Biomedical subjects

R Murugan

Publications and source records attributed to R Murugan.

At least 19 recordsLinked to original sources

PubMed2006

Effect of alcohols on binding of camphor to cytochrome P450cam: spectroscopic and stopped flow transient kinetic studies.

Addition of alcohols to cytochrome P450cam (CYP101) was shown to release the substrate camphor from the heme pocket of the enzyme. The release of the substrate was found to be caused both due to increased solubility of the substrate in solution in presence of alcohol and due to change in the tertiary structure of the active site of the enzyme. The far-UV CD and near-UV CD spectra reveal that addition of alcohols to cytochrome P450cam cause a small change in the secondary structural elements but a significant change in the tertiary structural organization of this enzyme. The CD spectra at the heme region at various concentrations of alcohols indicate a substantial change in the tertiary structural organization around the heme moiety too. The equilibrium constant associated with the binding of camphor to Cyt P450cam is strongly dependent on the concentration of alcohols and the corresponding free energy associated with the binding is found to scale linearly with the concentration of alcohols. Kinetic experiments on binding of camphor to Cyt P450cam show that both k(on) and k(off) rate constants are strongly affected by addition of alcohols suggesting that alcohol expel camphor out of the heme cavity of Cyt P450cam by affecting tertiary structure of Cyt P450cam as well as by modifying the solubility properties of camphor in aqueous medium.

Alcohols↗

PubMed2006

Effect of external fluctuations on the affinity-specificity negative correlation in DNA-probe interactions.

We show that the site-specific interaction of a probe DNA with the template DNA can be well modeled as an unbiased random jump process, where the probe molecule first nonspecifically binds to the template DNA and then searches for the specific site via unbiased random jump motion on the template DNA. By investigating the effects of increasing the jump size, and the fluctuations in the position of the specific site and the fluctuations in the specific site interval on the affinity-specificity negative correlation, we show that (1) increasing the jump size will in turn increase the affinity of the probe toward its target site on the template DNA, however, with a limiting value--the maximum affinity condition; (2) the degree of supercoiling or condensation of the template DNA as well as the electrostatic interactions between the probe and the template in turn control the jump size associated with the dynamics of the probe on the template DNA; (3) under a maximum specificity condition (therefore with minimum affinity), by introducing an external fluctuation in the relative position of the target site on the template DNA with respect to the probe, one can still improve the affinity rate; (4) on the other hand, one can improve the specificity of the probe toward the target site on the template DNA by introducing external fluctuations in the target-site interval. Finally, we propose the design strategies and optimum experimental conditions to simultaneously enhance the affinity as well as the specificity of probe toward its target site on the template DNA.

Binding Sites↗

PubMed2006

Stochastic transcription initiation: Time dependent transcription rates.

The noise in the central process such as transcription, replication and translation of the genomic DNA is very important since it can directly affect the phenotypic and behavioral aspects of an organism as well as the entire cellular function. Here we develop a model on the transcription process based on the assumption that the initiation of the transcription is a stochastic event and the transcription rates may be time dependent random quantities. We derive the central measure properties i.e. mean and the variance, of the distribution of the transcription rates. Our results show that the Fano factor which is a measure of deviation from the Poisson distribution associated with the fluctuations in the number of mRNA molecules deviates from unity due to the randomness in the transcription rates. However when the RNA polymerase molecule searches for the promoter sequences on the DNA lattice by random jumps, the Fano factor approaches the Poisson limit as the jump size associated with the RNA polymerase increases. Since the jump size associated with dynamics of RNAP molecule is positively correlated with the degree of super coiling of DNA, we argue that the super coiled or close-packed structure of DNA might have evolved to keep the noises at the transcriptional level in a minimum.

DNA-Directed RNA Polymerases↗

PubMed2005

Critical jump sizes in DNA-protein interactions.

Interaction of a protein molecule with a specific-site on the DNA lattice can be modeled as an unbiased random jump process. Here we show that there exists a critical jump size (kc) beyond which site-specific association of a protein molecule with a DNA lattice cannot be facilitated. The maximum achievable association rate is predicted to be approximately 10(10) mol-1 s-1. This critical jump size scales with the total length of DNA lattice (N) as kc proportional, variantN2/3. Beyond kc the mean first passage time MFPT (denoted as T) required for the protein molecule to target the specific-site follows a linear scaling law as T proportional, variantN rather than the usual T proportional, variantN2 scaling law. On the basis of these results we argue that the evolution of the super coiled structures of the genomic DNA must be a consequence of the existence of this critical jump sizes. We finally show that the random jump method of searching for the specific-site by the protein molecule on the DNA lattice itself introduce an abstract linear type potential favoring the site-specific association rate.

Binding Sites↗

PubMed2005

Production of ultra-fine bioresorbable carbonated hydroxyapatite.

