Clinical Characteristics and Predictors of Mortality in Elderly Patients Hospitalized with COVID-19 in Bangladesh: A Multicenter, Retrospective Study.

Purpose: Elderly patients are at high risk of fatality from COVID-19. The present work aims to describe the clinical characteristics of elderly inpatients with COVID-19 and identify the predictors of in-hospital mortality at admission. Materials and Methods: In this retrospective, multicenter cohort study, we included elderly COVID-19 inpatients (n = 245) from four hospitals in Sylhet, Bangladesh, who had been discharged between October 2020 and February 2021. Demographic, clinical, and laboratory data were extracted from hospital records and compared between survivors and nonsurvivors. We used univariable and multivariable logistic regression analysis to explore the risk factors associated with in-hospital death. Principal Results. Of the included patients, 202 (82.44%) were discharged and 43 (17.55%) died in hospital. Except hypertension, other comorbidities like diabetes, chronic kidney disease, ischemic heart disease, and chronic obstructive pulmonary disease were more prevalent in nonsurvivors. Nonsurvivors had a higher prevalence of leukocytosis (51.2 versus 30.7; p=0.01), lymphopenia (72.1 versus 55; p=0.05), and thrombocytopenia (20.9 versus 9.9; p=0.07). Multivariable regression analysis showed an increasing odds ratio of in-hospital death associated with older age (odds ratio 1.05, 95% CI 1.01-1.10, per year increase; p=0.009), thrombocytopenia (OR = 3.56; 95% CI 1.22-10.33, p=0.019), and admission SpO2 (OR 0.91, 95% CI 0.88-0.95; p=0.001). Conclusions: Higher age, thrombocytopenia, and lower initial level of SpO2 at admission are predictors of in-hospital mortality in elderly patients with COVID-19.

Case-Control Study to Evaluate Zinc Deficiency as a Risk Factor for Oxygen Requirement in Patients with COVID-19.

Zinc is one of the important trace elements of blood. It helps in maturation of immune system in our body. In the past studies originate the relationship between viral disease and serum zinc deficiency. This was a multicenter case-control study to measure the serum zinc level of COVID-19 patients with different respiratory supports and to evaluate the Odds Ratio (OR) and Relative risk (RR) of zinc deficiency for oxygen requirement for COVID-19 patients. Study places were COVID-19 unit of three tertiary hospitals of Sylhet, a Northern district of Bangladesh. There were 30 controls and 90 cases in this study. The mean zinc level of cases (53-38mcg/dl) was significantly (p=0.000072) lower than the level of controls (73-23mcg/dl). The mean zinc level of the COVID-19 patients required oxygen (49-33mcg/dl) was significantly (p=0.0054) lower than the patients were not treated by oxygen therapy (64-51mcg/dl). The RR of getting affected by COVID-19 was 1-91 for the low zinc level people. Among the COVID-19 affected participants the lower zinc level people had a RR of 1-93 to receive oxygen supplementation. Lower zinc level people are more likely to be affected by COVID-19 in comparison to the normal zinc level people. Among the COVID-19 patients the lower zinc level people had nearly double (RR 1-93) risk of becoming hypoxic and eventually prone for oxygen support.

Evaluating the Respiratory Support Pattern and Relationship with Initial Oxygen Saturation of the COVID Isolation Unit Patients in Sylhet.

