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Biomedical subjects

R Banerjee

Publications and source records attributed to R Banerjee.

At least 19 recordsLinked to original sources
PubMed2003

Artificial vitreous replacements.

Vitreoretinal pathologies remain the significant leading cause of blindness, after cataract, throughout the world. Biomaterials as a whole, and vitreous substitutes in particular, have had a major role to play in vitreoretinal surgery. History has witnessed the advent of vitreous substitutes from collagen and hyaluronic acid to gases, silicone oils and perfluorocarbon liquids and now to polymer hydrogels. Such a long list only underscores the fact that we have been found wanting in our quest for an ideal vitreous substitute. This review focuses on the merits and demerits of various vitreous substitutes, which have been tried to date, clinical as well as experimental. The properties required of an ideal substitute and the performance of the vitreous substitutes with respect to these criteria have been described. Finally, the concluding note dwells on the future directions for biomaterial research in the posterior segment of the eye and undermines the role of tissue engineering in vitreoretinal regeneration.

Biocompatible Materials↗
PubMed2002

Purification and characterization of maltooligosaccharide-forming amylase from Bacillus circulans GRS 313.

A maltooligosaccharide-forming amylase that hydrolyzes starch into maltotriose and maltopentaose was found in the culture filtrate of a strain of Bacillus circulans GRS 313 isolated from local soil. The enzyme was purified by organic solvent fractionation, Sephadex G-100 gel filtration and CM-Sephadex column chromatography. Optimum pH and temperature of amylase were evaluated using response surface methodology (RSM) and were found to be 48 degrees C and 4.9, respectively. The enzyme was stable up to 60 degrees C and its pH stability was in the range of 5.0-8.0. The Km and Vmax of the amylase with starch were 11.66 mg/ml and 68.97 U, respectively, and the energy of activation, Ea, was 7.52 kcal/mol. Dextrin inhibited the enzyme competitively, with a Ki of 6.1 mg/ml, and glucose caused noncompetitive inhibition with a Ki of 9.5 mg/ml. The enzyme was inhibited by Hg2+, Mn2+, Fe3+ and Cu2+ and enhanced by Co2+ and Mg2+. EDTA reversed the inhibitory effect of the metals. Paper chromatographic and high-performance liquid chromatography analysis of the products of the amylolytic reaction showed the presence of maltotriose, maltotetraose, maltopentaose, maltose and glucose in the starch hydrolysate.

Amylases↗
PubMed2002

Controlling the reactivity of radical intermediates by coenzyme B(12)-dependent methylmalonyl-CoA mutase.

Adenosylcobalamin or coenzyme B(12)-dependent enzymes are members of the still relatively small group of radical enzymes and catalyse 1,2-rearrangement reactions. A member of this family is methylmalonyl-CoA mutase, which catalyses the isomerization of methylmalonyl-CoA to succinyl-CoA and, unlike the others, is present in both bacteria and animals. Enzymes that catalyse some of the most chemically challenging reactions are the ones that tend to deploy radical chemistry. The use of radical intermediates in an active site lined with amino acid side chains that threaten to extinguish the reaction by presenting alternative groups for abstraction poses the conundrum of how the enzymes control their reactivity. In this review, insights into this issue that have emerged from kinetic, mutagenesis and structural studies are described for methylmalonyl-CoA mutase.

Acyl Coenzyme A↗
PubMed2001

Characterization of NO binding to human cystathionine beta-synthase: possible implications of the effects of CO and NO binding to the human enzyme.

