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Biomedical subjects

R Banerjee

Publications and source records attributed to R Banerjee.

At least 55 records · Page 3Linked to original sources
PubMed2000

Thromboviscometry as a tool for evaluation of thrombotic risk in systemic hypertension.

In the present study, thromboviscometry was used to analyse the dynamic coagulation of blood in patients with severe systemic hypertension. Fibrinogen levels and whole blood viscosity, corrected for 45% haematocrit, were also monitored. The efficacy of thromboviscometry as an adjunct diagnostic tool, for determination of thrombogenic potential, was compared with that of detection of fibrinogen levels in the blood. Twenty-five cases of severe systemic hypertension (HT) in the 40 to 50-year age group were compared with 50 age and sex-matched normal controls (NC). The changes in whole blood viscosity were monitored with time at a constant shear rate, in a concentric cylinder viscometer, during the clotting process. The total thrombus formation time was significantly less in the HT group when compared with NC (238.9 +/- 38.72 s vs 315.1 +/- 32.93 s, P < 0.0005). The time required for a sudden increase in viscosity during clotting was also significantly lower in the HT group (205.9 +/- 34.37 s vs 272.9 +/- 28.83 s, P < 0.0005) and the overall rate of increase of thrombus viscosity was significantly higher in HT (245.2 +/- 36.44 centiPoise/s vs 183.6 +/- 16.32 centiPoise/s, P < 0.0005). There was, however, no significant change in the fibrinogen levels of the two groups. Thus, thromboviscometry was a more sensitive indicator of the thrombogenic potential of blood in HT than fibrinogen levels. The increased thrombogenic potential of hypertensive blood could be due to acceleration of the initial part of the coagulation process during the activation of factor Xa and the formation of thrombin.

Adult↗
PubMed2000

Effects of carcinogen-induced transcription factors on the activation of hepatitis B virus expression in human hepatoblastoma HepG2 cells and its implication on hepatocellular carcinomas.

To elucidate the molecular mechanisms involved in the action of common carcinogens, which can act as important cofactors in modulating hepatitis B virus-mediated hepatocellular carcinogenesis, we have investigated the influence of aflatoxin B(1) (AFB), a potent liver carcinogen, as well as benzo[a]pyrene (BP) and 4-aminobiphenyl (4-ABP), carcinogens in cigarette smoke, on the induction of various transcription factors in human hepatoblastoma HepG2 cells. DNA electrophoretic mobility shift assays were performed with nuclear extracts from HepG2 cells treated with 10 micromol/L AFB, 40 micromol/L BP, or 300 micromol/L 4-ABP for 6 and 24 hours. Eight- and 6-fold increases in nuclear transcription factor kappaB (NF-kappaB), and 5- and 10-fold increases in activated protein (AP-1) transcription factor were observed with 24 hours AFB and BP treatments, respectively, whereas 4-ABP treatment resulted in an approximately 4-fold induction of both NF-kappaB and AP-1. Moreover, 4-ABP gave the strongest NF-kappaB activation in 6 hours of treatment. Four- and 10-fold activation of stress protein was detected by a consensus heat shock factor (HSF) sequence binding probe, with AFB and BP treatments, respectively. DNA adducts were observed by immunoassays in HepG2 cells treated with AFB and BP but not with 4-ABP. Increased human hepatitis B virus (HBV) surface antigen (HBsAg) synthesis was detected in AFB- and BP-treated HepG2 cells following transfection with recircularized HBV DNA. These data suggest that certain carcinogen-induced transcription factors may influence viral carcinogenesis and initiate hepatocellular carcinomas (HCC).

Carcinogens↗
PubMed2000

Analysis of dynamic surface properties of therapeutic surfactants and lung phospholipids.