COVID-19 is the devastating pandemic of the century predominantly fatal due to its respiratory failure nature. Severe and critical patients need oxygen supplementation in different forms. This cross-sectional study was conducted in four tertiary hospitals of Sylhet, Bangladesh from November 2020 to March 2021. All the patients admitted in the COVID-19 isolation units and fulfill the selection criterion were enrolled in this study. The primary objective of this study was to evaluate different types of respiratory supports and its relationship with initial oxygen saturation (SpO2). Total 481 patients were enrolled. There was a male predominance (65.00%) in the participants. Highest number of participants was from 61-70 years age group. Number of ventilated patients were significantly high (p<0.001) in the COVID-19 patient group. The initial SpO2 and hospital staying period of COVID-19 positive and negative group did not show any significant difference but these two parameters showed significant difference among died and survived group (p<0.001). Nearly one fourth patients (24.94%) of total patients were treated in ICU with high flow nasal cannula (HFNC), non-invasive ventilation (NIV) and mechanical ventilation. Among the ICU admitted patients nearly one-fourth (24.16%) patients were treated with mechanical ventilation. Mortality rate was 62.00% for ventilated patients, 70.60% for NIV patients and 15.80% for the HFNC patients.

Impacts of selenium and vitamin E supplementation on mRNA of heat shock proteins, selenoproteins and antioxidants in broilers exposed to high temperature.

The study was carried out to investigate the effect of dietary selenium (Se) and vitamin E (VE) supplementation on mRNA level of heat shock proteins, selenoproteins, and antioxidant enzyme activities in the breast meat of broilers under summer heat stress conditions. A total of 200 male broilers (Ross 308) of 1 day age were randomly separated into 4 groups in a complete randomized design and were given a basal diet (Control, 0.08 mg Se/kg diet) or basal diet supplemented with VE (250 mg/kg VE), sodium selenite (0.2 mg/kg Se), or Se + VE (0.2 mg/kg Se + 250 mg/kg VE) to investigate the expression of key antioxidant and heat shock protein (HSP) genes under high temperature stress. Dietary Se, VE and Se + VE significantly enhanced the activities and mRNA levels of catalase as well as superoxide dismutase (SOD) but decreased the mRNA levels of HSP70 and HSP90. Se alone or combined with VE increased the concentration of selenoprotein P and selenoproteins mRNA level and decreased the expression of HSP60. In addition, Se and Se + VE significantly enhanced the glutathione peroxidase (GPx) activity and the expression of GPx1 and GPx4 in breast muscle tissues. It is noteworthy that all the treatments significantly decreased malondialdehyde (MDA) level in the breast meat. Overall results showed that Se in combination with VE has maximal effects to mitigate heat stress. Based on given results it can be recommended that Se + VE are a suitable dietary supplement for broilers to ameliorate the negative effects of summer heat stress conditions.

Paraquat induced acute kidney injury and lung fibrosis: a case report from Bangladesh.

BACKGROUND: Since Bangladesh government issued a ban on the use of highly toxic WHO Class I pesticides, annual consumption of herbicides like Paraquat have been sharply increasing in the markets. Paraquat poisoning is an emerging public health threat and its high mortality rate is responsible for a significant number of deaths. Diagnostic limitations and unavailable sample at presentation have resulted in under-reporting and lack of awareness among the treating physicians, making Paraquat poisoning one of the most neglected toxicological emergencies. Herein, we present a case of Paraquat induced multi-organ failure and emphasis on pitfalls in the management. CASE PRESENTATION: An 18-years-old healthy male was admitted in Sylhet M.A.G Osmani Medical College Hospital with history of attempted suicide by Paraquat ingestion. On admission, he had high serum creatinine but otherwise asymptomatic. He was discharged on day 10 when his renal functions returned to normal. But On day 15, he started having respiratory symptoms-unresponsive to any of the local treatments he received, and by day 30, he developed overt lung fibrosis. We present sequential blood picture, radiographs and CT scans demonstrating Paraquat induced kidney and lung injury over the course of 30 days. CONCLUSION: Paraquat poisoning can lead to death and fatal long-term consequences. All cases of Paraquat poisoning, regardless of symptoms, must be hospitalized and observed for early detection of complications. Distribution of Paraquat should be restricted and/or banned as 38 other countries have done so, which we believe will greatly reduce poisoning related mortality.

Clinical and urodynamic outcomes in children with anorectal malformation subtype of recto-bladder neck fistula.

