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Biomedical subjects

A Banerjee

Publications and source records attributed to A Banerjee.

At least 19 recordsLinked to original sources
PubMed2000

Identification of a cysteine residue in the active site of nitroalkane oxidase by modification with N-ethylmaleimide.

The flavoprotein nitroalkane oxidase catalyzes the oxidative denitrification of primary or secondary nitroalkanes to the corresponding aldehydes or ketones with production of hydrogen peroxide and nitrite. The enzyme is irreversibly inactivated by treatment with N-ethylmaleimide at pH 7. The inactivation is time-dependent and shows first-order kinetics for three half-lives. The second-order rate constant for inactivation is 3.4 +/- 0.06 m(-)(1) min(-)(1). The competitive inhibitor valerate protects the enzyme from inactivation, indicating an active site-directed modification. Comparison of tryptic maps of enzyme treated with N-[ethyl-1-(14)C]maleimide in the absence and presence of valerate shows a single radioactive peptide differentially labeled in the unprotected enzyme. The sequence of this peptide was determined to be LLNEVMCYPLFDGGNIGLR using Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cysteine residue was identified as the site of alkylation by ion trap mass spectrometry.

Amino Acid Sequence↗
PubMed2000

Biocatalytic preparation of a chiral synthon for a vasopeptidase inhibitor: enzymatic conversion of N(2)-

[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01) is a key chiral intermediate for the synthesis of Omapatrilat (BMS-186716), a new vasopeptidease inhibitor under development. By using a selective enrichment culture technique we have isolated a strain of Sphingomonas paucimobilis SC 16113, which contains a novel L-lysine epsilon-aminotransferase. This enzyme catalyzed the oxidation of the epsilon-amino group of lysine in the dipeptide dimer N(2)-[N[phenyl-methoxy)-carbonyl] L-homocysteinyl] L-lysine)1,1-disulphide (BMS-201391-01) to produce BMS-199541-01. The aminotransferase reaction required alpha-ketoglutarate as the amino acceptor. Glutamate formed during this reaction was recycled back to alpha-ketoglutarate by glutamate oxidase from Streptomyces noursei SC 6007. Fermentation processes were developed for growth of S. paucimobilis SC 16113 and S. noursei SC 6007 for the production of L-lysine epsilon-amino transferase and glutamate oxidase, respectively. L-lysine epsilon-aminotransferase was purified to homogeneity and N-terminal and internal peptides sequences of the purified protein were determined. The mol wt of L-lysine epsilon-aminotransferase is 81 000 Da and subunit size is 40 000 Da. L-lysine epsilon-aminotransferase gene (lat gene) from S. paucimobilis SC 16113 was cloned and overexpressed in Escherichia coli. Glutamate oxidase was purified to homogeneity from S. noursei SC 6003. The mol wt of glutamate oxidase is 125 000 Da and subunit size is 60 000 Da. The glutamate oxiadase gene from S. noursei SC 6003 was cloned and expressed in Streptomyces lividans. The biotransformation process was developed for the conversion of BMS-201391-01 to BMS-199541-01 by using L-lysine epsilon-aminotransferase expressed in E. coli. In the biotransformation process, for conversion of BMS-201391-01 (CBZ protecting group) to BMS-199541-01, a reaction yield of 65-70 M% was obtained depending upon reaction conditions used in the process. Phenylacetyl or phenoxyacetyl protected analogues of BMS-201391-01 also served as substrates for L-lysine epsilon-aminotransferase giving reaction yields of 70 M% for the corresponding BMS-199541-01 analogs. Two other dipeptides N-[N[(phenylmethoxy)carbonyl]-L-methionyl]-L-lysine (BMS-203528) and N,2-[S-acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (BMS-204556) were also substrates for L-lysine epsilon-aminotransferase. N-alpha-protected (CBZ or BOC)-L-lysine were also oxidized by L-lysine epsilon-aminotransferase.

Journal Article↗
PubMed2000

Enzymatic acetylation of 10-deacetylbaccatin III to baccatin III by C-10 deacetylase from Nocardioides luteus SC 13913.

