Search PubMed⌕ Search

Biomedical subjects

D Dasgupta

Publications and source records attributed to D Dasgupta.

At least 37 records · Page 2Linked to original sources
PubMed1998

Interaction of the antitumor antibiotic chromomycin A3 with glutathione, a sulfhydryl agent, and the effect upon its DNA binding properties.

Chromomycin A3 (CHR), an anticancer antibiotic, blocks macromolecular synthesis via reversible interaction with DNA only in the presence of divalent cations like Mg2+. In the absence of DNA, the antibiotic forms a dimer: Mg2+ complex [(CHR)2Mg2+]. It is the DNA-binding ligand. The antibiotic has potential reactive centers that could interact with GSH, the most abundant non-protein thiol in eukaryotic cells and a putative cofactor involved in the activation of many antibiotics in vivo. To understand the mode of action of CHR in vivo, we studied the interactions of CHR and the (CHR)2Mg2+ complex with GSH and the association of the resultant complexes with DNA by means of absorption, fluorescence, and circular dichroism spectroscopy. The novel finding was that GSH interacts non-covalently with CHR without a chemical modification of the antibiotic. The interaction was reversible in nature. The results are reported in two parts: the interaction of CHR with GSH in the absence and presence of Mg2+, and the effect of this interaction on the DNA-binding properties of the antibiotic. CHR forms a single type of complex with GSH. In contrast, (CHR)2Mg2+ forms two different types of complexes with GSH: a low GSH complex at approximately 12 mM GSH and a high GSH complex at > or = 16 mM GSH. Binding and thermodynamic parameters for the reversible association of the complexes with DNA demonstrated that they bind differently to the same DNA. The thermodynamic parameters indicate that the presence of GSH alters the mode of binding of the (CHR)2Mg2+ complex with DNA. The (CHR)2Mg2+ complex binds to DNA via an entropy-driven process, whereas in the presence of GSH the association is enthalpy-driven. The significance of these results in the understanding of the molecular basis of action of the antibiotic is discussed.

Antibiotics, Antineoplastic↗
PubMed1998

Enhancement of transcriptional activity of T7 RNA polymerase by guanidine hydrochloride.

T7 RNA polymerase shows an increase in processive transcription in the presence of low concentrations of guanidine hydrochloride (GdnCl) upto 60 mM, which is not observed when the enzyme is treated with urea. Higher concentrations of the denaturant lead to a progressive loss in the processive transcriptional activity of the enzyme. We have attempted to explain the above phenomenon in terms of the structural change in the enzyme. Fluorescence and CD studies suggest that the tertiary structure of the native enzyme undergoes an alteration upon addition of low concentration of guanidine hydrochloride. This is also indicated from the decreased susceptibility of the enzyme to limited proteolysis by trypsin.

Bacteriophage T7↗
PubMed1998

Pseudo-affinity column chromatography based rapid purification procedure for T7 RNA polymerase.

Based on the observation that T7 RNAP binds reversibly to the polyaromatic sulphonated triazine dye, cibacron blue, we have developed a rapid purification procedure for T7 RNAP. It employs chromatography of the ammonium sulfate fraction through a blue sepharose column, which has the dye coupled to the solid sepharose support. The enzyme can be eluted by 2M NaCl or 1M NaCl together with 1 mM UTP. These methods are compared with another purification procedure using ion-exchange column chromatography. All of them yield essentially pure T7 RNAP with high specific activity.

Chromatography, Affinity↗
PubMed1998

Chemical modification of 3-HBA-6-hydroxylase by phenylglyoxal: kinetic and physicochemical studies on the modified enzyme.

