Biomedical subjects
Publications and source records attributed to C Mukhopadhyay.
Salmonella osteomyelitis of the rib is a rare clinical entity. In our case, a muhidrug resistant Salmonella enterica serotype Typhi was isolated from an immuno-competent patient with osteomyclitis of the ribs, who was treated earlier with ciprotloxacin for typhoid fever. The patient was successfully treated for osteomyclitis with intravenous ceftriaxone.
Human erythrovirus B19 (B19), previously known as parvovirus B19, is a small spherical, non-enveloped single stranded DNA virus. It has been shown to cause a wide spectrum of clinical conditons including various hematological disorders. We report here for the first time from Inida a case of pure red cell aplasia in a 45-year-old female for last 7 years due to chronic persistent B19 infection leading to myelodysplasia after 4 years. Her sera were positive for two times 4 months apart for B19 IgM and B19 DNA at the initial stage. Presently the patient is on repeated blood transfusion on every 15-20 days.
Complex carbohydrates linked to glycoproteins are recently being implicated to play a variety of biological roles. The lack of well-resolved crystallographic coordinates of the carbohydrates makes it difficult to assess the contributions of the glycan chain on protein structure and dynamics. We have modeled two different oligosaccharides NeuNAc2Gal3Man3GlcNAc5Fuc and Man3GlcNAc4 to generate two glycosylation variants of major histocompatibility complex (MHC) class I glycoprotein. Molecular dynamics simulations of the isolated fourteen- and seven-residue oligosaccharides have been done in vacuo and in solution. The dynamics of the two glycoforms of MHC class I protein have been simulated in solution in the free as well as in the peptide-bound form. Good agreement between the calculated solution conformations of the oligosaccharides in isolated and conjugated forms and the average conformations obtained from x-ray or NMR data was observed for most of the glycosidic linkages. These molecular dynamics simulations of the isolated glycan chains and the glycoconjugates reveal the details of the conformational flexibility of the glycan chains; they also provide atomic level details of protein-carbohydrate interactions and the effect of the ligand binding on the carbohydrate structure and dynamics. It was found that though there is some flexibility in some of the glycosidic linkages in the isolated oligosaccharides, in the protein-conjugated form the linkages adopt more restricted conformations. The glycan chains protrude out into the solvent and might hinder the lateral association of the proteins. The presence of the bulky glycan chains does not affect the average backbone fold of the protein but induces local changes in protein structure and dynamics. It has been noted that the extent of the changes depends upon the nature of the attached glycan chain. The glycan chains do not appear to influence the peptide binding property of the protein directly, but may stabilize the protein residues that are involved in ligand binding.
Molecular dynamics simulation of ganglioside GD1a attached to the upper layer of a fully hydrated lipid bilayer of dimyristoyl phosphatidyl choline (DMPC) at room temperature under periodic boundary conditions was performed. The time average conformation of GD1a reveals that the terminal sialic acid is more exposed into the solvent than the internal branched one. Many interresidual contacts between N-acetyl galactosamine-internal branched sialic acid; external Gal-external sialic acid; N-acetyl galactosamine-internal gal are also observed. The conformation of the GD1-hexasaccharide is stabilized by a number of intra molecular hydrogen bonds that were previously observed experimentally. The simulation results indicate that the presence of a single GD1a molecule has local effects on the bilayer. A local disorder in the arrangement of the acyl chains as well as the head groups is evident in the upper layer due to the presence of GD1a.
A total of 7,904 persons visiting University Hospital of Banaras Hindu University (BHU), Varanasi, were screened for HIV antibody by ELISA and/or rapid test. The overall seroprevalence of HIV (3.17%) in this area was higher than that of Uttar Pradesh and India as a whole. The seroprevalence of HIV/AIDS among the high risk group (HRG) (6.42%) being significantly higher than low risk group (LRG) (0.37%). In the LRG, the prevalence among medical and surgical patients; and ANC (antenatal cases) was 1.17% and 0.14%, respectively. However, HIV infection was not detected in HCWs (healthcare workers), BHU students and foreigners. Among HRG, CSWs (commercial sex workers) had the highest prevalence (14.0%) and STD (sexually transmitted diseases) patients, the lowest prevalence (1.99%). The STD patients had significantly lower prevalence rate as compared to others in HRG. Majority of the HIV positive were found to be within the age group 15-44 years, with heterosexual mode as the main route of transmission. Seven HIV positive children were of 2-5 yr old age group. Majority of seropositive women (62.52%) were working in a low income jobs and were mainly infected by their spouses who was mostly migrating labourers of lower socioeconomic group and with less than primary level of education. Married females, being mainly the spouses of HIV positive males had the highest seropositivity (60.25%). These population people were directly transmitting the infection from the metropolitan cities to the rural areas of this region. Prevalence of HIV-2 was negligible as compared to HIV-1, the actual reason is yet to be explored.