Baccatin III is a polycyclic diterpene which can be used for the semi-synthesis of paclitaxel and analogs. An enzymatic process was developed for the conversion of 10-deacetylbaccatin III (10-DAB) to baccatin III without requiring protection of the 7-hydroxyl group. A C-10 deacetylase from Nocardioides luteus SC 13912 was immobilized on diethylaminoethyl cellulose (DEAE-Cellulose) and the immobilized enzyme was used in the biotransformation process. The reaction was catalyzed using vinyl acetate as acyl donor at ambient temperature and at pH 7. A reaction yield of 51% was obtained.

Journal Article↗
PubMed2000

Adenosine preconditioning reduces both pre and postischemic arrhythmias in human myocardium.

INTRODUCTION: Consistently, clinical series record supraventricular tachyarrhythmias in approximately 30% of patients following coronary artery bypass surgery (CABG). Ischemic preconditioning and adenosine preconditioning (Ado-PC) decrease postischemia/reperfusion (I/R) myocardial stunning, infarct size, and pharmacologically induced arrhythmias in all species including man. We hypothesized that adenosine preconditioning would decrease spontaneous pre- and postischemic atrial arrhythmias in human myocardium. The purposes of this study were to determine the effect of in vivo and in vitro Ado-PC on atrial arrhythmias. METHODS: Human atrial trabeculae were harvested from CABG patients, placed in organ baths, and paced (1 Hz). Developed force (DF) was recorded during simulated I/R (30/45 min). Prior to I/R, trabeculae were treated with Ado (125 microM) for 5 min (in vitro), or patients were treated with Ado (12 mg iv) 5 min (in vivo) prior to harvest of trabeculae. Contraction frequency >4 Hz (defined as atrial tachyarrhythmias) was recorded in all groups pre- and postischemia. RESULTS: Control trabeculae exhibited increased tachyarrhythmias pre- and postischemia. In vivo and in vitro Ado-PC suppressed both pre- and postischemic arrhythmias. CONCLUSIONS: Adenosine preconditioning suppresses the frequency of pre- and postischemic tachyarrhythmias against an ischemia/reperfusion insult in human myocardium. This antiarrhythmic effect occurs with both in vitro and in vivo administration of adenosine. Preconditioning with adenosine prior to elective ischemia/reperfusion is a promising strategy of reducing spontaneous atrial arrhythmias in patients undergoing myocardial revascularization.

Adenosine↗
PubMed2000

Dentine caries excavation: a review of current clinical techniques.

Since the invention and application of rotary instruments, the operative treatment of carious lesions has often resulted in considerable removal of tooth structure. More recently, newer techniques for removal of carious dentine have been developed in an attempt to minimise this excessive tissue loss. The following article reviews and discusses some of the techniques available to excavate demineralised dentine clinically. These methods can be classified as mechanical and non-mechanical, rotary and non-rotary and include: dental handpieces/burs, manual excavators, air-abrasion, air-polishing, ultrasonication, sono-abrasion, chemo-mechanical methods, lasers and enzymes. The advantages and disadvantages of each technique are discussed.

Air↗
PubMed2000

Identification of an essential tyrosine residue in nitroalkane oxidase by modification with tetranitromethane.

The flavoprotein nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with production of nitrite and hydrogen peroxide. The enzyme is irreversibly inactivated by incubation with tetranitromethane, a tyrosine-directed reagent, at pH 7.3. The inactivation is time-dependent and shows first-order kinetics for two half-lives of inactivation. Further inactivation can be achieved upon a second addition of tetranitromethane. A saturation kinetic pattern is observed when the rate of inactivation is determined versus the concentration of tetranitromethane, indicating that a reversible enzyme-inhibitor complex is formed before irreversible inactivation occurs. Values of 0.096 +/- 0.013 min(-1) and 12.9 +/- 3.8 mM were determined for the first-order rate constant for inactivation and the dissociation constant for the reversibly formed complex, respectively. The competitive inhibitor valerate protects the enzyme from inactivation by tetranitromethane, suggesting an active-site-directed inactivation. The UV-visible absorbance spectrum of the inactivated enzyme is perturbed with respect to that of the native enzyme, suggesting that treatment with tetranitromethane resulted in nitration of the enzyme. Comparison of tryptic maps of nitroalkane oxidase treated with tetranitromethane in the presence and absence of valerate shows a single peptide differentially labeled in the inactivated enzyme. The spectral properties of the modified peptide are consistent with nitration of a tyrosine residue. The amino acid sequence of the nitrated peptide is L-L-N-E-V-M-C-(NO(2)-Y)-P-L-F-D-G-G-N-I-G-L-R. The possible role of this tyrosine in substrate binding is discussed.