INTRODUCTION: Patients with anorectal malformations (ARMs) have a high incidence of genitourinary anomalies. Those with a recto-bladder neck fistula may represent a high-risk group, but their long-term urologic outcomes are poorly described. OBJECTIVE: To evaluate the clinical and urodynamic outcomes in a large cohort of patients with an ARM subtype of recto-bladder neck fistula. MATERIALS AND METHODS: A retrospective cohort study was performed of patients who had been treated for a recto-bladder neck fistula at the present institution since 2007. The primary outcomes were the ability to achieve urinary continence after 4 years of age, and development of a mildly decreased glomerular filtration rate (GFR) or worse (<89 ml/min/1.73 m2). Continence was defined as the ability to store urine for 3-4 h during the day and 8 h overnight without leakage. RESULTS: Demographic and clinical data are provided in the Summary Table. The most recent urodynamic findings included the presence of detrusor overactivity in 30 (75%) patients, median leak point pressure of 56.0 cmH2O (range, 14-140), median functional cystometric capacity at 40 cmH2O of 125.5% age-expected capacity (range, 36-473%), and median maximum cystometric capacity of 131.0% age-expected capacity (range, 44-473%). A mildly decreased GFR or worse developed in 13 (24%) patients. Of the 52 (78%) patients who were followed by pediatric urology at the present institution with a median follow-up of 30.9 months (range, 0.0-86.8), 35 (67%) were at least 4 years of age and could be assessed for continence. Continence was achieved in five (14%) patients voiding spontaneously and 15 (43%) performing CIC. Recurrent urinary tract infections (UTI) (OR 0.70, P = 0.006) were an independent predictor of incontinence, while urethral anomalies (OR 1.40, P = 0.03) were an independent predictor of chronic kidney disease (CKD) on multiple logistic regression analysis. DISCUSSION: The findings favorably compared with other studies, but were more robust due to the size of the cohort and breadth of urologic evaluation. Limitations included the retrospective design at a single institution. Incomplete clinical data and misclassification of continence may have lead to bias. CONCLUSIONS: This large cohort of patients with an ARM subtype of recto-bladder neck fistula had a high incidence of genitourinary anomalies. They were rarely able to achieve continence with spontaneous voiding alone and were at risk of developing CKD, both of which were likely multifocal in origin. Long-term urologic follow-up is warranted for patients with a recto-bladder neck fistula.

Optimal formation of genetically modified and functional pancreatic islet spheroids by using hanging-drop strategy.

BACKGROUND: Rejection and hypoxia are important factors causing islet loss at an early stage after pancreatic islet transplantation. Recently, islets have been dissociated into single cells for reaggregation into so-called islet spheroids. Herein, we used a hanging-drop strategy to form islet spheroids to achieve functional equivalence to intact islets. METHODS: To obtain single islet cells, we dissociated islets with trypsin-EDTA digestion for 10 minutes. To obtain spheroids, we dropped various numbers of single cells (125, 250, or 500 cells/30 µL drop) onto a Petri dish, that was inverted for incubation in humidified air containing 5% CO(2) at 37 °C for 7 days. The aggregated spheroids in the droplets were harvested for further culture. RESULTS: The size of the aggregated islet spheroids depended on the number of single cells (125-500 cells/30 µL droplet). Their morphology was similar to that of intact islets without any cellular damage. When treated with various concentrations of glucose to evaluate responsiveness, their glucose-mediated stimulation index value was similar to that of intact islets, an observation that was attributed to strong cell-to-cell interactions in islet spheroids. However, islet spheroids aggregated in general culture dishes showed abnormal glucose responsiveness owing to weak cell-to-cell interactions. Cell-to-cell interactions in islet spheroids were confirmed with an anti-connexin-36 monoclonal antibody. Finally, nonviral poly(ethylene imine)-mediated interleukin-10 cytokine gene delivered beforehand into dissociated single cells before formation of islet spheroids increased the gene transfection efficacy and interleukin-10 secretion from islet spheroids >4-fold compared with intact islets. CONCLUSION: These results demonstrated the potential application of genetically modified, functional islet spheroids with of controlled size and morphology using an hanging-drop technique.