Homocysteine is a key junction metabolite that can be converted to cystathionine in a reaction catalyzed by the heme and pyridoxal phosphate-dependent cystathionine beta-synthase. The heme has unusual spectroscopic properties and the axial ligands have been assigned as histidine and cysteine, respectively. Its role in the protein is not obvious from the chemistry of the beta-replacement reaction that is catalyzed. We have characterized the binding of the gaseous signaling molecule, NO, to cystathionine beta-synthase and examined its effect on the reactions catalyzed by the truncated dimeric form of the enzyme, W409X, which is a natural variant. Binding of NO appears to result in the formation of a five-coordinate ferrous nitrosyl species in which both endogenous ligands have been lost. This is in contrast to CO binding which is reported to displace the thiolate ligand and form a six-coordinate species. NO binds to the full-length enzyme with a K(d) of 281+/-50 microM and to the truncated enzyme with a K(d) of 350+/-44 microM. Binding of NO to the full-length enzyme inhibits activity with a K(i) of 320+/-60 microM. These studies demonstrate that as with CO, perturbation of the heme environment by NO is communicated to the active site with concomitant inhibition of enzyme activity, and suggests a regulatory role for heme in cystathionine beta-synthase.

Carbon Monoxide↗
PubMed2001

Mercuric chloride-induced spin or ligation state changes in ferric or ferrous human cystathionine beta-synthase inhibit enzyme activity.

Cystathionine beta-synthase is a key heme and pyridoxal phosphate-dependent enzyme involved in homocysteine metabolism in humans. The role of the recently discovered heme in this protein remains an important open question. The axial ligands to the heme in both the ferrous and ferric states have been assigned as cysteine and histidine residues, respectively. In this study, we have examined the effect of ligation and spin state changes in the heme on the activity of the enzyme. Treatment of the ferric enzyme with HgCl2 results in the conversion of six-coordinate low-spin heme to five-coordinate high-spin heme and is paralleled by a loss of activity. In contrast, treatment of the ferrous enzyme with HgCl2 results in replacement of the cysteine ligand by an unidentified sixth ligand and retention of the six-coordinate state, and is also accompanied by loss of enzyme activity. Treatment of the five-coordinate HgCl2-treated enzyme with thiols, such as homocysteine, results in reversion to a six-coordinate state. Resonance Raman spectroscopy with 34S-labeled enzyme reveals the return of the endogenous thiol ligand under these conditions and rules out direct coordination by the thiolate of homocysteine to the heme.

Cystathionine beta-Synthase↗
PubMed2001

Design, synthesis, and transfection biology of novel cationic glycolipids for use in liposomal gene delivery.

The molecular structure of the cationic lipids used in gene transfection strongly influences their transfection efficiency. High transfection efficiencies of non-glycerol-based simple monocationic transfection lipids with hydroxyethyl headgroups recently reported by us (Banerjee et al. J. Med. Chem. 1999, 42, 4292-4299) are consistent with the earlier observations that the presence of hydroxyl functionalities in the headgroup region of a cationic lipid contributes favorably in liposomal gene delivery. Using simple sugar molecules as the source of multiple hydroxyl functionalities in the headgroup region of the transfection lipids, we have synthesized four novel simple monocationic transfection lipids, namely, 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol (1), 1-deoxy-1-[methyl(ditetradecyl)ammonio]-D-arabinitol (2), 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol (3) and 1-deoxy-1-[methyl(dioctadecyl)ammonio]-D-arabinitol (4), containing hydrophobic aliphatic tails and the hydrophilic arabinosyl or xylose sugar groups linked directly to the positively charged nitrogen atom. Syntheses, chemical characterizations, and the transfection biology of these novel transfection lipids 1-4 are described in this paper. Lipid 1, the xylosyl derivative, showed maximum transfection on COS-1 cells. All the lipids showed transfection with cholesterol as colipid and not with dioleoylphosphatidylethanolamine (DOPE). Radioactive quantitation of free and complexed DNA combined with ethidium bromide exclusion measurements suggest that though nearly 70% of the DNA exists as complexed DNA, the DNA may not have condensed as was observed with other cationic lipids. Presence of additional (more than two) hydroxyl functionalities in the headgroup of the cationic lipids appears to have improved the transfection efficiency and made these lipids less cytotoxic compared to two-hydroxyl derivatives.

Animals↗
PubMed2001

Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation.