Amino Acid Sequence↗
PubMed2000

Scanning electron microscopic observations of human dentine after mechanical caries excavation.

OBJECTIVES: The structural integrity and surface characteristics of dentine remaining after caries excavation may be relevant to the subsequent bonding of adhesive restorative materials to the prepared cavity. This in vitro investigation aimed to analyse the different surface characteristics of the dentine cavity floor created after preparation using five different mechanical and chemo-mechanical methods of excavation: hand excavation, slow-speed bur, sono-abrasion, air-abrasion and Carisolv gel. METHODS: Ten cavities were prepared using each excavation method in extracted teeth with occlusal carious lesions. Epoxy resin replicas of the 50 cavities were manufactured from silicone impressions and then analysed using secondary electron scanning electron microscopy (SEM) to ascertain the surface characteristics of the dentine at the cavity floor. RESULTS AND CONCLUSIONS: Results from the 50 cavities examined suggested that each alternative excavation technique produced a different and characteristic dentine surface. Carisolv gel was the only method examined that consistently removed the smear layer during excavation to leave exposed dentine tubules at the end of cavity preparation.

Air Abrasion, Dental↗
PubMed2000

Beta-carotene prolongs survival, decreases lipid peroxidation and enhances glutathione status in transplantable murine lymphoma.

Carotenoids of dietary origin have recently been the subject of renewed research interest because of epidemiological evidence indicating an inverse relationship between intake of carotenoids-rich plant substances and risk of certain cancers. This study was attempted to understand the biological actions of dietary beta-carotene (BC) on Dalton's lymphoma (DL), a rapidly proliferating transplantable tumor, in effecting the survival of the lymphoma-bearing mice. The glutathione (GSH) level and the extent of lipid peroxidation in the liver, kidney and brain were monitored in BC-treated (100 mg/kg food) mice transplanted with DL. These markers showed substantial alterations during the whole length of tumor progression in lymphoma-bearing mice without BC supplementation. When treated with BC, both malondialdehyde contents (evidence of lipid peroxidation) and the GSH levels in different organs were found to be closer to normal values in the initial period of tumor progression. BC-mediated protection against lipid peroxidation was maximally found to be in hepatic tissue throughout the study following DL transplantation. This was fairly reflected in the higher BC concentration in hepatic tissue of BC-treated lymphoma group compared to untreated lymphoma control. Significantly higher survival time (51-55 days) was observed in BC-treated animals in comparison to their untreated DL counterparts (35-38 days). The prolonged survival observed in the BC-supplemented animals may be attributed to the higher resistance offered by animals receiving BC towards lipid peroxidation-related tissue injury.

Animals↗
PubMed2000

Protein kinase C zeta isoform is critical for proliferation in human glioblastoma cell lines.

Previous studies have confirmed that proliferation in glioblastoma cell lines can be blocked by non-isoform specific protein kinase C (PKC) inhibitors, e.g calphostin C, staurosporine. However, the exact mechanism of PKC involvement is poorly understood. The aim of this study was to explore the role of specific PKC isoforms in the aberrant growth of glioblastoma. Identification of the isoform(s) critical for proliferation in glioblastoma would present a better target for the design of chemotherapeutic strategies. To this end, we screened expression on PKC isoforms in four human glioblastoma cell lines both when proliferating and in a quiescent state using western assays. PKC isoforms alpha, beta, betaII and zeta were found to be expressed in all cell lines. PKC epsilon was detected in three out of four cell lines and PKC eta was detected in one out of four cell lines. Quiescence of growth resulted in down-regulation of PKC epsilon. We examined the role of these isoforms by studying the effect of PKC isoform-specific inhibitors bisindolylmaleimide-I and Gö6976 on proliferation in a panel of four human glioblastoma cell lines. Inhibition of PKC alpha and epsilon had no effect on proliferation, suggesting that previous studies targeting PKC alpha may not be of therapeutic benefit. More significantly, it was shown that inhibition of PKC zeta blocked proliferation. This suggests that the inhibition of PKC zeta may be an important chemotherapeutic target for arresting growth in glioblastoma.

Blotting, Western↗
PubMed2000

Selective mitochondrial KATP channel opening controls human myocardial preconditioning: too much of a good thing?