Ionic-substituted hydroxyapatite (HAp) based materials may be a better choice than pure HAp owing to their similarity in chemical composition with biological apatite. The present study reports a process for the production of carbonated hydroxyapatite (CHAp) using microwaves. The CHAp was evaluated for its phase purity, chemical homogeneity, functionality, morphology, and solubility. The CHAp thus obtained was compared with a pure HAp and a biological apatite, which provides quite an interesting insight into the carbonate substitution. The in vitro ionic dissolution rates determined under physiological conditions clearly demonstrate the soluble nature of CHAp compared to HAp. The overall results indicate that the processed CHAp has increased resorption relative to pure HAp and has a chemical composition corresponding to some extent with that of biological apatite.

Apatites↗

PubMed2005

On the origin of affinity--specificity negative correlation in DNA--probe interactions.

Affinity (the fastness) and specificity (the correctness) are the two important factors that decide the efficiency of a nucleic acid probe to target its specific site on a DNA lattice. DNA-probe interactions differ from protein-ligand interactions in a way that here the specificity and the affinity of the interactions correlate negatively with each other. We present a simple phenomenological theory to explain the negative correlation between the specificity and the affinity of the probe towards its target site on the template DNA under solution conditions. We show that a simple random jump model can explain this fact and we also predict that the negative correlation between the affinity and specificity diminishes as the temperature increases or the viscosity of the medium decreases. Moreover, the length of target DNA and the distance between the initial position of the probe on the template DNA lattice and the target site increases the magnitude of affinity-specificity negative correlation. These results are consistent with experimental observations. Finally we propose practical strategies to coherently improve the specificity and the affinity with respect to important molecular biological techniques such as PCR and Southern blotting.

Affinity Labels↗

PubMed2005

Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering.

Efficacy of aligned poly(l-lactic acid) (PLLA) nano/micro fibrous scaffolds for neural tissue engineering is described and their performance with random PLLA scaffolds is compared as well in this study. Perfectly aligned PLLA fibrous scaffolds were fabricated by an electrospinning technique under optimum condition and the diameter of the electrospun fibers can easily be tailored by adjusting the concentration of polymer solution. As the structure of PLLA scaffold was intended for neural tissue engineering, its suitability was evaluated in vitro using neural stem cells (NSCs) as a model cell line. Cell morphology, differentiation and neurite outgrowth were studied by various microscopic techniques. The results show that the direction of NSC elongation and its neurite outgrowth is parallel to the direction of PLLA fibers for aligned scaffolds. No significant changes were observed on the cell orientation with respect to the fiber diameters. However, the rate of NSC differentiation was higher for PLLA nanofibers than that of micro fibers and it was independent of the fiber alignment. Based on the experimental results, the aligned nanofibrous PLLA scaffold could be used as a potential cell carrier in neural tissue engineering.

Animals↗

PubMed2004

Role of substrate on the conformational stability of the heme active site of cytochrome P450cam: effect of temperature and low concentrations of denaturants.

The effect of 1R-camphor on the conformational stability of the heme active site of cytochrome P450cam has been investigated. The absorption spectra of the heme moiety showed the presence of two hitherto unknown intermediates formed at low urea concentrations or during small temperature perturbations. The corresponding thermodynamic parameters were obtained by global fitting of the experimental data to a generalized sequential unfolding model at different wavelengths, which showed that the active conformation of the enzyme is stabilized by binding of the substrate at the active site. Circular-dichroism spectra of the enzyme in the visible- and far-UV region were studied to identify the critical range of denaturant concentration and the temperature at which the tertiary structure around the heme center was affected with almost no change in the secondary structure of the enzyme. This critical range of urea concentration was 0-2.8 M in the presence of camphor and 0-1.5 M in the absence of camphor. The tertiary structure of the enzyme was found to undergo conformational change in the temperature range 20-60 degrees C in the presence of the substrate and 20-47 degrees C in its absence. The spectral assignments of the intermediate species of the heme active site with the intact secondary structure of the enzyme were made by deconvolution of the Soret absorption spectra, and the results were analyzed to determine stabilization of the heme active-site geometry by 1R-camphor. Results showed that subtle conformational changes due to melting of the tertiary contacts in the active site lead to formation of intermediates which are coordinatively similar to the native enzyme. Analogous intermediate species might be responsible for leakage in the redox catalytic cycle of the enzyme.

Binding Sites↗

PubMed2004

DNA-protein interactions under random jump conditions.

We model the site-specific association of a protein molecule with DNA as a random walk with random jumps. Results show that the simultaneous occurrence of processes such as sliding, hopping, and intersegmental transfer can facilitate the diffusion-controlled site-specific association rate. We have also shown that sliding would dominate at lower DNA length, whereas at higher lengths hopping and intersegmental transfer would dominate. Apart from this, we predict that the association rate is directly proportional to the size of nonspecific DNA that flanks the specific site. These results are consistent with the experimental observations.

