Serum magnesium in diabetic retinopathy: the association needs investigation.

BACKGROUND: Magnesium has an essential role in glucose metabolism, and hypomagnesaemia is common in diabetes mellitus. However, the relationship between serum magnesium and diabetic retinopathy is poorly understood. AIM: To determine the association between serum magnesium levels and retinopathy in type 2 diabetic patients with normal renal function and to correlate it with severity of retinopathy. METHODS: This cross-sectional observational study was conducted in a semi-urban tertiary-care teaching hospital. Clinicodemographic profile and serum magnesium levels were determined in patients with type 2 diabetes mellitus (DM) with (group 1) and without (group 2) retinopathy. Serum magnesium levels were correlated with the presence and severity of retinopathy. RESULTS: Of 104 type 2 DM patients, 50 had retinopathy. Younger age, longer duration of disease and poorer glycaemic control (p < 0.05) were found to be associated with retinopathy. The mean serum magnesium levels in patients with retinopathy and those without retinopathy were 1.63 ± 0.30 mg/dL and 1.76 ± 0.22 mg/dL, respectively (p = 0.029). Reduced serum magnesium was associated with elevated fasting sugars (p = 0.019) and female gender (p = 0.037). On comparative analysis of patients with sight-threatening diabetic retinopathy (STDR), non-STDR and no retinopathy by ANOVA test, patients with STDR had significantly lower serum magnesium (1.55 ± 0.33 mg/dL) (p = 0.031). CONCLUSION: Serum magnesium levels were lower in patients with diabetic retinopathy. Patients with STDR had lower serum magnesium compared with those without STDR. SUMMARY: Serum magnesium, studied extensively for its role in glucose metabolism, was found to be lower in patients with diabetic retinopathy compared with those without retinopathy. Sight-threatening diabetic retinopathy had significantly lower levels of serum magnesium.

Evaluation of Circulating Plasma VEGF-A, ET-1 and Magnesium Levels as the Predictive Markers for Proliferative Diabetic Retinopathy.

Diabetic retinopathy (DR) is the most common cause for preventable blindness in India. The onset of micro and macrovascular complications in T2DM is multifactorial and difficult to predict. The status of micronutrients, several inflammatory cytokines, elevated triacylglycerols, oxidative stress etc., are being studied extensively. Hypomagnesemia plays a pivotal role in worsening of insulin resistance. Although, Vascular Endothelial Growth Factor-A (VEGF-A) and Endothelin-1 (ET-1) are known to be elevated in DR, yet few reports cite their role, especially in Indian population. In this study, we included thirty subjects with T2DM in each of the three groups namely, T2DM cases without retinopathy, Non Proliferative DR (NPDR) and Proliferative DR (PDR) retinopathy. The glycemic status, circulating plasma VEGF-A, ET-1 levels, serum magnesium and lipids were estimated and compared among the groups. An ROC was drawn to evaluate VEGF-A, ET-1 and serum magnesium levels as the predictive markers for PDR. On comparison VEGF-A, ET-1 and serum magnesium levels showed a significant difference among the three groups. PDR cases had higher circulating levels of VEGF-A, ET-1 and low serum magnesium levels when compared to others. ROC for VEGF-A and ET-1 showed an optimum cut-off of 1521 ng/ml (AUC 0.975) and 16 pg/ml (AUC 0.96) respectively. A negative ROC was drawn to check the lower cut-off limit for serum magnesium; we documented an optimum cut off of 1.7 mg/dl (AUC 0.837). ET-1, VEGF-A and serum Magnesium levels are significantly altered in PDR and can be used as the predictive markers of PDR.

Evaluation of Preanalytical Quality Indicators by Six Sigma and Pareto`s Principle.