Biostability and disinfectant by-product formation in drinking water blended with UF-treated filter backwash water.

The overall objective of this study was to investigate the impact of blending membrane-treated water treatment plant (WTP) residuals with plant-filtered water on finished water quality in terms of biostability and disinfectant by-product (DBP) formation. Filter backwash water (FBWW) was treated with a pilot-scale ultrafiltration (UF) membrane to produce permeate that was blended with plant-finished water. The batch studies involved storing samples for a specified time with a disinfectant residual to simulate residence time in the distribution system. Both chlorinated and non-chlorinated FBWW streams were evaluated, and the experimental design incorporated free chlorine, monochloramine, and chlorine dioxide in parallel to a model system that did not receive a disinfectant dose. The results of the study found that blending 10% UF-treated FBWW with plant-filtered water did not have an impact on water biostability as monitored with heterotrophic plate counts (HPCs) or DBP concentrations as monitored by TTHM and HAA5 concentrations. However, the presence of preformed THM and HAA species found in chlorinated FBWW streams may result in higher levels of initial DBP concentrations in blended water matrices, and could have a significant impact on finished water quality in terms of meeting specific DBP guidelines or regulations.

Use of fungi for the bioconversion of rice straw into cellulase enzyme.

Cellulase production was carried out by solid state bioconversion (SSB) method using rice straw, a lignocellulosic material and agricultural waste, as the substrate of three Trichoderma spp. and Phanerochaete chrysosporium in lab-scale experiments. The results were compared to select the best fungi among them for the production of cellulase. Phanerochaete chrysosporium was found to be the best among these species of fungi, which produced the highest cellulase enzyme of 1.43 IU/mL of filter paper activity (FPase) and 2.40 IU/mL of carboxymethylcellulose activity (CMCase). The "glucosamine" and "reducing sugar" parameters were observed to evaluate the growth and substrate utilization in the experiments. In the case of Phanerochaete Chrysosporium, the highest glucosamine concentration was 1.60 g/L and a high concentration of the release of reducing sugar was measured as 2.58 g/L obtained on the 4th day of fermentation. The pH values were also recorded. The range of the pH was about 5.15 to 5.56 in the case of Phanerochaete Chrysosporium.

Direct and indirect inactivation of Microcystis aeruginosa by UV-radiation.

Excessive algal growth in drinking water sources like lakes and reservoirs is responsible for filter-clogging, undesirable taste and odor, disinfection-by-product formation and toxin generation. Although various methods are currently being used to control algal bloom, their successes are limited. Some water utilities routinely use copper sulfate to control excessive algal growth. But there is a growing concern against its use mainly because it is non-specific to target algae and kills many non-target species. In this study, the scope of using UV-radiation to control algal growth was assessed using Microcystis aeruginosa as test species. A UV-dose of 75 mW s cm(-2) was found to be lethal to M. aeruginosa. A smaller dose of 37 mW s cm(-2) prevented growth for about 7 days. It was found that UV-radiation may increase the specific gravity of the cells and thus may adversely affect the ability of the cells to remain in suspension. Three days after a UV-dose of 75 mW s cm(-2), almost all the cells settled to the bottom of the incubation tubes, whereas all the unirradiated cells remained in suspension. It was also observed that UV-radiation on algal extracellular products has a significant residual effect and can contribute to algal growth control. The extent of residual effect depends on the UV-dose and can continue even for 7 days. UV-radiation was found to produce H2O2 in the microM level concentration. But at such level, H2O2 itself is not likely to cause the residual effect that was found in this study.

No evidence for increased oxidative damage to lipids, proteins, or DNA in Huntington's disease.