Highly regioselective electrophilic substitution of dibenzofluorene was achieved and the nitro derivative was transformed to a variety of new anticancer agents.
The structure and stereochemistry of a new terpenoid ester, nardostachysin (1), isolated from the rhizomes of Nardostachys jatamansi, were established as the 7',8'-dihydroxy-4'-methylene hexahydrocyclopenta[c]pyran-1'-one-8'-methyl ester of 7, 9-guaiadien-14-oic acid, by spectral and chemical studies.
Protein nitration and lipid peroxidation are implicated in the pathogenesis of atherosclerosis; however, neither the cellular mediators nor the reaction pathways for these events in vivo are established. In the present study, we examined the chemical pathways available to monocytes for generating reactive nitrogen species and explored their potential contribution to the protein nitration and lipid peroxidation of biological targets. Isolated human monocytes activated in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide ((*)NO) metabolism, nitrate apolipoprotein B-100 tyrosine residues and initiate LDL lipid peroxidation. LDL nitration (assessed by gas chromatography-mass spectrometry quantification of nitrotyrosine) and lipid peroxidation (assessed by high-performance liquid chromatography with online tandem mass spectrometric quantification of distinct products) required cell activation and NO(2)(-); occurred in the presence of metal chelators, superoxide dismutase (SOD), and scavengers of hypohalous acids; and was blocked by myeloperoxidase (MPO) inhibitors and catalase. Monocytes activated in the presence of the exogenous (*)NO generator PAPA NONOate (Z-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2- diolate) promoted LDL protein nitration and lipid peroxidation by a combination of pathways. At low rates of (*)NO flux, both protein nitration and lipid peroxidation were inhibited by catalase and peroxidase inhibitors but not SOD, suggesting a role for MPO. As rates of (*)NO flux increased, both nitrotyrosine formation and 9-hydroxy-10,12-octadecadienoate/9-hydroperoxy-10,12-octadecadieno ic acid production by monocytes became insensitive to the presence of catalase or peroxidase inhibitors, but they were increasingly inhibited by SOD and methionine, suggesting a role for peroxynitrite. Collectively, these results demonstrate that monocytes use distinct mechanisms for generating (*)NO-derived oxidants, and they identify MPO as a source of nitrating intermediates in monocytes.
Protein A (PA), a cell wall constituent of Staphylococcus aureus, has got the unique property of binding with the Fc fragment of IgG from various species. The sequence data indicate five highly homologous Fc-binding regions in protein A. Computer sequence analysis provided the tryptic and chymotryptic fragments of IgG-binding domains of protein A. Molecular modeling in conjunction with molecular mechanical calculation has been used to search for the smallest possible proteolytic fragments of PA, still retaining Fc-binding activity. A 20-residue peptide (typtic fragment) and a 16-residue peptide (chymotryptic fragment) have been indicated, by molecular modeling studies, to possess IgG-binding affinity comparable to that of the B domain of Protein A. Binding of a 20-residue peptide has been substantiated experimentally by immunoprecipitation, capillary electrophoresis, and circular dichroism spectroscopic analyses.
Glycoprotein-glycans have recently been implicated to play a variety of functional roles. The same glycan chain have been found complexed with proteins of diverse functions. In this article two such glycan chains found attached to Fc regions of immunoglobulin G and immunoglobulin M have been studied. An extensive simulated annealing procedure have been adopted to arrive at a low-energy minimum of the two oligosaccharides. Molecular dynamics simulations have been performed to study the flexibility of the glycosidic linkages. It was found that both glycan chains can undergo conformational transitions and adopt folded and extended conformations. The two beta (1-2) linkages of complex-type glycan had been found to prefer different conformational regime and the terminal fucose linked to the GlcNAc residue drastically modifies the GlcNAc beta (1-4) GlcNAc linkage conformation. In the high-mannose type glycan chain alpha (1-3) linkages can induce flexibility in addition to the alpha (1-6) linkages. The results have been compared with recent experimental nmr and fluorescence energy transfer data.