Exogenous surfactant is a specialized biomaterial used for substitution of the lipoprotein mixture normally present in lungs--pulmonary surfactant. Respiratory Distress Syndrome is a disease of preterm infants mainly caused by a deficiency of mature lung surfactant. Pulmonary surfactant is known to stabilize small alveoli and prevent them from collapsing during expiration due to its unique surface properties. A pulsating bubble surfactometer was used for in vitro analysis of surface parameters of therapeutic surfactants and of test formulations to be used for exogenous therapy in Respiratory Distress Syndrome. Surface parameters that were considered for comparison were minimum surface tension (gamma(min)) at three different frequencies (20, 40 and 60 cpm), adsorption at two extreme bubble radii (Rmin and Rmax), stability index at the three frequencies, recruitment index and the surface viscoelastic parameters. Survanta, ALEC and Exosurf were compared with formulations consisting of the main phospholipids of pulmonary surfactant, namely dipalmitoyl phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) as well as binary mixtures of these phospholipids in the ratio 2:3. Survanta performed much better than the non-protein therapeutic surfactants in all parameters and at all three frequencies. Exosurf had a very low stability index and a very low modulus of surface dilatational elasticity at all three frequencies. The test compounds showed a frequency dependence in their performance. At 20 cpm, PC:PG (2:3) was the best test combination. It achieved a gamma(min) and stability index equivalent to Survanta at this frequency. None of the test compounds were comparable to Survanta at 40 and 60 cpm. These findings may have important therapeutic implications for exogenous surfactants.

Humans↗
PubMed2000

tert-butyl N-[2-[N-(N, N'-dicyclohexylureidocarbonylethyl)carbamoyl]prop-2-yl]carbamate.

The title compound, C(25)H(44)N(4)O(5), exhibits a turn with the main chain reversing direction, held together by an intramolecular N-H.O hydrogen bond. In the urea fragment, a notable amide C-N bond between the carboxyl C and the tertiary N atom shows marked single-bond character [1.437 (2) A]. The dihedral angle of the beta-alanyl residue, centrally located in the turn, is gauche [69.2 (2) degrees ]. The packing is mediated by two intermolecular hydrogen bonds and van der Waals contacts involving the methyl moieties and the cyclohexyl rings.

Crystallography, X-Ray↗
PubMed2000

Granulocytic ehrlichiosis in mice deficient in phagocyte oxidase or inducible nitric oxide synthase.

Mice deficient in phox (gp91(phox-/-)) or NOS2 (NOS2(-/-)) were infected with the agent of human granulocytic ehrlichiosis (HGE) to evaluate the importance of these pathways in the eradication of HGE bacteria. NOS2(-/-) mice had delayed clearance of the HGE agent in comparison to control or gp91(phox-/-) mice, suggesting that reactive nitrogen intermediates play a role in the early control of HGE.

Animals↗
PubMed2000

Exogenous surfactant therapy and mucus rheology in chronic obstructive airway diseases.

Exogenous surfactant is a specialized biomaterial used for substitution of the lipoprotein mixture normally present in the lungs-pulmonary surfactant. Respiratory Distress Syndrome is a disease of preterm infants mainly caused by pulmonary immaturity as evidenced by a deficiency of mature lung surfactant. Pulmonary surfactant is known to stabilize small alveoli and prevent them from collapsing during expiration. However, apart from alveoli, surfactant also lines the narrow conducting airways of the tracheobronchial tree. This paper reviews the role of this surfactant in the airways and its effect on mucus rheology and mucociliary clearance. Its potential role as a therapeutic biomaterial in chronic obstructive airway diseases, namely asthma, chronic bronchitis, and respiratory manifestations of cystic fibrosis, are discussed. This paper also attempts to elucidate the exact steps in the pathogenic pathway of these diseases which could be reversed by supplementation of exogenous surfactant formulations. It is shown that there is great potential for the use of present day surfactants (which are actually formulated for use in Respiratory Disease Syndrome) as therapy in the aforementioned diseases of altered mucus viscoelasticity and mucociliary clearance. However, for improved effectiveness, specific surfactant formulations satisfying certain specific criteria should be tailor-made for the clinical condition for which they are intended. The properties required to be fulfilled by the optimal exogenous surfactant in each of the above clinical conditions are enumerated in this paper.