Binding Sites↗

PubMed2004

Fabrication of nano-structured porous PLLA scaffold intended for nerve tissue engineering.

Nerve tissue engineering (NTE) is one of the most promising methods to restore central nerve systems in human health care. Three-dimensional distribution and growth of cells within the porous scaffold are of clinical significance for NTE. In this study, an attempt was made to develop porous polymeric nano-fibrous scaffold using a biodegradable poly(L-lactic acid) (PLLA) for in vitro culture of nerve stem cells (NSCs). The processing of PLLA scaffold has been carried out by liquid-liquid phase separation method. The physico-chemical properties of the scaffold were fully characterized by using differential scanning calorimetry and scanning electron microscopy. These results confirmed that the prepared scaffold is highly porous and fibrous with diameters down to nanometer scale. As our nano-structured PLLA scaffold mimics natural extracellular matrix, we have intended this biodegradable scaffold as cell carrier in NTE. The in vitro performance of NSCs seeded on nano-fibrous scaffold is addressed in this study. The cell cultural tests showed that the NSCs could differentiate on the nano-structured scaffold and the scaffold acted as a positive cue to support neurite outgrowth. These results suggested that the nano-structured porous PLLA scaffold is a potential cell carrier in NTE.

Animals↗

PubMed2004

Coupling of therapeutic molecules onto surface modified coralline hydroxyapatite.

Surface modification and coupling of therapeutic molecules, tetracycline, onto coralline hydroxyapatite (CHA) and their in vitro evaluations were described in this study. Initially, CHA was graft polymerized with glycidylmethacrylate (GMA) using redox initiators and subsequently coupled to tetracycline through epoxy groups. The CHA grafted with polyGMA (CHA-g-PGMA) was characterized by Fourier transform infrared spectroscopy and powder X-ray diffraction (XRD) for proof of grafting. The absorption peaks pertaining to epoxy and ester carbonyl groups were observed for the graft polymer due to PGMA grafting. The XRD results signified that there was no secondary phase in the apatite lattice and crystallinity was also not affected by grafting, which suggested that the PGMA chains were grafted only on the surface of CHA. Drug loading and releasing was evaluated and found that CHA-g-PGMA exhibited higher loading efficiency than CHA. The in vitro release of tetracycline was performed in phosphate buffered saline under physiological condition and the release profiles showed that the tetracycline-containing graft polymer releases the drug for prolonged period as compared to CHA. Based on the experimental results, CHA-g-PGMA appears to be a promising biomaterial for drug delivery.

Adsorption↗

PubMed2004

Bioresorbable composite bone paste using polysaccharide based nano hydroxyapatite.

Aim of this study concerns to the development of bioresorbable composite materials for bone repair and regeneration. Despite nano hydroxyapatite (HA) has wide range of medical applications, particles mobilization and slow resorbable nature limits its use in certain applications particularly, periodontal and alveolar ridge augmentation. To enhance its usage, we have prepared HA composite bone paste with a natural polysaccharide, chitosan, using wet chemical method at low temperature. The prepared composites were analyzed by various physicochemical methods and suggesting that the nano HA crystallites are well intact with the chitosan macromolecules. FT-IR results are indicating the existence of hydroxyl and amide groups in addition to the characteristic peaks of nano HA in the composite paste. The physical nature of paste form implies that it would be highly beneficial for the particle immobilization upon implantation. In vitro physiological stability and solubility of the composite was performed in phosphate buffered saline under physiological condition and found that the rate of resorbability of composite was quite higher than nano HA. These findings suggest that the HA/chitosan composites may have a great impact on human health care systems as bioresorbable bone substitute.

Absorbable Implants↗

PubMed2003

A theory on the origin of cooperativity in DNA renaturation kinetics.

Theoretical proof for the existence of cooperativity (experimental evidences have already reported in our earlier work: Biochem. Biophys. Res. Commun., 293 (2002) 870) in the phenomenon of DNA renaturation has been obtained using a generalized birth-death master equation approach. Results have shown that no special searching mechanism (i.e. a facilitated mechanism) was necessary for complementary DNA strands to find their correct-contacts but searching by a 'random jump' method was enough to explain not only the observed cooperativity phenomenon but also the magnitude of correct-contact-forming rate constant. Impact of cooperativity phenomenon on length of primers and their specificity in polymerase chain reaction has also been discussed.

Base Pairing↗

PubMed2003

A stochastic model on DNA renaturation kinetics.

A simple stochastic model on DNA renaturation kinetics in the presence and absence of cooperativity have been developed [the corresponding deterministic models have been explicitly treated in our previous work. Biochem Biophys Res Commun 293 (2002) 870-873]. Theoretical mean and variance of number of bases in single-stranded DNA (ssDNA), (which is of course a random variable) have been calculated and compared with the experimental values. The results showed that only the cooperative model correctly predicted the time t(m) at which variance becomes maximum whereas, the non-cooperative model overestimated it and thus proved the validity of the cooperative model. Some of the applications of this cooperative theory in resolving the problems of the central dogma of life, PCR etc. have also been discussed.