Methionine synthase is a key enzyme in the methionine cycle that catalyzes the transmethylation of homocysteine to methionine in a cobalamin-dependent reaction that utilizes methyltetrahydrofolate as a methyl group donor. Cob(I)alamin, a supernucleophilic form of the cofactor, is an intermediate in this reaction, and its reactivity renders the enzyme susceptible to oxidative inactivation. In bacteria, an NADPH-dependent two-protein system comprising flavodoxin reductase and flavodoxin, transfers electrons during reactivation of methionine synthase. Until recently, the physiological reducing system in mammals was unknown. Identification of mutations in the gene encoding a putative methionine synthase reductase in the cblE class of patients with an isolated functional deficiency of methionine synthase suggested a role for this protein in activation (Leclerc, D., Wilson, A., Dumas, R., Gafuik, C., Song, D., Watkins, D., Heng, H. H. Q., Rommens, J. M., Scherer, S. W., Rosenblatt, D. S., and Gravel, R. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3059-3064). In this study, we have cloned and expressed the cDNA encoding human methionine synthase reductase and demonstrate that it is sufficient for supporting NADPH-dependent activity of methionine synthase at a level that is comparable with that seen in the in vitro assay that utilizes artificial reductants. Methionine synthase reductase is a soluble, monomeric protein with a molecular mass of 78 kDa. It is a member of the family of dual flavoproteins and is isolated with an equimolar concentration of FAD and FMN. Reduction by NADPH results in the formation of an air stable semiquinone similar to that observed with cytochrome P-450 reductase. Methionine synthase reductase reduces cytochrome c in an NADPH-dependent reaction at a rate (0.44 micromol min(-1) mg(-1) at 25 degrees C) that is comparable with that reported for NR1, a soluble dual flavoprotein of unknown function, but is approximately 100-fold slower than that of P-450 reductase. The K(m) for NADPH is 2.6 +/- 0.5 microm, and the K(act) for methionine synthase reductase is 80.7 +/- 13.7 nm for NADPH-dependent activity of methionine synthase.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2001

Identification of volatile selenium compounds produced in the hydride generation system from organoselenium compounds.

We report a novel aqueous derivatization of selenomethionine (Semet), selenoethionine (Seet) and trimethylselenonium ion (TmSe) by NaBH4 and HCI to volatile selenium species, namely, diethyldiselenide (DeDSe), dimethyldiselenide (DMDSe), dimethylselenide (DmSe) and ethylhydrogenselenide (ESeH), in the hydride generation (HG) system. The volatile selenium compounds produced in the HG system were on-line trapped and concentrated in a U-tube that was immersed in the liquid nitrogen trap. The trapped volatile Se compounds were volatilized at 80 degrees C in a water bath, and 50-500 microL of volatile gas was injected into the GC/AED and GC/MS, respectively. It has been established that DmSe, DmDSe, and DeDSe are the predominant Se compounds that are produced in the HG system from TmSe, Semet, and Seet, respectively, followed by ESeH from Seet. Analytical methods previously employed have stated that these compounds are inactive in the HG system. Prior decomposition of Semet, Seet, and TmSe to selenous acid is essential before HG. To the best of our knowledge, current findings for the production and identification of volatile selenium compounds in the HG system are new and different from existing reports; hence, direct estimation of Semet, Seet, and TmSe is possible when coupling with a HG system using a suitable Se-specific detector.

Journal Article↗
PubMed2001

Pyridoxal phosphate binding sites are similar in human heme-dependent and yeast heme-independent cystathionine beta-synthases. Evidence from 31P NMR and pulsed EPR spectroscopy that heme and PLP cofactors are not proximal in the human enzyme.

