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Biomedical subjects

R A Kumar

Publications and source records attributed to R A Kumar.

At least 19 recordsLinked to original sources
PubMed2007

Predictive value of IL-18 and SC5b-9 for neurocognitive dysfunction after cardiopulmonary bypass.

BACKGROUND: Neurological injury after cardiopulmonary bypass (CPB) continues to be a major problem after cardiac surgery. The aim of this study was to investigate the predictive value of Interleukin-18 (IL-18) and SC5b-9 as biochemical markers of neurocognitive dysfunction after cardiac surgery. METHODS: A total of 30 patients undergoing elective cardiac surgery using CPB were recruited. Blood samples were obtained for IL-18 and SC5b-9 concentrations before induction, 24, 48, 72, 96 and 120 h post-CPB and 6 weeks after operation. In addition, patients underwent a standard battery of neuropsychometric tests before operation and at day 5 and 6 weeks after operation. RESULTS: Serum concentration of IL-18, but not SC5b-9, was significantly different between patients with and without neurocognitive dysfunction; serum IL-18 concentration significantly increased in patients with neurocognitive dysfunction (P = 0.018). Neurological outcome was significantly dependent on peak difference in IL-18 concentration at day 5 (P = 0.033), but not on peak difference in SC5b-9 concentration (P = 0.16). Eight patients had neurocognitive dysfunction at day 5 and three had neurocognitive dysfunction at 6 weeks. In a very small number of patients, no significant association was demonstrated between IL-18 or SC5b-9 concentrations and neurocognitive dysfunction at 6 weeks. CONCLUSIONS: IL-18 has the potential as a useful marker of neurological dysfunction, requiring further investigation.

Aged↗
PubMed2006

Mutation and evolutionary analyses identify NR2E1-candidate-regulatory mutations in humans with severe cortical malformations.

Nuclear receptor 2E1 (NR2E1) is expressed in human fetal and adult brains; however, its role in human brain-behavior development is unknown. Previously, we have corrected the cortical hypoplasia and behavioral abnormalities in Nr2e1(-/-) mice using a genomic clone spanning human NR2E1, which bolsters the hypothesis that NR2E1 may similarly play a role in human cortical and behavioral development. To test the hypothesis that humans with abnormal brain-behavior development may have null or hypomorphic NR2E1 mutations, we undertook the first candidate mutation screen of NR2E1 by sequencing its entire coding region, untranslated, splice site, proximal promoter and evolutionarily conserved non-coding regions in 56 unrelated patients with cortical disorders, namely microcephaly. We then genotyped the candidate mutations in 325 unrelated control subjects and 15 relatives. We did not detect any coding region changes in NR2E1; however, we identified seven novel candidate regulatory mutations that were absent from control subjects. We used in silico tools to predict the effects of these candidate mutations on neural transcription factor binding sites (TFBS). Four candidate mutations were predicted to alter TFBS. To facilitate the present and future studies of NR2E1, we also elucidated its molecular evolution, genetic diversity, haplotype structure and linkage disequilibrium by sequencing an additional 94 unaffected humans representing Africa, the Americas, Asia, Europe, the Middle East and Oceania, as well as great apes and monkeys. We detected strong purifying selection, low genetic diversity, 21 novel polymorphisms and five common haplotypes at NR2E1. We conclude that protein-coding changes in NR2E1 do not contribute to cortical and behavioral abnormalities in the patients examined here, but that regulatory mutations may play a role.

Animals↗
PubMed2002

Intrafamilial correlation of clinical manifestations in neurofibromatosis 2 (NF2).

Measuring correlation in clinical traits among relatives is important to our understanding of the causes of variable expressivity in Mendelian diseases. Random effects models are widely used to estimate intrafamilial correlations, but such models have limitations. We incorporated survival techniques into a random effects model so that it can be used to estimate intrafamilial correlations in continuous variables with right censoring, such as age at onset. We also describe a negative-binomial gamma mixture model to determine intrafamilial correlations of discrete (e.g., count) data. We demonstrate the utility of these methods by analyzing intrafamilial correlations among patients with neurofibromatosis 2 (NF2), an autosomal-dominant disease caused by mutations of the NF2 tumor-suppressor gene. We estimated intrafamilial correlations in age at first symptom of NF2, age at onset of hearing loss, and number of intracranial meningiomas in 390 NF2 nonprobands from 153 unrelated families. A significant intrafamilial correlation was observed for each of the three features: age at onset (0.35; 95% confidence interval (CI) 0.23-0.47), age at onset of hearing loss (0.51; 95% CI, 0.35-0.64), and number of meninginomas (0.29; 95% CI, 0.15-0.43). Significant correlations were also observed for age at first symptom within NF2 families with truncating mutations (0.41; 95% CI, 0.06-0.68) or splice-site mutations (0.29; 95% CI, 0.03-0.51), for age at onset of hearing loss within families with missense mutations (0.67; 95% CI, 0.18-0.89), and for number of meningiomas within families with splice-site mutations (0.39; 95% CI, 0.13-0.66). Our findings are consistent with effects of both allelic and nonallelic familial factors on the clinical variability of NF2.