Animals↗
PubMed2000

Zoonoses and bats: a look from human health viewpoint.

Bats are the most abundant and most widely distributed mammals on the Earth after humans. Except Antarctica and some small remote islands, they are prevalent worldwide. Although the actual role played by bats as reservoir or in disseminating zoonoses is still enigmatic a multitude of zoonotic diseases are known to be associated with bats. Such diseases including viral, bacterial, parasitic, fungal and rickettsial diseases, reported from all over the world including India have been briefed in this article as an informative approach because dramatically increased and diversified human activities during the last few decades perturbing natural ecosystems are enough to compel public health personnel to have an investigative look at these flying mammals.

Animals↗
PubMed2000

Effects of clove oil-phospholipid mixtures on rheology of gum tragacanth - possible application for surfactant action on mucus gel simulants.

The present study evaluates the effectiveness of specialised biomaterials consisting of clove oil- phospholipid mixtures as possible substitute surfactants in diseases of altered mucus viscosity by studying their effect on the viscosity of mucus gel simulants in vitro. Test surfactants consisting of phospholipid-clove oil mixtures in the ratio of 1 part of oil to 9 parts of phospholipid were prepared. The phospholipids used were dipalmitoyl phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) and binary mixtures of PC: PE and PC: PG in the ratio of 2 parts of PC to 3 parts of PE or PG. The effects of the phospholipid-clove oil mixtures on the viscosity of mucus gel simulant (MGS: a polymeric gel consisting predominantly of gum tragacanth and simulating respiratory mucus), was studied by application of steady shear rates ranging from 0.512 to 51.2/s in a concentric cylinder viscometer at 37 degrees C. The change in MGS viscosity, after incubation with surfactants, was found to have a non-Newtonian character and to follow the power law model with R2 values >0.8. The addition of clove oil-phospholipid mixtures caused a decrease in the MGS viscosity when compared with the effect of the phospholipid alone at low shear rates in case of PC, PG and PCPG. The combination of PC : PG with clove oil caused ratios of change in MGS viscosity < 1 i.e., caused a decrease in the MGS viscosity. PC: PG with clove oil was capable of lowering MGS viscosity and should be further researched as possible therapies for diseases of altered mucus rheology.

Biocompatible Materials↗
PubMed2000

Effects of exercise on rheological and microcirculatory parameters.

The physiological changes occurring during exercise and its possible consequences have been receiving considerable attention lately. In this paper, we studied the changes in hemorheological and microcirculatory parameters, before and after the exercise, in the subjects undergoing mild exercise (n = 20). A cycle ergometer adjusted at 2.5 kilopounds was used for 15 minutes. The whole blood viscosity showed a significant increase after exercise at all shear rates (0.512-51.2/s) except at the high shear rate (94.5/s). However, the significant level was more (P < 0.005) at low shear rates (0.512-4.39/s). A significant elevation in plasma viscosity was observed after the exercise (P < 0.0008). Red cell rigidity showed a significant increase after the exercise (P < 0.001) while red cell aggregation and hematocrit failed to show any significant change. Microcirculatory studies showed a significant increase in the basal perfusion level after exercise (P < 0.0002) when compared to the resting state value. There was a significant decrease in reactive hyperaemia perfusion index after exercise (P < 0.0007). Hence, it is evident from this study that short-term exercise significantly alters hemorheological and microcirculatory parameters.

Adult↗
PubMed1999

An efficient method for simultaneous isolation of biologically active transcription factors and DNA.