Algorithms↗

PubMed2003

Competitive model on denaturant-mediated protein unfolding.

A denaturant-mediated protein unfolding model, which is different from already existing ones based on the assumption that denaturant competes for water molecules to interact and thus reduces water-protein interactions, which leads to unfolding phenomenon, has been developed with a detailed mathematical justification. Theoretical results suggested that the parameter (m(u)) obtained from the usual linear extrapolation model must be a linear function of the number of bound water molecules (n) on protein with a zero intercept. However, application of this theory to a set of proteins for which m(u) values for urea denaturation are already known showed that m(u) was a linear function of n but with a nonzero intercept. Finally this nonzero intercept was attributed to binding of denaturant to protein at n = 0. Detailed investigation of this factor showed that average equilibrium constant for binding of urea with aromatic side chains (generally nonpolar side chains) was k(b) approximately 0.65 +/- 0.45 mol(-1), which agreed well with earlier experimental estimations, and also suggested that an integrated approach was necessary to avoid discrepancy in deltaG(H(2)O) estimated from different models.

Binding, Competitive↗

PubMed2003

Graft polymerization of glycidylmethacrylate onto coralline hydroxyapatite.

Graft polymerization of glycidylmethacrylate (GMA) onto coralline hydroxyapatite (CHA) was carried out using potassium persulfate (K2S2O8) and sodium metabisulfite (Na2S2O5) as initiators in aqueous medium. To optimize the reaction conditions for getting maximum grafting yield, the concentrations of backbone, monomer, initiator, temperature and time were varied. The percent grafting was found to increase initially and then gradually decrease with respect to reaction parameters. The results obtained imply that the optimum temperature and time was 60 degrees C and 180 min, respectively, to obtain higher grafting yield. The grafting results have been discussed and a mechanism involved in the grafting of GMA onto CHA is described. The grafted materials were analyzed with Fourier transform infrared (FT-IR) spectroscopy and X-ray diffraction (XRD) measurements. The results of FT-IR confirmed the presence of epoxy group on the grafted CHA. The XRD pattern showed that there was no secondary phase in the apatite lattice due to chemical modification.

Biocompatible Materials↗

PubMed2002

Revised theory on DNA renaturation kinetics and its experimental verification.

Many molecular biology and genomics techniques (including C(0)t analysis, PCR, and HPLC) depend upon the renaturation of DNA in solution. Traditionally, the renaturation process has been treated as a second-order kinetics reaction with the concentration of the DNA as the order parameter. However, second-order kinetics only describes complementary strand recognition and not the intramolecular "zipping" of complementary strands once recognition has been established. And also we cannot use simply the DNA concentration as the order parameter. Here I present an improved model that takes both phenomena into account using "mol-bases" as the order parameter. The model was tested experimentally and found to provide a more accurate description of the observed data than previous DNA renaturation models based solely on second-order kinetics.

DNA↗

PubMed2001

Thermo-Raman studies on NaH(2)PO(4)-H(2)O for dehydration, condensation, and phase transformation.

Thermo-Raman spectroscopy was applied to study the thermal properties of NaH(2)PO(4)-2H(2)O from room temperature up to 600 degrees C. Raman spectra recorded at every degree of the temperature interval by monitoring the internal modes gave the microscopic picture of dehydration of NaH(2)PO(4)-2H(2)O, the condensation of NaH(2)PO(4), and the phase transformation of NaPO(3). The spectral variation observed could distinguish two steps of dehydration, resulting in the formation of NaH(2)PO(4)-H(2)O and NaH(2)PO(4) as compositional species from NaH(2)PO(4)-2H(2)O. The thermo-Raman intensity (TRI) and differential thermo-Raman intensity (DTRI) thermograms also showed two steps of dehydration in the temperature range from 42 to 52 degrees C and from 60 to 72 degrees C with a maximum rate at 48 and 68 degrees C, respectively. Furthermore, condensation resulted in sodium dihydrogen diphosphate (Na(2)H(2)P(2)O(7)) and sodium metaphosphate (NaPO(3)) in two steps at 212-224 and 260-360 degrees C, respectively. Clear evidence in the spectral variation representing two phase transformations of NaPO(3) at the temperature of 345 and 515 degrees C with three different polymorphs (phase III, phase II, and phase I of NaPO(3) in increasing order of temperature) was also observed. The thermal methods thermogravimetry, differential thermogravimetry, differential thermal analysis, and differential scanning calorimetry also supported the results but could not give a clear idea due to the lack of direct structural information.

Journal Article↗