Preanalytical steps are the major sources of error in clinical laboratory. The analytical errors can be corrected by quality control procedures but there is a need for stringent quality checks in preanalytical area as these processes are done outside the laboratory. Sigma value depicts the performance of laboratory and its quality measures. Hence in the present study six sigma and Pareto principle was applied to preanalytical quality indicators to evaluate the clinical biochemistry laboratory performance. This observational study was carried out for a period of 1 year from November 2015-2016. A total of 1,44,208 samples and 54,265 test requisition forms were screened for preanalytical errors like missing patient information, sample collection details in forms and hemolysed, lipemic, inappropriate, insufficient samples and total number of errors were calculated and converted into defects per million and sigma scale. Pareto`s chart was drawn using total number of errors and cumulative percentage. In 75% test requisition forms diagnosis was not mentioned and sigma value of 0.9 was obtained and for other errors like sample receiving time, stat and type of sample sigma values were 2.9, 2.6, and 2.8 respectively. For insufficient sample and improper ratio of blood to anticoagulant sigma value was 4.3. Pareto`s chart depicts out of 80% of errors in requisition forms, 20% is contributed by missing information like diagnosis. The development of quality indicators, application of six sigma and Pareto`s principle are quality measures by which not only preanalytical, the total testing process can be improved.

Putative Role of Cardio Metabolic Risk Among Poorly Controlled Asthmatics in South Indian Population.

Mortality and morbidity attributed to asthma remains to be the biggest nightmare worldwide. Hence, the study was aimed to compare the cardio metabolic risk factors as assessed by Body mass index (BMI), waist hip ratio (WHR), serum triacylglycerol and uric acid in well controlled and poorly controlled asthmatics and to correlate these parameters with the severity of asthma. A case control study was conducted on 90 subjects who were segregated into well controlled asthmatics (n = 30) and poorly controlled asthmatics (n = 30) who were diagnosed based on Global initiative for Asthma management guidelines and healthy volunteers (n = 30). Centrifuged fasting venous blood samples were used for biochemical analysis, pulmonary function test, BMI, and waist hip ratio (WHR) were measured. The statistical analysis was done using SPSS version 17. There was a significant increase in BMI, WHR, lipid profile, serum uric acid and decrease in forced expiratory volume (FEV1), forced vital capacity (FVC), and FEV1/FVC in poorly controlled asthmatics. There was a significant association between FEV1 and serum uric acid, BMI and Triacylgycerol in poorly controlled asthmatics. Poorly controlled asthmatics have greater risk of developing cardiometabolic problems. Serum uric acid can be used as one of the severity markers in asthma to assess cardio metabolic risk.

Association of Glycemic Status with Bone Turnover Markers in Type 2 Diabetes Mellitus.

CONTEXT: Type 2 diabetes mellitus has profound implications on the skeleton. Even though bone mineral density is increased in type 2 diabetes mellitus patients, they are more prone for fractures. The weakening of bone tissue in type 2 diabetes mellitus can be due to uncontrolled blood sugar levels leading to high levels of bone turnover markers in blood. AIMS: The aim of this study is to find the association between glycemic status and bone turnover markers in type 2 diabetes mellitus. SETTINGS AND DESIGN: This case-control study was carried out in a tertiary health care hospital. SUBJECTS AND METHODS: Fifty clinically diagnosed type 2 diabetes mellitus patients in the age group between 30 and 50 years were included as cases. Fifty age- and gender-matched healthy nondiabetics were included as controls. Patients with complications and chronic illness were excluded from the study. Depending on glycated hemoglobin (HbA1c) levels, patients were grouped into uncontrolled (HbA1c >7%, n = 36) and controlled (HbA1c <7%, n = 14) diabetics. Based on duration of diabetes, patients were grouped into newly diagnosed, 1-2 years, 3-5 years, and >5 years. Serum osteocalcin (OC), bone alkaline phosphatase (BAP), acid phosphatase (ACP), and HbA1c levels were estimated. OC/BAP and OC/ACP ratio was calculated. STATISTICAL ANALYSIS USED: Student's t-test, analysis of variance, and Chi-square tests were used for analysis. Receiver operating characteristic (ROC) curve analysis was done for OC/BAP and OC/ACP ratios. RESULTS: Serum OC, HbA1c, and OC/BAP ratio were increased in cases when compared to controls and were statistically significant (P < 0.001). OC/ACP ratio was decreased in type 2 diabetes mellitus and was statistically significant (P = 0.01). In patients with >5-year duration of diabetes, HbA1c level was high and was statistically significant (P < 0.042). BAP levels were high in uncontrolled diabetics but statistically not significant. ROC curve showed OC/BAP ratio better marker than OC/ACP ratio. CONCLUSIONS: Uncontrolled type 2 diabetes mellitus affects bone tissue resulting in variations in bone turnover markers. Bone turnover markers are better in predicting recent changes in bone morphology and are cost effective.