It has been proposed that mitochondrial dysfunction and excitotoxic mechanisms lead to oxidative damage in the brain of Huntington;s disease patients. We sought evidence that increased oxidative damage occurs by examining postmortem brain material from patients who had died with clinically and pathologically diagnosed Huntington's disease. Oxidative damage was measured using methods that have already demonstrated the presence of increased oxidative damage in Parkinson's disease, Alzheimer's disease, and senile dementia of the Lewy body type. No alterations in the levels of lipid peroxidation (as measured by lipid peroxides and thiobarbituric acid-malondialdehyde adducts) were found in the caudate nucleus, putamen, or frontal cortex of patients with Huntington's disease compared with normal controls. Similarly, there were no elevations in the levels of 8-hydroxyguanine or of a wide range of other markers of oxidative DNA damage. Levels of protein carbonyls in these tissues were also unaltered. Our data suggest that oxidative stress is not a major component of the degenerative processes occurring in Huntington's disease, or at least not to the extent that occurs in other neurodegenerative disorders.

Plasma levels of neuroexcitatory amino acids in patients with migraine or tension headache.

Plasma amino acids were analysed in patients with migraine with (9) and without (80) aura, in patients with tension headache (14) and in controls (62). The neuroexcitatory amino acids glutamic acid, glutamine, glycine, cysteic acid and homocysteic acid were elevated in migraine patients while total thiols (cysteine/cystine) were reduced. Patients with tension headache had values which were similar to those of controls. Tryptophan was elevated in migraine patients without aura only. Studies on two patients showed that the raised resting excitatory amino acid levels became still further elevated during a migraine attack. These results show that high concentrations of neurotransmitter amino acids occur normally in migraine patients and suggest that this profile may be a contributory factor in migraine attacks. Tension headache, however, has different biochemical parameters.

Aspergillus fungal mass detected by transesophageal echocardiography.

Cardiac fungal infections have become more prevalent and are being diagnosed with increasing frequency. The most common infective organism is Candida albicans, followed by Aspergillus fumigatus and Cryptococcus. Cardiac involvement is usually associated with endocarditis, myocarditis, pericarditis, or intracardiac fungal mass. Early diagnosis is imperative, as these patients have poor outcome once there is cardiac involvement. In this report we describe a patient in whom an intracardiac mass was detected with transesophageal echocardiography and confirmed to be aspergillus fungal ball at surgery.

Platelet sulphotransferase activity, plasma sulphate levels and sulphation capacity in patients with migraine and tension headache.

Activity of both the M- and P-forms of sulphotransferase (ST) was measured in platelets from patients with migraine, tension headache and controls. Mean PST values were 0.065 +/- 0.023 and 0.057 +/- 0.052 nmol/mg protein/min for migraine patients with and without aura. The corresponding values for tension headache and controls were 0.122 +/- 0.059 and 0.127 +/- 0.093 nmol/mg protein/min respectively (p < 0.05). Mean MST values were not different for any of the groups, and MST and PST activities measured in two patients during a migraine attack were not significantly altered from baseline levels. Mean plasma inorganic sulphate concentrations and paracetamol metabolites were not significantly different in any of the groups studied. The results suggest that PST activity may be a factor in the aetiology of migraine.

IgG anticardiolipin antibody titer > 40 GPL and the risk of subsequent thrombo-occlusive events and death. A prospective cohort study.