The inactivation of 3-HBA-6-hydroxylase isolated from Micrococcus species by phenylglyoxal and protection offered by 3-HBA against inactivation indicate the presence of arginine residue at or near the substrate binding site. The loss of enzyme activity was time and concentration dependent and displayed pseudo-first order kinetics. A 'n' value of 0.9 was obtained thus suggesting the modification of a single arginine residue per active site which led to the loss of enzyme activity. The enzyme activity could be restored by extensive dialysis at neutral pH. Quenching of the intrinsic fluorescence and reduction in the ellipticity value at 280 nm in the near-UV CD spectrum of the enzyme was noticed after its treatment with phenylglyoxal. These observations probably imply distinct perturbations in the environment of adjacent aromatic amino acid residues such as tryptophan as a consequence of arginine modification.

Chemical Phenomena↗
PubMed1997

Differential interactions of the Mg2+ complexes of chromomycin A3 and mithramycin with poly(dG-dC) x poly(dC-dG) and poly(dG) x poly(dC).

The interaction of the two anticancer antibiotics, chromomycin A3 and mithramycin, with the polynucleotides poly(dG-dC) x poly(dC-dG), representative of B-DNA, and poly(dG) x poly(dC), representative of A-DNA, in the presence of Mg2+ is studied by spectroscopic techniques such as absorbance, fluorescence, and dircular dichroism (CD). The studies were done with both drug x Mg2+ complexes, I and II, having 1:1 and 2:1 stoichiometries with respect to drug and Mg2+, respectively [Aich, P., Sen, R., & Dasgupta, D. (1992) Biochemistry 31, 2988-2997]. The objective of the present work is 2-fold. First, an attempt is made to understand the structural basis of the ligand-DNA interaction, particularly the role of DNA backbone conformation with its groove size and the accessibility of the 2-amino group in the minor groove of guanosine. Second, the role of the antibiotic saccharide moieties in the association with DNA was studied. For this purpose, the spectroscopic characterization of the binding was done followed by the evaluation of binding parameters and associated thermodynamics. Analysis of the observed thermodynamics for the ligand-DNA interactions in terms of the different structures of the polynucleotides was done. The salient results are as follows. Complex I does not discriminate significantly among the A- and B-forms of DNA when it binds to them in an entropy-driven process. On the other hand, complex II for both drugs recognizes B- and A-forms of DNA in different ways. This observation implies that the sequence specificity shown by this complex is a sequel to the difference in the parameters such as groove size and accessibility of the guanosine amino group. Another important finding is that binding with the same polynucleotide is not comparable for the complex II of the two drugs. It emphasizes the involvement of the sugar moieties, when the drug x Mg2+ complex binds to DNA. The presence of an acetoxy group in the sugars of chromomycin A3 imparts some distinctive specific features of the association of the chromomycin dimer x Mg2+ complex with DNA. Finally, the results are compared with those available from NMR studies of different drug-oligonucleotide complexes under conditions where complex II is the ligand.

Chromomycin A3↗
PubMed1997

Conformational changes in plant Ins(1,4,5)P3 receptor on interaction with different myo-inositol trisphosphates and its effect on Ca2+ release from microsomal fraction and liposomes.