Urinary 8-hydroxydeoxyguanosine as a marker of oxidative stress induced genetic toxicity in oral cancer patients.

CONTEXT: Recently, non-communicable diseases have snatched the lead from infectious diseases in causing mortality. Of these, oral cancer accounts for a significant proportion of deaths. Every year in India significant percentage of newly diagnosed malignancy is oral cancer attributed to various reasons. AIMS: The aim of this study was to assess the extent of oxidative stress and its effect on modification of DNA by urinary nucleoside 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in oral cancer subjects. To see the relationship between the nucleoside 8-OHdG and antioxidant capacity ferric reducing ability plasma (FRAP) in oral cancer subjects. SETTINGS AND DESIGN: Case-control study included three groups with 60 volunteers, who were divided into 30 controls, and equal number of clinically diagnosed oral cancer male patients: (Subdivided into newly diagnosed [n = 15] and 1-year treatment follow-up oral cancer subjects [n = 15]). MATERIALS AND METHODS: A random urine sample was used for analysis of 8-OHdG concentration. Serum triglycerides, lipid peroxidation, protein thiols, and FRAP assay were performed by spectrophotometric technique. STATISTICAL ANALYSIS USED: Student's t-test and one-way analysis of variance were performed for group comparison and Pearson's correlation analysis were used. A P < 0.05 was considered the optimum level of significance. RESULTS: The urinary 8-OHdG and serum malondialdehyde levels were significantly elevated in newly diagnosed oral cancer subjects in their 1-year treatment compared to the control group (P < 0.05). A significant correlation was observed between urinary 8-OHdG and FRAP in both groups of oral cancer subjects. CONCLUSIONS: Urinary 8-OHdG can be a useful diagnostic marker of oxidative DNA damage in oral cancer subjects.

SERUM magnesium levels as an indicator of status of Diabetes Mellitus type 2.

CONTEXT: Magnesium deficiency is commonly associated with endocrine and metabolic disorders, especially with Diabetes Mellitus type 2 though the mechanism of hypomagnesemia in Diabetes Mellitus is not completely known. There is a close association between metabolic control of Diabetes Mellitus and impaired magnesium balance. AIMS: To estimate the serum levels of magnesium in patients of with Diabetes Mellitus type 2 and to find a correlation if any, with the duration and control (by estimating HbA1c) of Diabetes Mellitus type 2. MATERIALS AND METHODS: Fifty patients of Diabetes Mellitus type 2 were included in the study. Blood samples were analyzed for fasting and post prandial glucose, HbA1c and magnesium. The patients were grouped into three categories based upon their HbA1c levels into those with good control, need intervention and poor control. The three groups were compared with reference to their mean levels of blood glucose and magnesium. Association of serum magnesium levels with HbA1c, Fasting and postprandial blood glucose and duration of Diabetes Mellitus was also done. RESULTS: Serum magnesium levels were found to decline with rise in HbA1c levels and with duration of Diabetes Mellitus type 2. CONCLUSION: Hypomagnesemia is linked to poor control of Diabetes Mellitus type 2 and depletion of serum magnesium occurs exponentially with duration of disease.

Association of inflammatory sialoproteins, lipid peroxides and serum magnesium levels with cardiometabolic risk factors in obese children of South Indian population.

The Incidence of childhood obesity and metabolic syndrome is increasing even in rural and semi-urban regions of India. Adipose tissue mass secretes several inflammatory proteins, which could potentially alter the metabolic processes, leading to several complications at the later stages of life. With limited studies on protein bound sialic acid (PBSA) as a marker of oxidative stress mediated inflammation in obese children, this study was aimed to assess and correlate PBSA with lipid peroxidation and other cardiometabolic risk factors like Insulin Resistance (IR), serum magnesium, and high sensitive C reactive Protein (hsCRP) levels in order to provide an insight into the degree of systemic inflammation and oxidative stress. This study included 62 obese children (≥95% percentile of the CDC chart) and 60 non obese controls. This study documents significant higher levels of PBSA, IR, Malondialdehyde (MDA), hsCRP and uric acid in obese children (p<0.001). PBSA was associated with IR, hsCRP, uric acid, hypomagnesaemia. Higher degrees of oxidative stress, Insulin resistance and low serum magnesium levels were noted in obese children. PBSA and hsCRP levels were elevated and were associated with Insulin resistance in obese children of South Indian population.