BACKGROUND: Anticardiolipin antibodies (aCL) have been associated with an increased risk of stroke and thrombo-occlusive events. Little is known about the influence of aCL on recurrent thrombo-occlusive events. METHODS: Consecutively identified patients (n = 132) with focal cerebral ischemia [stroke = 112, transient ischemic attack (TIA) = 20] harboring aCL of at least 10 GPL units at the time of their index event were prospectively followed to estimate the effect of aCL titer on time to and risk of subsequent thrombo-occlusive events (stroke, TIA, deep venous thrombosis, pulmonary embolism, myocardial infarction) and death. On the basis of prior literature, we divided patients into those with aCL < or = 40 GPL (n = 111; mean age, 63 +/- 14 years; mean follow-up, 1.95 years) and those with aCL > 40 GPL (n = 21; mean age, 54 +/- 20 years; mean follow-up, 1.50 years). RESULTS: There was no difference between groups for prevalence of hypertension, diabetes mellitus, cigarette smoking, atrial fibrillation, prior TIA, or sex. The GPL > 40 group was younger (54 +/- 20 versus 63 +/- 14 years; P = .055), had more prior strokes [9/21 (48%) versus 27/111 (20%); P = .030], more frequent subsequent thrombo-occlusive events and death [15/21 (71%) versus 51/111 (48%); P = .030], and a shorter median time (years) to event (0.15 versus 0.61, log rank P = .005). The risk ratio for recurrent event and death with GPL > 40 obtained from Cox proportional hazards models, adjusted for prior strokes, prior TIAs, hypertension, diabetes mellitus, atrial fibrillation, and cigarette smoking was 1.9 (95% confidence interval, 1.0 to 3.5; P = .051). CONCLUSIONS: Our data suggest that subsequent thrombo-occlusive events and death after focal cerebral ischemia associated with IgG aCL may occur sooner and more frequently with GPL > 40.

Oxidative DNA damage in the parkinsonian brain: an apparent selective increase in 8-hydroxyguanine levels in substantia nigra.

Oxidative damage has been implicated in the pathology of Parkinson's disease (PD), e.g., rises in the level of the DNA damage product, 8-hydroxy-2'-deoxyguanosine, have been reported. However, many other products result from oxidative DNA damage, and the pattern of products can be diagnostic of the oxidizing species. Gas chromatography/mass spectrometry was used to examine products of oxidation and deamination of all four DNA bases in control and PD brains. Products were detected in all brain regions examined, both normal and PD. Analysis showed that levels of 8-hydroxyguanine (8-OHG) tended to be elevated and levels of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine) tended to be decreased in PD. The most striking difference was a rise in 8-OHG in PD substantia nigra (p = 0.0002); rises in other base oxidation/deamination products were not evident, showing that elevation in 8-OHG is unlikely to be due to peroxynitrite (ONOO-) or hydroxyl radicals (OH.), or to be a prooxidant effect of treatment with L-Dopa. However, some or all of the rise in 8-OHG could be due to a change in 8-OHG/FAPy guanine ratios rather than to an increase in total oxidative guanine damage.

A generalised increase in protein carbonyls in the brain in Parkinson's but not incidental Lewy body disease.

The degeneration of neurones in Parkinson's disease (PD) may involve oxidative stress. Previously, increased lipid peroxidation and oxidative DNA damage have been reported in parkinsonian substantia nigra. In the present study the protein carbonyl assay was used to assess oxidative protein damage in postmortem brain tissue from patients with PD and age-matched controls. In brain areas associated with PD, such as substantia nigra, caudate nucleus, and putamen, there was a significant increase in carbonyl levels. However, increased carbonyl levels were also found in areas of the brain not thought to be affected in PD. This perhaps suggests that protein carbonyl formation is related to therapy with L-DOPA, which can exert prooxidant properties in vitro. Consistent with this possibility, brain regions from individuals with incidental Lewy body disease (putative presymptomatic PD) showed no rise in carbonyls in any brain areas. Our data show that either oxidative protein damage occurs widely but late in PD brain, and/or that L-DOPA treatment contributes to protein oxidation.

Bioreductive activation of catechol estrogen-ortho-quinones: aromatization of the B ring in 4-hydroxyequilenin markedly alters quinoid formation and reactivity.