Adolescent↗
PubMed2002

Experimental studies on a new bioactive material: HAIonomer cements.

The lack of exotherm during setting, absence of monomer and improved release of incorporated therapeutic agents has resulted in the development of glass ionomer cements (GICs) for biomedical applications. In order to improve biocompatibility and biomechanically match GICs to bone, hydroxyapatite-ionomer (HAIonomer) hybrid cements were developed. Ultra-fine hydroxyapatite (HA) powders were produced using a new induction spraying technique that utilizes a radio-frequency source to spheriodize an atomized suspension containing HA crystallites. The spheriodized particulates were then held at 800 degrees C for 4 h in a carbolite furnace using a heating and cooling rate of 25 degrees C/min to obtain almost fully crystalline HA powders. The heat-treated particles were characterized and introduced into a commercial glass ionomer cement. 4 (H4), 12 (H12) and 28 (H28) vol% of fluoroalumino silicate were substituted by crystalline HA particles that were dispersed using a high-speed dispersion technique. The HAIonomer cements were subjected to hardness, compressive and diametral tensile strength testing based upon BS6039:1981. The storage time were extended to one week to investigate the effects of cement maturation on mechanical properties. Commercially available capsulated GIC (GC) and GIC at maximum powder:liquid ratio (GM) served as comparisons. Results were analyzed using factorial ANOVA/Scheffe's post-hoc tests and independent samples t-test at significance level 0.05. The effect of time on hardness was material dependent. With the exception of H12, a significant increase in hardness was observed for all materials at one week. A significant increase in compressive strength was, however, observed for H12 over time. At 1 day and 1 week, the hardness of H28 was significantly lower than for GM, H4, and H12. No significant difference in compression and diametral tensile strengths were observed between materials at both time intervals. Results show that HAIonomers is a promising material, which possess good mechanical properties. Potential uses of this new material include bone cements and performed implants for hard tissue replacement in the field of otological, oral-maxillofacial and orthopedic surgery.

Biocompatible Materials↗
PubMed2000

Genetics of anesthetic response: autosomal mutations that render Drosophila resistant to halothane.

Molecular mechanisms of anesthetic action are poorly understood. Genetic approaches to investigate mechanisms of anesthesia, although sparse and rather new, are turning out to be informative and add a new perspective. Before beginning a systematic investigation of anesthesia by this approach, it is necessary to have at hand a large collection of mutations in different loci that alter anesthetic response. We report here the isolation and characterization of six mutant autosomal lines that show a decreased sensitivity to the inhalation of anesthetic halothane. Two of these mutations, Omar(82) and Qajjem(211) are shown to map to separate loci on the third chromosome.

Anesthetics, Inhalation↗
PubMed1999

New features and enhancements in the X-PLOR computer program.

This article describes new methods for X-ray crystallographic refinement and nuclear magnetic resonance (NMR) structure determination that are available in the recent release of the X-PLOR software, X-PLOR 98.0. The major new features of the X-PLOR 98.0 software are: (i) the introduction of maximum likelihood methods (Pannu and Read, Acta Crystallogr 1996;A52:659-668) for X-ray crystallographic refinement with structure factor amplitude, intensity and phase probability targets, (ii) the addition of the Andersen thermal coupling method for temperature control during simulated annealing refinements, (iii) a new utility function for converting reflection data in to the X-PLOR format, (iv) validated scripts and performance enhancements for structure determination from NMR distance restraints using torsion angle dynamics, (v) fast code for direct nuclear Oberhauser effect (NOE) refinement using matrix doubling and gaussian quadratures, (vi) methodologies for using ambiguous restraint information to perform automated iterative peak assignment and structure determination (Nilges et al., J Mol Biol 1997;269: 408-422). Additional developments in methodology for refining crystal structures from poor initial models include the implementation of a fast adaptive bulk solvent scattering correction and an energy minimization routine that makes use of second derivative information. Trial crystallographic refinements with an energy minimization protocol that includes these enhancements indicate significantly improved convergence. The quality of the resulting models appears comparable to models obtained from refinement protocols that incorporate torsion angle dynamics. Test applications of the new energy minimizer to NMR structure refinement with using NOE calculations also show improved convergence, leading to more optimized final models.