A Comparative Study of Serum Uric Acid levels and Lipid Ratios in Coronary Artery Disease Patients.

INTRODUCTION: Coronary Artery Disease (CAD) appears to be common in the Indian population of different geographical origins, religions and languages. Measurement of lipid fractions and ratios are widely recommended for risk assessment. A few studies have shown that serum uric acid plays a role in the development of cardiovascular morbidity. Very few reports are cited linking serum uric acid with the lipid fraction in CAD. OBJECTIVES: To find the significance of non-HDL cholesterol, LDL-c/HDL-c ratio, TC/HDL ratio and serum uric acid level in CAD patients. SUBJECTS AND METHODOLOGY: In this study, we included fifty CAD patients as subjects and an equal number of controls. Both subjects and controls were assessed for anthropometric, physiological and biochemical parameters. RESULTS: The present study showed significant increased levels of total cholesterol (p=0.002), TAGs (p<0.001), HDL (p=0.005), LDL (p<0.006) and non-HDL cholesterol (p<0.001). LDL-c/HDL-c ratio (p<0.001) and TC/HDL ratio (p<0.001) in CAD patients (subjects) were also significant when compared to controls. Uric acid level in CAD patients was increased (p<0.001). CONCLUSION: Serum Uric Acid, TC/HDL and LDL/HDL ratios could be regarded as objective markers, in association with existing atherogenic dyslipidemia in patients with CAD.

Evaluation of Body Mass Index (BMI) Percentile cut-off Levels with Reference to Insulin Resistance: A Comparative Study on South Indian Obese and Non-Obese Adolescents.

OBJECTIVES: To investigate the relationship of Body Mass Index (BMI) percentile cut off with Homeostasis Model Assessment-Insulin Resistance (HOMA-IR), a surrogate marker for insulin resistance in obese, overweight and non-obese adolescents. STUDY DESIGN: A cross-sectional analysis of 120 adolescents (divided into sixty overweight/obese and an equal number of non-obese) was performed on nondiabetic, nonpregnant (11 to 18 year old school going) adolescents in south Indian (Puducherry) population. The main outcome measure of insulin resistance was calculated as HOMA-IR (greater than 3.16). Obesity was defined as per the BMI criteria, BMI greater than or equal to 95(th) percentile,overweight greater than or equal to 85(th) percentile and non-obese less than 84.9(th) percentile. All adolescents were subjected to analysis of anthropometric parameters that included weight, height and BMI (body mass index). Biochemical parameters, namely venous plasma glucose (fasting) and venous plasma insulin (fasting) were included for insulin resistance calculation by HOMA-IR. RESULTS: Having a BMI of greater than or equal to 85(th) percentilewas associated with high HOMA-IR levels. As the BMI percentile increased, HOMA-IR levels also increased. Prevalence of insulin resistance for a BMI percentile of less than 84.9 was nil. Prevalence of insulin resistance for a BMI percentile of 85-94.9 was 26%.Prevalence of insulin resistance for BMI of greater than or equal to 95(th) percentile was 64%. CONCLUSION: Insulin resistance is highly prevalent in obese and overweight adolescents as compared to that in non-obese adolescents. The onset of Impaired Fasting Glucose (IFG) is associated with the development of severe hyper-insulinaemia in obese adolescents and early primordial and primary prevention can thus alleviate the burden of future cardiometabolic disorders.

Planning an objective and need based curriculum: the logistics with reference to the undergraduate medical education in biochemistry.

PURPOSE: The medical education is recently being transformed into several domains in order to adapt to the need and the value based academics which is required for the quality doctors who serve the community. Presently, the biochemistry curricula for the graduate students of medicine have been questioned by as many experts, because of their multiple lacunae. In this review, we would like to highlight the scenario which is related to the existing biochemistry curricula for graduate medical students, which have been followed in several medical schools and universities and we also hope to share our ideas for implementing objective and pragmatic curricula. Evidence based research, wherein the articles which are related to innovative teaching-learning tools are collected and the pros and cons which are related to the different methods analyzed in biochemistry point of view. CONCLUSION: Rapid changes in the content of the curriculum may not be required, but a gradual introduction of the novel approach and the methods of teaching biochemistry can be adopted into the curriculum.