Two classes of cystathionine beta-synthases have been identified in eukaryotes, the heme-independent enzyme found in yeast and the heme-dependent form found in mammals. Both classes of enzymes catalyze a pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to produce cystathionine. The role of the heme in the human enzyme and its location relative to the PLP in the active site are unknown. (31)P NMR spectroscopy revealed that spin-lattice relaxation rates of the phosphorus nucleus in PLP are similar in both the paramagnetic ferric (T(1) = 6.34 +/- 0.01 s) and the diamagnetic ferrous (T(1) = 5.04 +/- 0.06 s) enzyme, suggesting that the two cofactors are not proximal to each other. This is also supported by pulsed EPR studies that do not provide any evidence for strong or weak coupling between the phosphorus nucleus and the ferric iron. However, the (31)P signal in the reduced enzyme moved from 5.4 to 2.2 ppm, and the line width decreased from 73 to 16 Hz, providing the first structural evidence for transmission to the active site of an oxidation state change in the heme pocket. These results are consistent with a regulatory role for the heme as suggested by previous biochemical studies from our laboratory. The (31)P chemical shifts of the resting forms of the yeast and human enzymes are similar, suggesting that despite the difference in their heme content, the microenvironment of the PLP is similar in the two enzymes. The addition of the substrate, serine, resulted in an upfield shift of the phosphorus resonance in both enzymes, signaling formation of reaction intermediates. The resting enzyme spectra were recovered following addition of excess homocysteine, indicating that both enzymes retained catalytic activity during the course of the NMR experiment.

Amino Acid Sequence↗
PubMed2001

Lack of abundance of cytoplasmic cyclosporin A-binding protein renders free-living Leishmania donovani resistant to cyclosporin A.

The majority of the effects of cyclosporin A (CsA) on cells is caused by the inhibition of phosphatase activity of calcineurin (CN) by the cyclophilin A (CyPA)-CsA complex formed in the cytoplasm. Although CsA inhibits the proliferation of a large number of parasites, not all are susceptible. The presence of structurally altered CyPA with lower affinity for CsA had been suggested to be the cause of resistance. We report here the identification and cloning of a high affinity CsA-binding protein (LdCyP) from Leishmania donovani, a trypanosomatid parasite that is naturally resistant to CsA. The translated LdCyP consists of 187 amino acids with a cleavable 21-amino acid hydrophobic NH(2)-terminal extension. Modeling studies confirmed that all the residues of human CyPs responsible for interaction with CsA are sequentially and conformationally conserved in LdCyP. The purified recombinant protein displayed biochemical parameters comparable to human CyPs. Reverse transcription-polymerase chain reaction analysis confirmed that LdCyP was abundantly expressed. Immunoblot experiments and direct CsA binding studies revealed that LdCyP located in the subcellular organelles constituted the bulk of the CsA binding activity present in L. donovani, whereas the level of binding activity in the cytosol was conspicuously low. CsA selectively facilitated the secretion of LdCyP in the culture medium. Based on these results, it is concluded that the insensitivity of L. donovani to CsA is probably due to the paucity of CsA binding activity in the cytoplasm of the parasite. We suggest that LdCyP, located in the secretory pathway, may function as a chaperone by binding to membrane proteins rather than as the mediator of CN inhibition.

Amino Acid Sequence↗
PubMed2001

Interaction of poliovirus-encoded 2C/2BC polypeptides with the 3' terminus negative-strand cloverleaf requires an intact stem-loop b.

The poliovirus-encoded, membrane-associated polypeptide 2C and its precursor, 2BC, is believed to be required for initiation and elongation of viral RNA synthesis. Previous studies have shown that the 2C polypeptide specifically interacts with the 3'-terminal sequence of poliovirus negative-strand RNA. This interaction is facilitated by the presence of the conserved sequence UGUUUU in the "stem a" within the 3'-terminal cloverleaf structure. We show that the 2C precursor, 2BC, also interacts with the 3'-terminal cloverleaf of the negative-strand RNA. We also demonstrate here that interaction of 2C/2BC with the negative strand 3'-terminal sequence not only depends on an intact "stem a" containing the UGUUUU sequence but is equally influenced by the presence of an intact "stem b" within the negative-strand cloverleaf. The results presented here suggest that the spatial configuration of stem a UGUUUU sequence with respect to an intact stem-b is crucial for 2C/2BC interaction with the 3'-terminal negative-strand cloverleaf structure.

Base Sequence↗
PubMed2001

Resonance Raman characterization of the heme cofactor in cystathionine beta-synthase. Identification of the Fe-S(Cys) vibration in the six-coordinate low-spin heme.