The interaction of the only reported plant inositol trisphosphate receptor with different myo-inositol trisphosphates (InsP3 species), namely Ins(1,4,5)P3, Ins(1,3,4)P3, Ins(1,5,6)P3, and Ins(2,4,5)P3, were studied to assess the extent of Ca2+ mobilization from microsomes/vacuoles as well as liposomes in vitro. Ins(1,4,5)P3 and Ins(2,4,5)P3 bind with the receptor with comparable affinities, as evidenced from their dissociation constants (Kd approx. 100 nM at 5 degrees C), whereas the interaction between Ins(1,3,4)P3/Ins(1,5,6)P3 and the receptor was not detected even with these ligands at 5 microM. Ins(1,3,4)P3/Ins(1,5,6)P3 isomers also do not elicit Ca2+ release from liposomes or microsomes/ vacuoles. The ability of any InsP3 to bind the receptor for Ins(1,4,5)P3 is a prime requirement for Ca2+ release. However, the comparison of binding affinities at a single temperature does not help to correlate it directly with the extent of Ca2+ release from the intracellular stores because the concentration of Ca2+ released by Ins(1,4,5)P3 as estimated over a period of 20 s is 3500 +/- 200 nM/mg of protein and is about 4-fold higher than that by Ins(2,4,5)P3 under identical conditions. To understand the role of the receptor conformation in Ca2+ release by different isomers, we have probed the conformational change of the receptor when the different isomers bind to it. Accessibility of the tryptophan residues in the free and Ins(1,4,5)P3/Ins(2,4,5)P3-bound receptor was monitored by a neutral fluorescence quencher, acrylamide. The resulting Stern-Volmer-type quenching plots of the internal fluorescence indicate a change in the conformation of the receptor on binding to Ins(1,4,5)P3 and Ins(2,4,5)P3. It is also detected when far-UV CD spectra (205-250 nm) of the free and ligand [Ins(1,4,5)P3/Ins(2,4,5)P3]-bound receptor are compared. The results from CD spectroscopic studies further indicate that the conformational changes induced by the two isomers are different in nature. When thermodynamic parameters, such as enthalpy (delta H), entropy (delta S) and free energy (delta G), for the formation of the two InsP3-receptor complexes are compared, a major difference in the extent of changes in enthalpy and entropy is noted. All these findings taken together support the proposition that it is the overall interaction leading to the requisite conformational change in the receptor that determines the potency of the InsP3 isomers in their abilities of Ca2+ mobilization from the intracellular stores or reconstituted liposomes.

Acrylamide↗
PubMed1996

Interaction of myoinositoltrisphosphate-phytase complex with the receptor for intercellular Ca2+ mobilization in plants.

One of the myoinositol trisphosphates produced by the phytase-myoinositol hexakisphosphate (InsP6) reaction is Ins(2,4,5)P3. That Ins(2,4,5)P3 can elicit Ca2+ mobilization from intracellular stores in plants [Samanta, S., Dalal, B., Biswas, S., & Biswas, B.B.(1993) Biochem. Biophys. Res. Commun. 191,427] prompted us to elucidate the mechanism. The InsP3 [Ins(1,4,5)P3/Ins(2,4,5)P3]-phytase complex has been found to interact with the receptor for InsP3 in vitro forming a ternary complex, and a nanomolar concentration of InsP3 is required. For enzymatic cleavage of InsP3 by phytase, micromolar concentrations are needed, and the affinities of the phytase for different myoinositol phosphates have been found to depend upon the number of phosphate groups present in the substrate. Fraction accessibility of tryptophan residues to a neutral fluorescence quencher, acrylamide in free and myoinositol phosphates bound phytase, as determined by Stern-Volmer plot, records a progressive decrease starting from InsP6 to InsP with the notable exceptions of both Ins (1,4,5)P3 and Ins(2,4,5)P3. This deviation from the trend of change in the accessibility of tryptophan residues in myoinositol phosphate bound phytase is recorded from the fact that there is a high affinity (dissociation constant of the nanomolar order) and noncatalytic binding site in phytase for the two isomers of InsP3. In the nanomolar range of concentrations, both isomers of InsP3 bind to a second site of phytase having about 40-fold higher affinity than the normal substrate binding site. InsP3, when bound to noncatalytic site in phytase is not hydrolyzed but induces a significant change in the conformation of phytase as assayed from the relative accessibility of tryptophan residues. This conformational change in phytase is recognized by the receptor for InsP3, because in absence of InsP3 no interaction between the receptor and phytase is detected. However, InsP3-phytase complex is a better elicitor of Ca2+ efflux from microsomal/vacuolar fractions than free InsP3. This is further confirmed by the fact that when Ins(1,3,4)P3-phytase complex can elicit Ca2+ efflux from intracellular stores, Ins(1,3,4)P3 per se is minimally effective.

6-Phytase↗
PubMed1996

Conformational changes of E. coli RNA polymerase during transcription initiation.