Crystallography, X-Ray↗
PubMed1999

Probing cell-surface architecture through synthesis: an NMR-determined structural motif for tumor-associated mucins.

Cell-surface mucin glycoproteins are altered with the onset of oncogenesis. Knowledge of mucin structure could be used in vaccine strategies that target tumor-associated mucin motifs. Thus far, however, mucins have resisted detailed molecular analysis. Reported herein is the solution conformation of a highly complex segment of the mucin CD43. The elongated secondary structure of the isolated mucin strand approaches the stability of motifs found in folded proteins. The features required for the mucin motif to emerge are also described. Immunocharacterization of related constructs strongly suggests that the observed epitopes represent distinguishing features of tumor cell-surface architecture.

Antigens, CD↗
PubMed1999

Biodegradation of tannic acid by Citrobacter freundii isolated from a tannery effluent.

A bacterial strain capable of utilizing tannic acid as sole carbon source was isolated from the effluent of a tannery and was identified as Citrobacter freundii. This organism could grow at concentrations as high as 5% (w/v) of tannic acid and produced extracellular tannase to hydrolyze the same. When grown in minimal medium containing 1% tannic acid (w/v) at 30 degrees C, this strain produced 1.87 U/ml of tannase at 6 h. At that time, tannic acid degradation products, namely glucose and gallic acid, were detectable in the culture filtrate; the other intermediate metabolites formed were pyrogallol (extracellular) and pyruvate (intracellular). 2-hydroxymuconic acid is presumed to form as a result of ortho-cleavage of pyrogallol. The proposed biochemical pathway for the degradation of tannic acid by Citrobacter freundii is: Tannic acid-->[Glucose + Gallic acid]-->Pyrogallol -->2-hydroxymuconic acid -->[?]-->Pyruvate.

Biodegradation, Environmental↗
PubMed1998

Presence and formation of heme and occurrence of certain heme proteins in the filarial parasite Setaria digitata.

There is currently renewed interest in the biological significance of heme proteins. The most common heme proteins include hemoglobin, myoglobin, cytochromes, and redox enzymes such as catalase and peroxidase. Setaria digitata is a cattle filarial parasite, which is devoid of typical cytochrome systems. However, studies showed activities of delta Aminolevulinate synthase (ALAS), delta Aminolevulinate dehydratase (ALAD), and heme oxygenase in appreciable amounts, suggesting the presence of necessary equipment for the biosynthesis of heme. This is further confirmed by the end product inhibition of ALAS by heme and the observation of the death of the parasite by succinyl acetone, an inhibitor of the biosynthesis of heme. Though typical cytochrome systems are absent, microsomal cytochrome P 450 and elevated levels of heme containing enzymes such as catalase and peroxidase are present in the parasite. A unique hemoglobin is also detected which shows a difference in biological functions from the host system and that of the much-studied nematode parasite Ascaris sum.

5-Aminolevulinate Synthetase↗
PubMed1998

Beta-sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: implications for Alzheimer's therapy.

Inhibition of cerebral amyloid beta-protein deposition seems to be an important target for Alzheimer's disease therapy. Amyloidogenesis could be inhibited by short synthetic peptides designed as beta-sheet breakers. Here we demonstrate a 5-residue peptide that inhibits amyloid beta-protein fibrillogenesis, disassembles preformed fibrils in vitro and prevents neuronal death induced by fibrils in cell culture. In addition, the beta-sheet breaker peptide significantly reduces amyloid beta-protein deposition in vivo and completely blocks the formation of amyloid fibrils in a rat brain model of amyloidosis. These findings may provide the basis for a new therapeutic approach to prevent amyloidosis in Alzheimer's disease.

Alzheimer Disease↗
PubMed1997

Structure, recognition and adaptive binding in RNA aptamer complexes.

Novel features of RNA structure, recognition and discrimination have been recently elucidated through the solution structural characterization of RNA aptamers that bind cofactors, aminoglycoside antibiotics, amino acids and peptides with high affinity and specificity. This review presents the solution structures of RNA aptamer complexes with adenosine monophosphate, flavin mononucleotide, arginine/citrulline and tobramycin together with an example of hydrogen exchange measurements of the base-pair kinetics for the AMP-RNA aptamer complex. A comparative analysis of the structures of these RNA aptamer complexes yields the principles, patterns and diversity associated with RNA architecture, molecular recognition and adaptive binding associated with complex formation.