Serum lipid peroxides and magnesium levels following three months of treatment with pioglitazone in patients with type-2 diabetes mellitus.

BACKGROUND: Higher levels of lipid peroxidation and hypomagnesaemia are frequently associated with Type 2 Diabetes mellitus (T2DM). Addressing these issues would definitely help us in preventing or prolonging the onset of pathogenesis of micro and macrovascular complications. Pioglitazone is used as a trusted insulin sensitizer and an adjuvant to the conventional oral hypoglycemic agents. This study was planned to explore the effects of pioglitazone on oxidative stress, serum magnesium, blood pressure, hepato-biliary and renal systems in addition to its effects on glycemic control. METHODS: Sixty-three T2DM cases, who were started on pioglitazone were included in this study. All the physiological and biochemical parameters were estimated prior to and following three months of therapy with pioglitazone. RESULTS: There was significant improvement in the glycemic control, serum magnesium and MDA levels with p values of 0.000, 0.023 and 0.000 respectively. Pioglitazone did not have any significant effects on the serum lipids and blood pressure in T2DM cases following three months of treatment. We did not observe any pronounced changes in hepato-biliary enzymes, serum urea and creatinine levels reaffirming safety of pioglitazone in T2DM. CONCLUSION: Three-month duration of treatment with Pioglitazone in T2DM cases helps in alleviating the levels of lipid peroxides, besides being associated with improved serum magnesium status and glycemic control.

Status of serum magnesium in type 2 diabetes mellitus with particular reference to serum triacylglycerol levels.

UNLABELLED: Magnesium (Mg) is the fourth most abundant cation in the body and the second most common intracellular cation. The association between hypomagnesemia and insulin resistance (IR) in diabetes mellitus has been documented earlier. However, we wanted to study whether the extracellular status of magnesium (Mg) could be a biochemical mediator between hypertriacylglycerolemia and diabetes mellitus. AIMS: To find out the probable association among HbA1c, triacylglycerol (TG) and magnesium levels - a predictor of vascular complications in T(2)DM. MATERIALS AND METHODS: Thirty patients who had attended the diabetic clinics during the period of this study were included. All the samples were analysed for glucose, TG, Mg, and HbA1c. For statistical analysis, SPSS 17 package was used. RESULTS: Serum TG (236.67*, 195.06) and HbA1C (9.97*, 8.57) levels were independently compared between the two groups and were significantly high in group A subjects (Mg<1.2mg/dl) as compared to group B subjects (Mg>1.2mg/dl) (*p-value<0.05), respectively. DISCUSSION AND CONCLUSION: Hypertriacylglycerolemia was pronounced in type 2 diabetes mellitus patients with accompanying hypomagnesemia. This compared well with that of the glycemic control. Low Mg levels, high TG levels in association with enhanced HbA1c levels could thus serve as a reliable biochemical indicator of insulin status and action without resorting to the usage of criteria for insulin sensitivity and resistance.

Circulating MicroRNAs as Biomarkers in Health and Disease.

In the recent years, circulating nucleic acids have emerged as new biomarkers. Among these, microRNAs(miRNA) have evolved as promising and potential markers of both physiological and pathological conditions. MiRNA are transcribed from DNA like the other mRNA molecules. Their secretions and functions have to be still explored in humans, though many theories have been proposed. It is a small non coding RNA which plays an important role in the regulation of the gene expression, cell-cell communication, cell division and apoptosis. MiRNAs are stable and tissue specific and they can be identified and quantitated, which make them ideal biomarkers. This review highlights the secretion,mechanism of action and the role of miRNA in the diagnosis and the management of different disease conditions.

How stiff is DNA?