Escherichia coli RNA polymerase-promoter complex undergoes a multistep process to initiate transcription. We have employed fluorescence spectroscopic approaches to detect the conformational states of the enzyme during this multistep process. A fluorescence assay based on the measurement of fluorescence of free and promoter-bound enzyme as a function of temperature within the range of 4 to 37 degrees C showed that, starting with initial 'closed complex', there are conformationally two distinct intermediate states of the polymerase till it attains the final form required for transcription initiation. The equilibrium from closed complex (RPc) to open complex (RPo) consists of at least the following two intermediate complexes: [formula: see text] Higher order structure of RNAP in each of these complexes was probed by means of measurement of accessibilities of the tryptophan fluorophores to the acrylamide. In the next part of the study, TbGTP, a fluorescent substrate, has been used to probe the state of active site in the enzyme for the complexes RPc, RPi1, RPi2 and RPo, respectively. From the comparison of changes in the parameters such as, fluorescence polarization anisotropy of TbGTP and its accessibility to the neutral quencher, acrylamide, in free and promoter-bound enzyme, we have further substantiated the first part of our results. Together these results suggest that formations of RPc and RPi1 do not involve radical conformational changes in the enzyme, while the enzyme undergoes major change in conformation in the steps RPil-->RPi2 and RPi2-->RPo. The strong tryptophan promoter cloned in plasmid pDR720 was chosen as a model promoter in these studies.

Binding Sites↗
PubMed1995

Role of magnesium ion in mithramycin-DNA interaction: binding of mithramycin-Mg2+ complexes with DNA.

Mithramycin is an anticancer drug that blocks macromolecular synthesis via reversible interaction with the DNA template in the presence of bivalent metal ions such as Mg2+. The role of Mg2+ in this antibiotic-DNA interaction is not clear. We approached the problem in two steps via studies on the interactions between (i) mithramycin and Mg2+ and (ii) mithramycin-Mg2+ complex(es) and DNA. Spectroscopic techniques such as absorption, fluorescence, and CD were employed for the purpose. From equilibrium and kinetic studies, we earlier reported that MTR forms two different types of complexes with Mg2+ [Aich, P., & Dasgupta, D. (1990) Biochem. Biophys. Res. Commun. 173, 689]. The two complexes are referred to as complex I (with 1:1 stoichiometry in terms of mithramycin: Mg2+) and complex II (with 2:1 stoichiometry in terms of mithramycin: Mg2+). In this report, we have further characterized these complexes by fluorescence spectroscopy. Interactions of these complexes with calf thymus DNA were examined to elucidate their binding. Evaluation of binding parameters (intrinsic binding constant and stoichiometry) from spectrophotometric and fluorimetric titrations suggests that the complexes bind differently to the same DNA. Measurement of van't Hoff enthalpies for the interaction of the two ligands and DNA shows that the complex I-DNA interaction is exothermic, in contrast to the endothermic nature of the complex II-DNA interaction. This could originate from a difference in the molecular nature of the interactions between the complexes and calf thymus DNA. Our studies to detect the nature of the groove via which these complexes bind to DNA suggest that both complexes approach via the minor groove of the DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Synthesis of a photoaffinity taxol analogue and its use in labeling tubulin.

A photoaffinity analogue of taxol, N-([3,5-3H]-4-azidobenzoyl)-N-debenzoyltaxol (7), was synthesized and used to photolabel microtubules. Approximately 20% of the noncovalently bound analogue becomes covalently bound upon irradiation at 300 nm. Incorporated label was stable to a 50% ethanol solution and sodium dodecyl sulfate. About 80% of the incorporated label was found in the beta-subunit and 20% in the alpha-subunit. Incorporation did not occur into unpolymerized tubulin, consistent with the fact that taxol binds only to polymerized tubulin, and was decreased by the presence of taxol. Little or no nonspecific labeling occurs. This analogue is currently being used to identify taxol binding site(s) on tubulin.

Affinity Labels↗
PubMed1994

Intrinsic fluorescence of E. coli RNA polymerase as a probe for its conformational changes during transcription initiation.