Adenosine Monophosphate↗
PubMed1997

Solution structure of the calicheamicin gamma 1I-DNA complex.

Calicheamicin gamma 1I is an enediyne antibiotic possessing antitumour activity associated with its ability to bind and following activation, affect double-strand cleavage at oligopyrimidine-oligopurine tracts on DNA. Footprinting and chemical modification studies have identified the (T-C-C-T).(A-G-G-A) sequence as a preferred calicheamicin gamma 1I binding site and established the importance of the 5'-guanine residue as critical for high affinity binding. The sequence specificity of intermolecular recognition has been identified with the aryltetrasaccharide component of the drug together with an important contribution from the iodine atom on the thiobenzoate ring to the affinity of complex formation. Calicheamicin gamma 1I binds to the minor groove of the DNA duplex and in the process positions the enediyne ring to abstract hydrogen atoms from partner strands leading to double-strand cleavage. We report on the solution structure of the calicheamicin gamma 1I-DNA hairpin duplex complex containing a central (T-C-C-T).(A-G-G-A) segment based on a combined analysis of NMR and molecular dynamics calculations including intensity refinement in a water box. The refined solution structures of the complex provide a molecular explanation of the sequence specificity of binding and cleavage by this member of the enediyne family of antitumor antibiotics. Calicheamicin gamma 1I binds to the DNA minor groove with its aryltetrasaccharide segment in an extended conformation spanning the (T-C-C-T).(A-G-G-A) segment of the duplex. Further, the thio sugar B molecule and the thiobenzoate ring C molecule are inserted in an edgewise manner deep into the minor groove with their faces sandwiched between the walls of the groove. A range of intermolecular hydrophobic and hydrogen-bonding interactions account for the sequence specific recognition in the complex. These include critical intermolecular contacts between the iodine and sulfur atoms of the thiobenzoate ring of the drug with the exposed exocyclic amino protons of the 5' and 3'-guanine bases, respectively, of the A-G-G-A segment on the DNA. The bound aryltetrasaccharide in turn positions the enediyne ring deep in the minor groove such that the pro-radical carbon centers of the enediyne are proximal to their anticipated proton abstraction sites. Specifically, the pro-radical C-3 and C-6 atoms are aligned opposite the abstractable H-5' (pro-S) and H-4' protons on partner strands across the minor groove, respectively, in the complex. The DNA duplex is right-handed with Watson-Crick base-pairing in the complex. The helix exhibits a B-DNA type minor groove width at the aryltetrasaccharide binding-site while there is widening of the groove at the adjacent enediyne binding-site in the complex. The DNA helix exhibits localized perturbations at the binding-site as reflected in imino proton complexation shifts and specific altered sugar pucker geometrics associated with complex formation. Sequence-specific binding of calicheamicin gamma 1I to the (T-C-C-T).(A-G-G-A) containing DNA hairpin duplex is favored by the complementarity of the fit through hydrophobic and hydrogen-bonding interactions between the drug and the floor and walls of the minor groove of a minimally perturbed DNA helix.

Aminoglycosides↗
PubMed1997

Solution structure of the esperamicin A1-DNA complex.

Esperamicin A1 is an enediyne antibiotic possessing antitumor activity associated with its ability to bind and, following activation, affect strand cleavage of DNA. We report on the solution structure of the esperamicin A1-d(C-G-G-A-T-C-C-G) duplex complex based on a combined analysis of NMR and molecular dynamics calculations including intensity refinement in a water box. The refined solution structures of the complex provide a molecular explanation of the sequence specificity for binding and cleavage by this member of the enediyne family of antitumor antibiotics. Esperamicin A1 binds to the DNA minor groove with its methoxyacrylyl-anthranilate moiety intercalating into the helix at the (G2-G3)-(C6'-C7') step. The methoxyacrylyl-anthranilate intercalator and the minor groove binding A-B-C+ risaccharide moieties rigidly anchor the enediyne in the minor groove such that the pro-radical centers of the enediyne are proximal to their anticipated proton abstraction sites. Specifically, the pro-radical C-3 and C-6 atoms are aligned opposite the abstractable H-5' (pro-S) proton of C6 and the H-1' proton of C6' on partner strands, respectively, in the complex. The thiomethyl sugar B residue is buried deep in an edgewise manner in the minor groove with its two faces sandwiched between the walls of the groove. Further, the polarizable sulfur atom of the thiomethyl group of sugar B residue is positioned opposite and can hydrogen-bond to the exposed amino proton of G3' in the complex. There is little perturbation away from a right-handed Watson-Crick base-paired duplex in the complex other than unwinding of the helix at the intercalation site and widening of the minor groove centered about the enediyne-binding and anthranilate intercalation sites. Sequence-specific binding of esperamicin A1 to the d(C-G-G-A-T-C-C-G) duplex is favored by the complementarity of the fit between the drug and the floor of the minor groove, good stacking between the intercalating anthranilate ring and flanking purine bases and intermolecular hydrogen-bonding interactions.