The natural stiffness of DNA, which contributes to the interactions of the many proteins involved in its biological processing and packaging, also plays an important role in modern nanotechnology. Here we report new Monte-Carlo simulations of deformable DNA molecules of potential utility in understanding the behavior of the long, double-helical polymer in the tight confines of a cell and in the design of novel nanomaterials and molecular devices. We directly determine the fluctuations in end-to-end extension associated with the conventional elastic-rod representation of DNA and with more realistic models that take account of the precise deformability of the constituent base-pair steps. Notably, the variance of end-to-end distance shows a quadratic increase with chain length in short chains of both types. We also consider the contributions to chain extension from the chemical linkages used to attach small molecular probes to DNA. The distribution of computed distances is sensitive to the intrinsic structure and allowed deformations of the tether. Surprisingly, the enhancement in end-to-end variance associated with the presence of the probe depends upon chain length, even when the probe is rigidly connected to DNA. We find that the elastic rod model of DNA in combination with a slightly fluctuating tether accounts satisfactorily for the distributions of end-to-end distances extracted from the small-angle X-ray scattering of gold nanocrystals covalently linked to the ends of short DNAs. There is no need to introduce additional structural fluctuations to reproduce the measured uptake in end-to-end fluctuations with chain length.

Properties of the Nucleic-acid Bases in Free and Watson-Crick Hydrogen-bonded States: Computational Insights into the Sequence-dependent Features of Double-helical DNA.

The nucleic-acid bases carry structural and energetic signatures that contribute to the unique features of genetic sequences. Here we review the connection between the chemical structure of the constituent nucleotides and the polymeric properties of DNA. The sequence-dependent accumulation of charge on the major- and minor-groove edges of the Watson-Crick base pairs, obtained from ab initio calculations, presents unique motifs for direct sequence recognition. The optimization of base interactions generates a propellering of base-pair planes of the same handedness as that found in high-resolution double-helical structures. The optimized base pairs also deform along conformational pathways, i.e., normal modes, of the same type induced by the binding of proteins. Empirical energy computations that incorporate the properties of the base pairs account satisfactorily for general features of the next level of double-helical structure, but miss key sequence-dependent differences in dimeric structure and deformability. The latter discrepancies appear to reflect factors other than intrinsic base-pair structure.

Feature extraction using molecular planes for fuzzy relational clustering of a flexible dopamine reuptake inhibitor.

Six rigid-body parameters (Shift, Slide, Rise, Tilt, Roll, Twist) are commonly used to describe the relative displacement and orientation of successive base pairs in a nucleic acid structure. The present work adapts this approach to describe the relative displacement and orientation of any two planes in an arbitrary molecule-specifically, planes which contain important pharmacophore elements. Relevant code from the 3DNA software package (Nucleic Acids Res. 2003, 31, 5108-5121) was generalized to treat molecular fragments other than DNA bases as input for the calculation of the corresponding rigid-body (or "planes") parameters. These parameters were used to construct feature vectors for a fuzzy relational clustering study of over 700 conformations of a flexible analogue of the dopamine reuptake inhibitor, GBR 12909. Several cluster validity measures were used to determine the optimal number of clusters. Translational (Shift, Slide, Rise) rather than rotational (Tilt, Roll, Twist) features dominate clustering based on planes that are relatively far apart, whereas both types of features are important to clustering when the pair of planes are close by. This approach was able to classify the data set of molecular conformations into groups and to identify representative conformers for use as template conformers in future Comparative Molecular Field Analysis studies of GBR 12909 analogues. The advantage of using the planes parameters, rather than the combination of atomic coordinates and angles between molecular planes used in our previous fuzzy relational clustering of the same data set (J. Chem. Inf. Model. 2005, 45, 610-623), is that the present clustering results are independent of molecular superposition and the technique is able to identify clusters in the molecule considered as a whole. This approach is easily generalizable to any two planes in any molecule.

Target-site preferences of Sleeping Beauty transposons.