A simple fluorimetric assay based on internal fluorescence of tryptophan residues of E. Coli RNA polymerase has been developed to ascertain the number of steps during conversion of closed complex of the polymerase-promoter (trp promoter cloned in plasmid pDR720) to open complex. Our results from measurement on relative ratio of fluorescence at 340 nm (lambda ex = 295 nm) for free and promoter-bound RNA polymerase as a function of temperature, within the range 4 degrees C to 37 degrees C, indicate following equilibria for the above conversion: R+P<-->RPc<-->RPi1<-->RPi2<-->RPo. Apart from detection of one more intermediate in terms of conformational states of the bound RNA polymerase, second feature of our studies is the examination of conformational state of the polymerase using accessibility of fluorophor, tryptophan residues, to a neutral quencher, acrylamide, as the probe. We observe that in terms of accessibility of tryptophan residues in protein, intermediate complex, RPi2, is conformationally most perturbed in comparison to free polymerase. Implications of these results are discussed and compared with the available reports from footprinting and gel retardation assays of RNA polymerase-promoter interactions.

Cloning, Molecular↗
PubMed1994

Refolding of denatured lactate dehydrogenase by Escherichia coli ribosomes.

Escherichia coli ribosomes were used to refold denatured lactate dehydrogenase from porcine muscle. This activity of ribosomes, unlike most of the chaperons, did not require the presence of ATP. The molar concentration of ribosomes required for this refolding was comparable with that of the enzyme. Restoration of the enzyme activity was demonstrated using assays for both the forward and backward reactions. Binding of the denatured enzyme to ribosomes and its refolding were fairly rapid processes as revealed by the time course of the reaction and inhibition of folding when the denatured enzyme was allowed to refold spontaneously for short times before the addition of ribosomes. This protein-folding activity was detected in 70 S ribosomes as well as its RNA, in 50 S particles and in 23 S rRNA. However, 30 S particles failed to refold the enzyme.

Escherichia coli↗
PubMed1993

One-stage "front" and "back" correction for rigid cervical kyphosis. A safer technique of correction for a rare case of adult-onset Still's disease.

This report describes a new safe technique for surgical correction of rigid cervical kyphosis, in a very rare case of adult-onset Still's disease. It differs from all the previously mentioned reports of similar corrective osteotomies, the essential difference being a combined anterior (front) and posterior (back) approach performed at one stage (under a single anesthesia). The "front" is a release procedure of an open-wedge variety, and the "back" is a closed-wedge type of osteotomy. Such a double procedure ensures a slow and controlled correction at the preoperatively determined level, rather than a snapshot, rapid, and often a violent correction, which may occur at an undesired level. This osteotomy is performed under general anesthesia after an endotracheal intubation by a special "guided" maneuver. This new technique, the authors believe, is only a more comfortable option for the surgeon, but also a safer alternative for the patient.

Adult↗
PubMed1993

Interaction of ribonucleotides with T7 RNA polymerase: probable role of GTP in transcription initiation.

Interaction of ribonucleotides (NTP where N = G, A, C or U) with bacteriophage T7 RNA polymerase (T7 RNAP) was studied by fluorescence emission spectroscopy of the enzyme. From the NTP-concentration-dependent quenching of fluorescence of the enzyme, apparent dissociation constants for NTP-T7 RNAP was found to be in following order: UTP>CTP>>ATP>GTP. Acrylamide quenching of tryptophan fluorescence of free and bound enzyme suggests a conformational change, particularly in the case of GTP (and ATP). This is the first report of high affinity binding of the enzyme with purine ribonucleotides in the absence of promoter. These results also suggest that GTP may induce a promoter-specific conformation of the enzyme. The observation could account for specific requirement of GTP in transcription initiation reported earlier (1-4).

Bacteriophage T7↗
PubMed1993

Tracheal tear during laryngopharyngectomy with gastric transposition.