Transcription factors play a crucial role in gene regulation during different stages of eukaryotic development as well as in controlling various cellular disorders involving the immune system. In order to study the role of cellular DNAs and the effects of certain biologically active regulatory proteins, which can affect gene expression, we have developed a rapid and efficient method for preparing highly purified DNAs as well as nuclear and cytoplasmic proteins, simultaneously. These DNAs and proteins can be effectively analyzed to determine their genetic integrity and binding motifs to specific DNA sequences, respectively. This protocol avoids the drastic use of mechanical shearing of cells, aggressive use of detergents or high speed ultracentrifugation steps, as well as facilitating the ease of collecting samples in a sequential and effective manner with minimal time lapse during processing. Such an approach permits the analysis of a large number of samples in a short time. The current technique uses a non-ionic detergent to isolate nuclei, and obtain the cytosolic extract, a low-ionic strength buffer to wash off the detergent and a high-salt buffer to extract nuclear proteins including transcription factors. The remainder of the cellular products are processed for DNA extraction. This method will be particularly useful to evaluate the time course effects of various cell signal transducing biological modifiers such as cytokines or mitogens, as well as drugs used in therapy, especially in infectious diseases and also in immunological or neoplastic disorders, with minimal physical contact to the laboratory personnel. This rapid DNA and protein isolation method can be widely used in various systems to analyze the modulation of DNA characteristics and transcriptionally active proteins as biomarkers in different human diseases.

Base Sequence↗
PubMed1999

Role of the dimethylbenzimidazole tail in the reaction catalyzed by coenzyme B12-dependent methylmalonyl-CoA mutase.

The recent structures of cobalamin-dependent methionine synthase and methylmalonyl-CoA mutase have revealed a striking conformational change that accompanies cofactor binding to these proteins. Alkylcobalamins have octahedral geometry in solution at physiological pH, and the lower axial coordination position is occupied by the nucleotide, dimethylbenzimidazole ribose phosphate, that is attached to one of the pyrrole rings of the corrin macrocycle via an aminopropanol moiety. In contrast, in the active sites of these two B12-dependent enzymes, the nucleotide tail is held in an extended conformation in which the base is far removed from the cobalt in cobalamin. Instead, a histidine residue donated by the protein replaces the displaced intramolecular base. This unexpected mode of cofactor binding in a subgroup of B12-dependent enzymes has raised the question of what role the nucleotide loop plays in cofactor binding and catalysis. To address this question, we have synthesized and characterized two truncated cofactor analogues: adenosylcobinamide and adenosylcobinamide phosphate methyl ester, lacking the nucleotide and nucleoside moieties, respectively. Our studies reveal that the nucleotide tail has a modest effect on the strength of cofactor binding, contributing approximately 1 kcal/mol to binding. In contrast, the nucleotide has a profound influence on organizing the active site for catalysis, as evidenced by the retention of the base-off conformation in the truncated cofactor analogues bound to the mutase and by their inability to support catalysis. Characterization of the kinetics of adenosylcobalamin (AdoCbl) binding by stopped-flow fluorescence spectroscopy reveals a pH-sensitive step that titrates to a pKa of 7.32 +/- 0.19 that is significantly different from the pKa of 3.7 for dimethylbenzimidazole in free AdoCbl. In contrast, the truncated cofactors associate very rapidly with the enzyme at rates that are too fast to measure. Based on these observations, we propose a model in which the base-on to base-off conformational change is slow and is assisted by the enzyme, and is followed by a rapid docking of the cofactor in the active site.

Benzimidazoles↗
PubMed1999

Proton transfer from histidine 244 may facilitate the 1,2 rearrangement reaction in coenzyme B(12)-dependent methylmalonyl-CoA mutase.