Human cystathionine beta-synthase (CBS) is an essential enzyme for the removal of the toxic metabolite homocysteine. Heme and pyridoxal phosphate (PLP) cofactors are necessary to catalyze the condensation of homocysteine and serine to generate cystathionine. While the role for the PLP cofactor is thought to be similar to that in other PLP-dependent enzymes that catalyze beta-replacement reactions, the exact role for the heme remains unclear. In this study, we have characterized the heme cofactor of CBS in both the ferric and ferrous states using resonance Raman spectroscopy. Positive identification of a cysteine ligand was achieved by global (34)S isotopic substitution which allowed us to assign the nu(Fe-S) for the six-coordinate low-spin ferric heme at 312 cm(-1). In addition, the CO adduct of ferrous CBS has vibrational frequencies characteristic of a histidine-heme-CO complex in a hydrophobic environment, and indicates that the Fe-S(Cys) bond is labile. We have also found that addition of HgCl(2) to the ferric heme causes conversion of the low-spin heme to a five-coordinate high-spin heme with loss of the cysteine ligand. The present spectroscopic studies do not support a reaction mechanism in which homocysteine binds directly to the heme via displacement of the Cys ligand in the binary enzyme complex, as had been previously proposed.

Binding, Competitive↗
PubMed2001

The coenzyme b12 analog 5'-deoxyadenosylcobinamide-gdp supports catalysis by methylmalonyl-coa mutase in the absence of trans-ligand coordination.

Methylmalonyl-CoA mutase is an 5'-adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to cobalt in solution, is replaced by His(610) donated by the active site. The role of the lower axial ligand in the trillion-fold labilization of the upper axial cobalt-carbon bond has been the subject of enduring debate in the model inorganic literature. In this study, we have used a cofactor analog, 5'deoxyadenosylcobinamide GDP (AdoCbi-GDP), which reconstitutes the enzyme in a "histidine-off" form and which allows us to evaluate the contribution of the lower axial ligand to catalysis. The k(cat) for the enzyme in the presence of AdoCbi-GDP is reduced by a factor of 4 compared with the native cofactor AdoCbl. The overall deuterium isotope effect in the presence of AdoCbi-GDP ((D)V = 7.2 +/- 0.8) is comparable with that observed in the presence of AdoCbl (5.0 +/- 0.6) and indicates that the hydrogen transfer steps in this reaction are not significantly affected by the change in coordination state of the bound cofactor. These surprising results are in marked contrast to the effects ascribed to the corresponding lower axial histidine ligands in the cobalamin-dependent enzymes glutamate mutase and methionine synthase.

Binding Sites↗
PubMed2001

Comparison of in vitro surface properties of clove oil-phospholipid suspensions with those of ALEC, Exosurf and Survanta.

Dipalmitoyl phosphatidylcholine, the main component of lung surfactant is ineffective as a replacement surfactant due to its poor adsorption. We studied clove oil as a possible additive for improving the surface activity of protein-free phospholipid suspensions. We added low doses of clove oil, to phospholipid suspensions and studied the surface properties by in vitro analysis using a pulsating bubble surfactometer and a Wilhelmy balance. Survanta, ALEC and Exosurf were used as controls for comparison. The test surfactants, which were phospholipid-oil suspensions at 1% concentration, in buffer containing either 2 or 5 mM calcium, were pulsated at 40 cpm in a pulsating bubble surfactometer. The phospholipids studied were dipalmitoyl phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), binary mixtures of PC:PE (2:3) and PC:PG (2:3). The addition of clove oil (CO) to each of the above phospholipids was in the ratio of nine parts of phospholipid to one part of oil. The presence of CO caused a significant improvement in the adsorption and minimum surface tension of all the phospholipid suspensions studied. The mixtures PC with CO, both in the presence of 2 and 5 mM calcium, and PCPE with CO at 2 mM calcium concentration had surface properties significantly better than those of ALEC and Exosurf and equivalent to those of Survanta. The addition of clove oil helps improve the surface properties of phospholipids.

Adsorption↗
PubMed2001

Genomic structures and population histories of linguistically distinct tribal groups of India.