Tracheal rupture occurred in 7 of 174 (4%) patients undergoing laryngopharyngectomy with gastric transposition. Tracheal tears were classified as proximal if they involved the upper two-thirds of the trachea (five patients), or distal if they extended into the lower one-third of the trachea (two patients) and their clinical features and management analyzed. Predisposing factors, including prior radiotherapy (three patients) and preoperative tracheostomy (1 patient) did not influence the site or severity of tracheal injury. Proximal tears were detected incidentally in four patients, but in one patient, manifested postoperatively with subcutaneous emphysema and pneumothorax. Distal tears manifested dramatically with a ventilatory leak. Adequate access for repair of distal tears may necessitate a right thoracotomy while proximal tears may be sutured through the cervical incision. Gastric transposition alone did not prevent air leak in two patients. Postoperative complications included prolapse of the stomach and bilateral pneumothoraces in one patient. Close interaction between the surgeon and the anesthesiologist ensured a successful outcome in six patients. There was one mortality.

Anastomosis, Surgical↗
PubMed1992

Interaction between antitumor antibiotic chromomycin A3 and Mg2+. I. Evidence for the formation of two types of chromomycin A3-Mg2+ complexes.

Chromomycin A3 (CHRA3) is an antitumor antibiotic which binds to Mg2+. In the present communication, we show, by means of equilibrium spectroscopic studies (such as absorption, fluorescence and circular dichroism), that two types of CHRA3-Mg2+ complexes (of 1:1 and 1.9:1 stoichiometries in terms of CHRA3:Mg2+, respectively) are formed depending on the concentrations of CHRA3 and Mg2+. The rate constant and activation energy for the formation of two complexes are different, thereby reinforcing the proposition that they are different molecular species. This observation is novel and significant in order to understand the anticancer property of the drug. It also provides explanation for earlier observations that site, affinity parameters and mode of interaction of the drug with DNA in the presence of Mg2+ depend on the relative concentration of Mg2+.

Chromomycin A3↗
PubMed1992

Role of magnesium ion in the interaction between chromomycin A3 and DNA: binding of chromomycin A3-Mg2+ complexes with DNA.

Chromomycin A3 is an antitumor antibiotic which blocks macromolecular synthesis via reversible interaction with DNA template only in the presence of divalent metal ions such as Mg2+. The role of Mg2+ in this antibiotic-DNA interaction is not well understood. We approached the problem in two steps via studies on the interaction of (i) chromomycin A3 and Mg2+ and (ii) chromomycin A3-Mg2+ complex(es) and DNA. Spectroscopic techniques such as absorption, fluorescence, and CD were employed for this purpose. The results could be summed up in two parts. Absorption, fluorescence, and CD spectra of the antibiotic change upon addition of Mg2+ due to complex formation between them. Analysis of the quantitative dependence of change in absorbance of chromomycin A3 (at 440 nm) upon input concentration of Mg2+ indicates formation of two types of complexes with different stoichiometries and formation constants. Trends in change of fluorescence and CD spectroscopic features of the antibiotic in the presence of Mg2+ at different concentrations further corroborate this result. The two complexes are referred to as complex I (with 1:1 stoichiometry in terms of chromomycin A3:Mg2+) and complex II (with 2:1 stoichiometry in terms of chromomycin A3:Mg2+), respectively, in future discussions. The interactions of these complexes with calf thymus DNA were examined to check whether they bind differently to the same DNA. Evaluation of binding parameters, intrinsic binding constants, and binding stoichiometry, by means of spectrophotometric and fluorescence titrations, shows that they are different. Distinctive spectroscopic features of complexes I and II, when they are bound to DNA, also support that they bind differently to the above DNA. Measurement of thermodynamic parameters characterizing their interactions with calf thymus DNA shows that complex I-DNA interaction is exothermic, in contrast to complex II-DNA interaction, which is endothermic. This feature implies a difference in the molecular nature of the interactions between the complexes and calf thymus DNA. These observations are novel and significant to understand the antitumor property of the antibiotic. They are also discussed to provide explanations for the earlier reports that in some cases appeared to be contradictory.

Animals↗