Methylmalonyl-CoA mutase is an adenosylcobalamin-dependent enzyme that catalyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA. This reaction results in the interchange of a carbonyl-CoA group and a hydrogen atom on vicinal carbons. The crystal structure of the enzyme reveals the presence of an aromatic cluster of residues in the active site that includes His-244, Tyr-243, and Tyr-89 in the large subunit. Of these, His-244 is within hydrogen bonding distance to the carbonyl oxygen of the carbonyl-CoA moiety of the substrate. The location of these aromatic residues suggests a possible role for them in catalysis either in radical stabilization and/or by direct participation in one or more steps in the reaction. The mechanism by which the initially formed substrate radical isomerizes to the product radical during the rearrangement of methylmalonyl-CoA to succinyl-CoA is unknown. Ab initio molecular orbital theory calculations predict that partial proton transfer can contribute significantly to the lowering of the barrier for the rearrangement reaction. In this study, we report the kinetic characterization of the H244G mutant, which results in an acute sensitivity of the enzyme to oxygen, indicating the important role of this residue in radical stabilization. Mutation of His-244 leads to an approximately 300-fold lowering in the catalytic efficiency of the enzyme and loss of one of the two titratable pK(a) values that govern the activity of the wild type enzyme. These data suggest that protonation of His-244 increases the reaction rate in wild type enzyme and provides experimental support for ab initio molecular orbital theory calculations that predict rate enhancement of the rearrangement reaction by the interaction of the migrating group with a general acid. However, the magnitude of the rate enhancement is significantly lower than that predicted by the theoretical studies.

Binding Sites↗
PubMed1999

Deletion of the regulatory domain in the pyridoxal phosphate-dependent heme protein cystathionine beta-synthase alleviates the defect observed in a catalytic site mutant.

The most common cause of severely elevated homocysteine or homocystinuria is inherited disorders in cystathionine beta-synthase. The latter enzyme is a unique hemeprotein that catalyzes pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine, thus committing homocysteine to catabolism. A point mutation, V168M, has been described in a homocystinuric cell line and is associated with a B(6)-responsive phenotype. In this study, we have examined the kinetic properties of this mutant and demonstrate that the mutation affects the PLP but not the heme content. The approximately 13-fold diminution in activity because of the mutation corresponds to an approximately 7-fold decrease in the level of bound PLP. This may be explained by half of the sites activity associated with cystathionine beta-synthase. The addition of PLP results in partial but not full restoration of activity to wild type levels. Elimination of the C-terminal quarter of the mutant protein results in alleviation of the catalytic penalty imposed by the V168M mutation. The resulting truncated protein is very similar to the corresponding truncated enzyme with wild type sequence and is now able to bind the full complement of both heme and PLP cofactors. These results indicate that the V168M mutation per se does not affect binding of PLP directly and that interactions between the regulatory C terminus and the catalytic N terminus are important in modulating the cofactor content and therefore the activity of the full-length enzyme. These studies provide the first biochemical explanation for the B(6)-responsive phenotype associated with a cystathionine beta-synthase-impaired homocystinuric genotype.

Catalytic Domain↗
PubMed1999

Novel series of non-glycerol-based cationic transfection lipids for use in liposomal gene delivery.

A novel series of nontoxic and non-glycerol-based simple monocationic transfection lipids containing one or two hydroxyethyl groups directly linked to the positively charged nitrogen atom were synthesized. The in vitro transfection efficiencies of these new liposomal gene delivery reagents were better than that of lipofectamine, a widely used transfection agent in cationic lipid-mediated gene transfer. The most efficient transfection formulation was observed to be a 1:1:0.3 mol ratio of DHDEAB (N, N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide):cholesterol:HDEAB (N-n-hexadecyl-N,N-dihydroxyethylammonium bromide) using a DHDEAB-to-DNA charge ratio (+/-) of 0.3:1. Observation of good transfection at charge ratios lower than 1 suggests that the amphiphile-DNA complex may have net negative charge. Our results reemphasize the important point that in cationic lipid-mediated gene delivery, the overall charge of the lipid-DNA complex need not always be positive. In addition, our transfection results also imply that favorable hydrogen-bonding interactions between the lipid headgroups and the cell surface of biological membranes may have some role for improving the transfection efficiency in cationic lipid-mediated gene delivery.

Animals↗
PubMed1999

Assignment of enzymatic functions to specific regions of the PLP-dependent heme protein cystathionine beta-synthase.