Despite emergence of bis-ANS as a major fluorescence probe of proteins structure, conformational and spectroscopic properties of protein/bis-ANS complexes remains largely unexplored. We have shown that fluorescence polarization of both ANS and bis-ANS is excitation wavelength dependent and this is a property of all protein-ANS/bis-ANS complexes studied. Bis-ANS excitation maximum is always more red shifted than the corresponding ANS complex. Even when corrected for the red shift, the bis-ANS complexes in some, but not all, cases show only a little lowering of polarization, suggesting modest additional depolarization in bis-ANS compared to ANS. Calculation of energy migration rate between the two rings suggests that energy migration rate should be high at all values of the naphthyl-naphthyl dihedral angle. Although, Molecular mechanics and dynamics calculations show that the lowest energy conformation of bis-ANS is when the two naphthalene rings are roughly perpendicular to each other, due to rapid energy migration this conformation should lead to dramatic lowering of emission anisotropy, unlike what is observed. Salt and temperature dependence of bis-ANS/protein interaction suggests little ionic interaction and pre-dominant interaction through hydrophobic aromatic rings. We conclude that bis-ANS binds to proteins through interaction with the aromatic rings and with two rings nearly parallel to each other.
Colchicine, a tricyclic alkaloid, has a remarkable range of biological activities. It binds with tubulin and prevents the formation of microtubules. This compound consists of a six membered aromatic ring (A ring), a seven membered troponoid ring (C ring) and another seven membered aliphatic ring (B ring). Using molecular mechanics and molecular dynamics simulations as tools, conformational analysis of colchicine and its several important analogs were done. Molecular mechanics studies show that conformational space of these molecules have one low energy region. Taking the low energy minima as the starting conformation, molecular dynamics simulation for 100 pico seconds is done for each of the analogs and molecular dynamics simulation in solution is done for three representative compounds colchicine,isocolchicine and A-C compound. Internal coordinate trajectories show that the value of the dihedral angle C9-C7-C1-C14 (phi), (C7-C1 bond connects the A and C ring), is within 40 degrees to 50 degrees for all the compounds with fluctuations less than 15 degrees. These calculations indicate that there is an overall similarity in the dynamically averaged structure of all the drugs. The A ring and B ring of the compounds are more or less rigid. The C ring is somewhat flexible, the average conformation and motional properties show overall similarity. The potential energy curve and dynamics behaviour of colchicine and isocolchicine suggests that the difference in binding property of colchine and isocolchicine may originate from the positional difference of carbonyl oxygen and methoxy group of C ring, which is the only difference in the structures of the two compounds and this has no effect on the motional property and average conformations of these two compounds. From our study it is proposed that the movements occuring at various positions of the drug molecules are significantly correlated. It is suggested that such correlated motion may play an important role in the biological property of these compounds.
The E. histolytica ferredoxin (EHFXD) is an iron-sulphur protein and is important in the control of E. histolytica because it donates an electron to and activates metronidazole (4) the most effective anti-amoebic drug. The knowledge of the three-dimensional structure of EHFXD can help to assist in rational design of anti-amebic drugs. Homology modeling of EHFXD has been done by using the knowledge of crystal structure of homologous Ferredoxin structures. It has been shown that EHFXD is more likely to contain 2[4Fe-4S] clusters and has a pseudodiad symmetrical fold. The fold is stabilised by hydrophobic patches as well as by intramolecular hydrogen bonds. The importance of two Leu residues in the N- and C-termi in bridging the two clusters has been emphasised. The electrostatic properties of the surface of EHFXD were examined and compared with that of other ferredoxins. It was observed that large regions of negative as well as positive electrostatic potential is a common feature of the [4Fe-4S] containing ferredoxins which might explain their biological role as both electron acceptor and electron donor molecules.
The morphogenesis of the external genitalia of human fetuses (16-250 mm crown-rump [CR] length, 6-26 weeks of gestation) obtained after medical termination of pregnancy were studied. Differential development (male/female) started after 50 mm CR length (9 weeks). At that time the external genitalia consisted of a cylindrical genital tubercle 2 mm in length with a visible coronary sulcus and glans and genital swellings on either side. A groove on the ventral aspect of the genital tubercle extended to the coronary sulcus; the lateral boundaries of this groove separated to form the urethral folds. In male fetuses the free edges of the urethral folds fused, starting from the proximal end, to form a tunnel over the ventral aspect of the phallus. The pelvic urethra opened into this tunnel, slightly distal to its origin. The mesodermal tissue forming the genital swellings migrated ventrally and then medially. As medial migration started, the skin in the midline between the genital swellings was raised up as a skin fold, which subsequently, as the genital swellings migrated further, became elevated. The proximal part of the tunnel formed by fusion of the urethral folds (proximal to the point of entry of the pelvic urethra) also was compressed and pushed out as the genital swellings fused in the midline over the root of the phallus. These changes took place at between 80 and 110 mm CR length (12-13 weeks' gestation); at this stage the phallus appeared short and was bent ventrally. With further growth and caudal migration of the scrotum, the phallus lost its ventral curvature. The appearance of the external genitalia at different gestational ages bore a close resemblance to that in children with hypospadias. We therefore conclude that hypospadias can be explained on the basis of an embryological arrest due to the absence of the required stimulus for male phenotypic development at the appropriate time.