There are various conflicting hypotheses regarding the origins of the tribal groups of India, who belong to three major language groups--Austro-Asiatic, Dravidian and Tibeto-Burman. To test some of the major hypotheses we designed a genetic study in which we sampled tribal populations belonging to all the three language groups. We used a set of autosomal DNA markers, mtDNA restriction-site polymorphisms (RSPs) and mtDNA hypervariable segment-1 (HVS-1) sequence polymorphisms in this study. Using the unlinked autosomal markers we found that there is a fair correspondence between linguistic and genomic affinities among the Indian tribal groups. We reconstructed mtDNA RSP haplotypes and found that there is extensive haplotype sharing among all tribal populations. However, there is very little sharing of mtDNA HVS-1 sequences across populations, and none across language groups. Haplogroup M is ubiquitous, and the subcluster U2i of haplogroup U occurs in a high frequency. Our analyses of haplogroup and HVS-1 sequence data provides evidence in support of the hypothesis that the Austro-Asiatic speakers are the most ancient inhabitants of India. Our data also support the earlier finding that some of the western Eurasian haplogroups found in India may have been present in India prior to the entry of Aryan speakers. However, we do not find compelling evidence to support the theory that haplogroup M was brought into India on an "out of Africa" wave of migration through a southern exit route from Ethiopia. On the contrary, our data raise the possibility that this haplogroup arose in India and was later carried to East Africa from India.

DNA, Mitochondrial↗
PubMed2001

NF1 deletions in S-100 protein-positive and negative cells of sporadic and neurofibromatosis 1 (NF1)-associated plexiform neurofibromas and malignant peripheral nerve sheath tumors.

Although plexiform neurofibroma (PN) is thought to represent a benign neoplasm with the potential for malignant transformation (malignant peripheral nerve sheath tumor; MPNST), its neoplastic nature has been difficult to prove due to cellular heterogeneity, which hampers standard molecular genetic analysis. Its mixed composition typically includes Schwann cells, fibroblasts, perineurial-like cells, and mast cells. Although NF1 loss of heterozygosity has been reported in subsets of PNs, it remains uncertain which cell type(s) harbor these alterations. Using a dual-color fluorescence in situ hybridization and immunohistochemistry technique, we studied NF1 gene status in S-100 protein-positive and -negative cell subpopulations in archival paraffin-embedded specimens from seven PNs, two atypical PNs, one cellular/atypical PN, and eight MPNSTs derived from 13 patients, seven of which had neurofibromatosis type 1 (NF1). NF1 loss was detected in four of seven PNs and one atypical PN, with deletions entirely restricted to S-100 protein-immunoreactive Schwann cells. In contrast, all eight MPNSTs harbored NF1 deletions, regardless of S-100 protein expression or NF1 clinical status. Our results suggest that the Schwann cell is the primary neoplastic component in PNs and that S-100 protein-negative cells in MPNST represent dedifferentiated Schwann cells, which harbor NF1 deletions in both NF1-associated and sporadic tumors.

Adolescent↗
PubMed2001

In vitro evaluation of surfactants with eucalyptus oil for respiratory distress syndrome.

The effects of low doses of eucalyptus oil (EO) were studied on the surface properties of phospholipid suspensions as exogenous surfactants, by in vitro analysis using a pulsating bubble surfactometer and a Wilhelmy balance. Survanta, ALEC and Exosurf, commonly used surfactants in therapy of respiratory distress syndrome were used as controls for comparison. The test surfactants, in Ringer's lactate at 1%, were pulsated at 40 cpm in the surfactometer. EO caused a significant improvement of adsorption of the surfactants. In the case of the binary mixture of dipalmitoylphosphatidylcholine and phosphatidylethanolamine (2:3), EO significantly improved the adsorption, stability and minimum surface tension obtained. This combination performed better than ALEC and Exosurf and was comparable to Survanta with respect to minimum surface tension attained. The re-spreading of a surface excess film of this mixture in a Wilhelmy balance was higher than that of ALEC and Exosurf. The ultrastructure of the EO enriched surfactants using cryogenic scanning electron microscopy showed easy facturability and formation of open membranous structures, which could have been associated with the improved surface activity.

Eucalyptus↗