Cystathionine beta-synthase is a unique heme protein that catalyzes a pyridoxal phosphate (or PLP)-dependent beta-replacement reaction. The reaction involves the condensation of serine and homocysteine and constitutes one of the two major avenues for detoxification of homocysteine in mammals. The enzyme is allosterically regulated by S-adenosylmethionine (AdoMet). In this study, we have characterized the kinetic, spectroscopic, and ligand binding properties of a truncated catalytic core of cystathionine beta-synthase extending from residues 1 through 408 in which the C-terminal 143 residues have been deleted. This is similar to a natural variant of the protein that has been described in a homocystinuric patient in which the predicted peptide is 419 amino acids in length. Truncation leads to the formation of a dimeric enzyme in contrast to the tetrameric organization of the native enzyme. Some of the kinetic properties of the truncated enzyme are different from the full-length form, most notably, significantly higher K(m)s for the two substrates, and loss of activation by AdoMet. This is paralleled by the absence of AdoMet binding to the truncated form, whereas four AdoMet molecules bind cooperatively to the full-length tetrameric enzyme with a K(d) of 7. 4 microM. Steady-state kinetic analysis indicates that the order of substrate addition is important. Thus, preincubation of the enzyme with homocysteine leads to a 2-fold increase in V(max) relative to preincubation of the enzyme with serine. Since the intracellular concentration of serine is significantly greater than that of homocysteine, the physiological significance of this phenomenon needs to be considered. Based on ligand binding studies and homology searches with protein sequences in the database, we assign residues 68-209 as being important for PLP binding, residues 241-341 for heme binding, and residues 421-469 for AdoMet binding.

Amino Acid Sequence↗
PubMed1999

Posttranscriptional regulation of mammalian methionine synthase by B12.

Methionine synthase is one of two key enzymes involved in the removal of the metabolite, homocysteine. Elevated homocysteine levels constitute a risk factor for cardiovascular diseases and for neural tube defects. In cell culture, the activity of methionine synthase is enhanced several-fold by supplementation with its cofactor, B12. The mechanism of this regulation is unknown, although it has been ascribed to a shift from apoenzyme to holoenzyme. Using sensitive assay techniques as well as a combination of Northern and Western analyses, we demonstrate that the effect of B12 on induction of methionine synthase activity is paralleled by an increase in the level of the enzyme. These studies exclude conversion of apoenzyme to holoenzyme as a basis for activation that had been described previously. Since the mRNA levels do not change during the same period that the methionine synthase levels increase, regulation of this protein by its cofactor must be exerted posttranscriptionally.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1999

Plasmepsin II, an acidic hemoglobinase from the Plasmodium falciparum food vacuole, is active at neutral pH on the host erythrocyte membrane skeleton.

Plasmepsin II, an aspartic protease from the human intraerythrocytic parasite Plasmodium falciparum, is involved in degradation of the host cell hemoglobin within the acidic food vacuole of the parasite. Previous characterization of enzymatic activities from Plasmodium soluble extracts, responsible for in vitro hydrolysis of erythrocyte spectrin, had shown that the hydrolysis process occurred at pH 5.0 and involved aspartic protease(s) cleaving mainly within the SH3 motif of the spectrin alpha-subunit. Therefore, we used a recombinant construct of the erythroid SH3 motif as substrate to investigate the involvement of plasmepsins in spectrin hydrolysis. Using specific anti-plasmepsin II antibodies in Western blotting experiments, plasmepsin II was detected in chromatographic fractions enriched in the parasite SH3 hydrolase activity. Involvement of plasmepsin II in hydrolysis was demonstrated by mass spectrometry identification of cleavage sites in the SH3 motif, upon hydrolysis by Plasmodium extract enzymatic activity, and by recombinant plasmepsin II. Furthermore, recombinant plasmepsin II digested native spectrin at pH 6.8, either purified or situated in erythrocyte ghosts. Additional degradation of actin and protein 4.1 from ghosts was observed. Specific antibodies were used in confocal imaging of schizont-infected erythrocytes to localize plasmepsin II in mature stages of the parasite development cycle; antibodies clearly labeled the periphery of the parasites. Taken together, these results strongly suggest that, in addition to hemoglobin degradation, plasmepsin II might be involved in cytoskeleton cleavage of infected erythrocytes.

Amino Acid Sequence↗