Aminoglycosides↗
PubMed1996

Conformational influences of glycosylation of a peptide: a possible model for the effect of glycosylation on the rate of protein folding.

Improved strategies for synthesis make it possible to expand the range of glycopeptides available for detailed conformational studies. The glycopeptide 1 was synthesized using a new solid phase synthesis of carbohydrates and a convergent coupling to peptide followed by deprotection. Its conformational properties were subjected to NMR analysis and compared with a control peptide 2 prepared by conventional solid phase methods. Whereas peptide 2 fails to manifest any appreciable secondary structure, the glycopeptide 1 does show considerable conformational bias suggestive of an equilibrium between an ordered and a random state. The implications of this ordering effect for the larger issue of protein folding are considered.

Carbohydrate Conformation↗
PubMed1996

RNA folding topology and intermolecular contacts in the AMP-RNA aptamer complex.

We report below on the NMR structural characterization of the complex between AMP and a 40-mer RNA aptamer in aqueous solution. Resonance assignments are based on multinuclear multidimensional NMR studies on complexes uniformly 13C, 15N-labeled with either AMP or the RNA aptamer. AMP binds to an internal loop (labeled G7-G8-A9-A10-G11-A12-A13-A14-C15-U16-G17) and bulge (G34 positioned opposite the internal loop) segment in the RNA aptamer, and our NMR study provides insights into features of the RNA folding topology and the molecular recognition events in the AMP binding pocket on the RNA. Specifically, the helical stems are extended by G-G mismatch formation from either direction into the internal loop/bulge segment of the RNA aptamer on complex formation. The internal loop adopts a unique fold with the purine ring of AMP intercalated between A10 and G11 in the complex. The G8-A9-A10-AMP segment adopts certain stacking features in common with a GNRA turn and is closed by the G7.G11 mismatch pair. The purine rings of A12 and G34 (syn) are stacked on each other and participate in stablizing the AMP intercalation site. A large number of intermolecular NOEs have been identified between the AMP ligand and the G8, A10, G11, G17, U18, and G34 residues on the RNA aptamer in the complex. The Watson-Crick edge of the AMP is oriented toward the exocyclic amino group of G8, suggestive of a hydrogen-bonding alignment between G8 and AMP in the complex. The AMP sugar ring is positioned in the minor groove of the rightward helical stem centered about the G17.G34 mismatch and U18.A33 Watson-Crick pairs. The AMP binds to one face of the folded internal loop/bulge segment of the RNA aptamer while the opposite face is capped by a stacked alignment of the A13-A14-C15-U16 segment located toward the 3'-end of the internal loop segment. Globally, the two helical stems of the RNA aptamer are aligned approximately orthogonal to each other with tertiary interactions centered about the internal loop/bulge segment generating the AMP binding site on the RNA.

Adenosine Monophosphate↗
PubMed1996

Structural basis of RNA folding and recognition in an AMP-RNA aptamer complex.

The catalytic properties of RNA and its well known role in gene expression and regulation are the consequence of its unique solution structures. Identification of the structural determinants of ligand recognition by RNA molecules is of fundamental importance for understanding the biological functions of RNA, as well as for the rational design of RNA Sequences with specific catalytic activities. Towards this latter end, Szostak et al. used in vitro selection techniques to isolate RNA sequences ('aptamers') containing a high-affinity binding site for ATP, the universal currency of cellular energy, and then used this motif to engineer ribozymes with polynucleotide kinase activity. Here we present the solution structure, as determined by multidimensional NMR spectroscopy and molecular dynamics calculations, of both uniformly and specifically 13C-, 15N-labelled 40-mer RNA containing the ATP-binding motif complexed with AMP. The aptamer adopts an L-shaped structure with two nearly orthogonal stems, each capped proximally by a G x G mismatch pair, binding the AMP ligand at their junction in a GNRA-like motif.

Adenosine Monophosphate↗