There is a clear association between excessive exposure to estrogens and the development of cancer in several tissues including breast and endometrium. The risk factors for women developing these cancers are all associated with longer estrogen exposure, as may be facilitated by early menses, late menopause and long-term estrogen replacement therapy. Equilenin (1,3,5(10),6,8-estrapentaen-3-ol-17-one) or its 17-hydroxylated analogs make up 15% of the most widely prescribed estrogen replacement formulation, Premarin, and yet there is very little information on the human metabolism of these estrogens. In this study, we synthesized the catechol metabolite of equilenin, 4-hydroxyequilenin, and examined how aromatization of the B ring affects the formation and reactivity of the o-quinone (3,5-cyclohexadien-1,2-dione). 4-Hydroxyequilenin-o-quinone is much more redox-active and longer-lived than the endogenous catechol estrone-o-quinones, which suggests that the mechanism(s) of toxicity of the former could be quite different. Interestingly, the rate of reduction of the 4-hydroxyequilenin-o-quinone is increased at least 13-fold in the presence of NAD(P)H:quinone oxidoreductase (DT-diaphorase). Once NADH is consumed however, the catechol auto-oxidized rapidly to the o-quinone. NADH consumption was accompanied by dicumarol-sensitive oxygen uptake both with the purified enzyme and with cytosol from human melanoma cells with high levels of DT-diaphorase activity. P450 reductase and rat liver microsomes also catalyzed NADPH consumption and oxygen uptake. 4-Hydroxyestrone-o-quinone was also rapidly reduced by NAD(P)H; however, this o-quinone does not auto-oxidize and once the o-quinone is reduced the reaction terminates. Including oxidative enzymes in the incubation completes the redox couple and 4-hydroxyestrone-o-quinone behaves like 4-hydroxyequilenin-o-quinone. These data suggest that reduction of estrogen-o-quinones may not result in detoxification. Instead this could represent a cytotoxic mechanism involving consumption of reducing equivalents (NADH/NADPH) as well as formation of superoxide and other reactive oxygen species leading to oxidative stress. Finally, we have compared the cytotoxicity of 4-hydroxyequilenin with that of the estrone catechols in human melanoma cells. 4-Hydroxyequilenin is 5-fold more toxic in these cells compared with 4-hydroxyestrone (ED50 = 7.8 versus 38 microM, respectively) suggesting that formation of the longer-lived redox-active 4-hydroxyequilenin-o-quinone was responsible for the cytotoxic differences. These results substantiate the conclusion that the involvement of quinoids in catechol estrogen toxicity depends on a combination of the rate of formation of the o-quinone, the lifetime of the o-quinone, and the electrophilic/redox reactivity of the quinoids.

Footprinting studies of DNA-sequence recognition by nogalamycin.

We have studied the DNA sequence binding preference of the antitumour antibiotic nogalamycin by DNase-I footprinting using a variety of DNA fragments. The DNA fragments were obtained by cloning synthetic oligonucleotides into longer DNA fragments and were designed to contain isolated ligand-binding sites surrounded by repetitive sequences such as (A)n.(T)n and (AT)n. Within regions of (A)n.(T)n, clear footprints are observed with low concentrations of nogalamycin (< 5 microM), with apparent binding affinities for tetranucleotide sequences which decrease in the order TGCA > AGCT = ACGT > TCGA. In contrast, within regions of (AT)n, the ligand binds best to AGCT; binding to TCGA and TGCA is no stronger than to alternating AT. Within (ATT)n, the preference is for ACGT > TCGA. Although each of these binding sites contains all four base pairs, there is no apparent consensus sequence, suggesting that the selectivity is affected by local DNA dynamic and structural effects. At higher drug concentrations (> 25 microM), nogalamycin prevents DNAse-I cleavage of (AT)n but shows no interaction with regions of (AC)n.(GT)n. Regions of (A)n.(T)n, which are poorly cut by DNase I, show enhanced rates of cleavage in the presence of low concentrations of nogalamycin, but are protected from cleavage at higher concentrations. We suggest that this arises because drug binding to adjacent regions distorts the DNA to a structure which is more readily cut by the enzyme and which is better able to bind further ligand molecules.