The Sleeping Beauty (SB) transposon is a Tc1/mariner family transposon that has applications in vertebrate animals for gene transfer, gene-tagging, and human gene therapy. In this study, we analyzed the target-site preferences of the SB transposon. At the genomic level, integration of SB transposons with respect to genes (exons and introns) and intergenic regions appears fairly random but not on a micro-scale. Although there appears to be a consensus sequence around the vicinity of the target sites, the primary sequence is not the determining factor for target selection. When integrations were examined over a limited topography, the sites used most often for integration did not match the consensus sequence. Rather, a unique deformation inherent in the sequence may be a recognition signal for target selection. The deformation is characterized by an angling of the target site such that the axis around the insertion site is off-center, the rotation of the helix (twisting) is non-uniform and there is an increase in the distance between the central base-pairs. Our observations offer several hypothetical insights into the transposition process. Our observations suggest that particular deformations of the double helix predicted by the V(step) algorithm can distinguish TA sites that vary by about 16-fold in their preferences for accommodating insertions of SB transposons.

Accurate representation of B-DNA double helical structure with implicit solvent and counterions.

High-resolution nuclear magnetic resonance (NMR) and crystallographic data have been taken to refine the force field used in the torsion angle space nucleic acids molecular mechanics program DUPLEX. The population balance deduced from NMR studies of two carcinogen-modified DNA conformers in equilibrium was used to fine tune a sigmoidal, distance-dependent dielectric function so that reasonable relative energies could be obtained. In addition, the base-pair and backbone geometry from high-resolution crystal structures of the Dickerson-Drew dodecamer was used to re-evaluate the deoxyribose pseudorotation profile and the Lennard-Jones nonbonded energy terms. With a modified dielectric function that assumes a very steep distance-dependent form, a deoxyribose pseudorotation profile with reduced energy barriers between C2'- and C3'-endo minima, and a shift of the Lennard-Jones potential energy minimum to a distance approximately 0.4 A greater than the sum of the van der Waals' radii, the sequence-dependent conformational features of the Dickerson-Drew dodecamer in both the solid state and the aqueous liquid crystalline phase are well reproduced. The robust performance of the revised force field, in conjunction with its efficiency through implicit treatment of solvent and counterions, provides a valuable tool for elucidating conformations and structure-function relationships of DNA, including those of molecules modified by carcinogens and other ligands.

Base sequence effects in bending induced by bulky carcinogen-DNA adducts: experimental and computational analysis.

The covalent binding of bulky mutagenic or carcinogenic compounds to DNA can lead to bending, which could significantly alter the interactions of DNA with critical replication and transcription proteins. The impact of adducts derived from the highly reactive bay region enantiomeric (+)- and (-)-anti-7,8-diol-9,10-epoxide derivatives of benzo[a]pyrene (BPDE) are of interest because the (+)-7R,8S,9S,10R-anti-BPDE enantiomer is highly tumorigenic in rodents, while the (-)-7S,8R,9R,10S-anti-BPDE enantiomer is not. Both (+)- and (-)-anti-BPDE bind covalently with DNA predominantly by trans addition at the exocyclic amino group of guanine to yield 10S (+)- and 10R (-)-trans-anti-[BP]-N(2)-dG adducts. We have synthesized a number of different oligonucleotides with single (+)- and (-)-trans-anti-[BP]-N(2)-dG adducts (G) in the base sequence context XG*Y, where X and Y are different DNA bases. The G* residues were positioned at or close to the center of 11 base pair ( approximately 1 helical turn) or 16 base pair ( approximately 1.5 turns) duplexes. All bases, except for X and Y and their partners, were identical. These sequences were self-ligated with T4 ligase to form multimers that yield a ladder of bands upon electrophoresis in native polyacrylamide gels. The extent of bending in each oligonucleotide was assessed by monitoring the decrease in gel mobilities of these linear, self-ligated oligomers, relative to unmodified oligonucleotides of the same base sequence. The extent of global bending was then estimated using a sequence-specific three-dimensional model from which the values of the base-pair step parameter roll adjacent to the lesion site could be extracted. We find that (+)-trans-anti-[BP]-N(2)-dG adducts are considerably more bent than the (-) isomers regardless of sequence and that A-T base pairs flanking the [BP]-N(2)-dG lesion site allow for local flexibility consistent with adduct conformational heterogeneity. Interestingly, the fit of computed versus observed gel mobilities using classical reptation treatments requires enhancement of unmodified DNA flexibility in gels, compared to aqueous salt solution. The differences in bending between the two stereoisomeric adduct duplexes and the observed base sequence context effects may play a significant role in the differential processing of these lesions by cellular replication, transcription, and repair enzymes.