Lambda-repressor dimers associate in solution to form tetramers and higher order structures. Dimer-dimer contact is also crucial in cooperative binding to adjacent operators. Fluorescence quenching studies indicate that the tryptophan 230 environment is significantly different in unliganded and adjacent operator-bound tetramers. Acrylodan attached to Cys 235, in a mutant F235C repressor, is also in different environments in the unliganded and adjacent operator bound tetramers. Thermodynamics of protein association, measured by fluorescence anisotropy, indicate that, whereas free repressor dimer association is strongly enthalpy driven, the single-operator (OR1)-bound repressor dimer association is largely entrophy driven with little change in enthalpy. Single-operator-bound dimer association to the corresponding tetramer does not lead to any significant change in tryptophan 230 environment, as was seen in the case of the free repressor. Data are also presented to support the contention that, under the conditions of this study, the free repressor association is predominantly from dimer to tetramer and then to octamer, unlike the dimer to octamer transition observed under a different condition. The results presented here point toward the conclusion that the lambda-repressor dimer-dimer interface is significantly different in the free and operator-bound states and that operator binding plays a crucial role in changing the nature of the dimer-dimer interface.
Ceruloplasmin is the principal carrier of copper in human plasma. It is an abundant protein that participates in the acute phase reaction to stress, but its physiological function(s) is unknown. An antioxidant activity of ceruloplasmin has been described, but recent evidence suggests that the protein may also exhibit potent pro-oxidant activity and cause oxidative modification of low density lipoprotein (LDL). The pro-oxidant activity is highly dependent on the structure of the protein; removal of a single one of the seven integral copper atoms, or a specific proteolytic cleavage event, completely suppresses LDL oxidation. This newly described pro-oxidant activity may help to explain epidemiological studies indicating that ceruloplasmin is an independent risk factor for cardiovascular disease.
alpha D-N-acetyl neuraminic acid (Neu5Ac, sialic acid) is a commonly occurring carbohydrate residue in various cell surface glycolipids and glycoproteins. This residue is linked terminally or internally to Gal residues via an alpha(2-->3) or alpha(2-->6) linkage. In the cell surface receptor, sialyl-LewisX, a terminal alpha(2-->3) linkage is present. Previous studies from our laboratory have shown that in solution LewisX adopts a relatively rigid structure. In order to model the Neu5Ac residue, vacuum molecular dynamics of this monosaccharide were compared with simulations that explicitly include solvent water. The dynamical average of the monosaccharide conformation obtained from the two simulations was similar. Vacuum calculations for the disaccharide Neu5Ac alpha(2-->3) Gal beta-O-methyl show that a number of low energy minima are accessible to this disaccharide. Molecular dynamics simulations starting from the low energy minima show conformational transitions with a time scale of 10-50 ps among several of the minima while large barriers between other minima prevent transitions on the time scale studied. Simulations of this disaccharide in the presence of solvent show fewer conformational transitions, illustrating a dampening effect of the solvent that has been observed in some other studies. Our results are most consistent with an equilibrium among multiple conformations for the Neu5Ac alpha(2-->3) Gal beta linkage.
A tetrasaccharide related to the blood group oligosaccharides, known as sialyl LewisX, has been proposed as the receptor for the lectin responsible for leukocyte adhesion named alternatively as E-selectin or ELAM-1. The 13C- and 1H-nmr spectra have been completely assigned for a tetrasaccharide model of this receptor, Neu5Ac alpha-(2-->3)-Gal beta-(1-->4)-[Fuc alpha-(1-->3)-]GlcNAc beta-NHAc. Quantitative nuclear Overhauser data (NOESY) have been recorded and analyzed by a complete spin matrix simulation method. Conformational space was exhaustively searched and all conformational models whose simulated NOESY spectra matched the experiment were found. Molecular mechanics and molecular dynamics calculations were carried out to test whether the experimental conformations are low energy and thus likely to represent true single conformations for the tetrasaccharide. It was concluded that while the LewisX trisaccharide portion of the compound adopts a single conformation, there is likely to be some flexibility about the Neu5Ac alpha-(2-->3)-linkage. A model featuring fast exchange between two different conformations of this linkage is found to be consistent with both the nmr experiments and the molecular dynamics simulations.