out with two groups of 20 subjects each acting as their own control, comparing presupplemention with postsupplemention. One group was supplemented with iron and the RDA level of vitamin C and the second group with iron and 260 mg/d vitamin C. SETTING: The International Antioxidant Research Centre, The Guy's, King's College and St Thomas's School of Biomedical Science, Guy's Campus, London. SUBJECTS: Forty normal healthy volunteers, recruited from the staff of the Medical School and Hospital in which two volunteers withdrew during the study. INTERVENTIONS: Subjects in both studies were randomly assigned to one of two groups (5 males and 5 females group) and received supplements containing iron (14 mg/d) and either 60 mg/d (Group A) or 260 mg/d (Group B) vitamin C for 12 wk. Blood samples were taken at 6 wk and 12 wk, and prior to supplementation and analysed for iron and antioxidant status (transferrin bound iron, vitamin C and E, and beta-carotene levels) in both studies. Samples from the first study were analysed for the susceptibility of LDL isolated from plasma to Cu2+-induced oxidation and samples from the second for platelet function. RESULTS: Transferrin-bound iron was significantly increased (P < 0.05) at 12 wk, in Group A subjects (from 14.9+/-5.3 micromol/1 to 19.5+/-2.3 micromol/l; mean+/-s.d.; n=19), whereas those in Group B showed a significant increase (P < 0.05) after 6 wk (from 15.8+/-4.5 micromol/l to 20.4+/-6.6 micromol/l; n = 19) which decreased at 12 wk (16.3+/-5.0 micromol/l). Plasma total ascorbate significantly increased from an initial level of 59.3+/-21.3 micromol/l to 87.6+/-29.0 micromol/l after 6 wk and 81.7+/-11.4 micromol/l after 12 wk following the Group B supplementation, but only after 12 wk in Group A (from 64.0+/-24.8 micromol/l to 77.2+/-13.2 micromol/l). Plasma alpha-tocopherol concentrations were significantly increased after 6 wk and 12 wk with both levels of supplementation (from 24.2+/-5.71 micromol/l Group A and 23.4+/-5.3 micromol/l Group B to 26.3+/-5.5 micromol/l and 25.71+/-4.7 micromol/1 respectively at 12wk). The mean lag phase to oxidation of low density lipoprotein (LDL) was significantly increased in subjects in Group B after 12 wk ingestion of iron and 260 mg vitamin C (from 80.0+/-14.8 min to 97.2+/-16.9 min; n = 9). Platelet sensitivity to ADP-induced aggregation was significantly decreased (P < 0.05) by 12 wk in Group A (from EC50 2.3 < or = 1.3 microM to 3.7+/-2.2 microM; n = 10), whereas those receiving higher vitamin C showed a significant decrease (P < 0.05; from EC50 1.9+/-0.6 microM to 3.1+/-1.8 microM) after 6wk which subsequently increased towards presupplemental levels (2.6+/-1.6 microM). Platelets from the latter subjects showed a significant reduction in ADP-induced ATP secretion at both 6wk and 12 wk. CONCLUSION: The results show modest beneficial effects on LDL oxidation and platelet function following supplementation with iron and vitamin C. No evidence for pro-oxidant effects was observed.

Serum total antioxidant activity after myocardial infarction.

Serum total antioxidant activity (TAA), albumin and uric acid were measured on admission, and for the next 2 days in 56 patients suffering myocardial infarction, 20 of whom received streptokinase. The 'antioxidant gap', the difference between the serum TAA and the sum of the serum albumin and uric acid activity, was calculated. No significant changes in serum total antioxidant activity were observed in either group of patients between admission, day 1 and day 2. However, a decline in the 'antioxidant gap' after myocardial infarction was associated with a significantly higher mortality.

An alteration leading to loss of ability to support phleomycin mutagenesis in the pKM101-derived plasmid pGW16 is located in or close to the mucAB genes.

The differences between the plasmid pKM101 and its derivative pGW16, which has lost the ability to support muc-dependent phleomycin mutagenesis, while retaining other muc-dependent phenotypes, have been further investigated. Deletion derivatives which retain only 10.8 kb (approximately one third) of the pKM101 genome but retain the muc genes have been constructed from both pKM101 and pGW16. The deletion derivatives confer protection and mutagenesis-enhancing properties similar to those of their respective parents, indicating that the lesion in the mutant plasmid pGW16 lies in or close to the muc genes. Differences in the muc-dependent phenotypes of strains containing pKM101 or pGW16 suggest that the pGW16 lesion results in either differential loss of function in the muc gene products, or constitutive expression of the muc gene products.