BACKGROUND: Paradoxically, patients with noninsulin-dependent diabetes mellitus experience a higher cardiovascular mortality rate than patients with insulin-dependent diabetes mellitus. We have shown that K(ATP) channel inhibition, with oral sulfonylureas, prevents myocardial preconditioning and may explain the paradox of cardiovascular death in patients with noninsulin-dependent diabetes mellitus. Cardiac preconditioning is an attractive protective strategy against any elective ischemia/reperfusion (I/R) injury. The relationship between the K(ATP) channels and human myocardial preconditioning has not previously been elucidated. METHODS: Human atrial trabeculae were harvested, placed in organ baths, and paced (1 Hz). Developed force was recorded during simulated 37 degrees C I/R (30/45 or 45/60 minutes). Before I/R, trabeculae were treated transiently with a selective mitochondrial K(ATP) channel opener for 5 minutes, followed by a 10-minute washout, or were exposed to the channel opener throughout ischemia. Recovery of function is expressed as percentage of baseline developed force. Conserved creatine kinase activity (units per gram of wet tissue) was measured at the end of reperfusion as an indicator of cellular protection. RESULTS: Transient mitochondrial K(ATP) channel opening provided protection from both I/R insults. Surprisingly, there was no protection afforded by continuous mitochondrial K(ATP) channel opening. CONCLUSIONS: Transient selective mitochondrial K(ATP) channel opening protects both viability and function of human myocardium against I/R injury, although prolonged opening of the mitochondrial K(ATP) channel does not. These results reinforce the concept of preconditioning as a transient event that must be completed before the onset of ischemia.

Adenosine Triphosphate↗
PubMed2000

Selective mitochondrial adenosine triphosphate-sensitive potassium channel activation is sufficient to precondition human myocardium.

OBJECTIVES: Recently, the mitochondrial adenosine triphosphate-sensitive potassium channel has been suggested to be the final common effector of myocardial preconditioning. The purpose of this study is to determine whether selective mitochondrial adenosine triphosphate-sensitive potassium channel activation alone can precondition human myocardium from an ischemia/reperfusion insult. METHODS: Isolated human right atrial trabeculae were placed in tissue baths, paced, and subjected to 30 minutes of normothermic hypoxia (ischemia) followed by 45 minutes of reoxygenation (reperfusion). Trabeculae were preconditioned with a selective mitochondrial adenosine triphosphate-sensitive potassium channel opener (diazoxide 30 micromol/L) or a nonselective purinergic agonist, adenosine (125 micromol/L), for 5 minutes (adenosine) followed by a 10-minute washout period. Developed force at end reperfusion (mean +/- standard error) was compared with baseline, and tissue creatine kinase and adenosine triphosphate levels were measured after ischemia/reperfusion. RESULTS: Trabeculae subjected to ischemia/reperfusion exhibited 30% +/- 2% of baseline developed force, whereas trabeculae subjected to selective adenosine triphosphate-sensitive potassium channel opening (diazoxide) and nonselective purinergic agonist (adenosine) recovered to 55% +/- 7% and 46% +/- 3% of baseline developed force, respectively. Tissue creatine kinase activity was preserved in both the diazoxide- and adenosine-treated trabeculae (5.4 +/- 12 and 5.4 +/- 14 micromol/L per gram wet tissue) compared with ischemia/reperfusion (1.8 +/- 0.2 U/mg wet tissue). Adenosine triphosphate levels at end reperfusion were also increased in the trabeculae treated with selective (diazoxide) and nonselective (adenosine) adenosine triphosphate-sensitive potassium channel opener (4.1 +/- 0.01 and 4. 4 +/- 0.2 micromol/L per gram wet tissue) compared with trabeculae subjected to ischemia/reperfusion (1.5 +/- 0.1 micromol/L per gram wet tissue). CONCLUSIONS: These results suggest that selective mitochondrial adenosine triphosphate-sensitive potassium channel activation preconditions human myocardium and the protection conferred is equal to that of adenosine preconditioning. Targeted openers of mitochondrial adenosine triphosphate- sensitive potassium channels promote constructive protection of myocellular energy levels, contractile function, and cellular viability in human myocardium after ischemia/reperfusion.

Adenosine↗
PubMed2000

Structural and interactional homology of clinically potential trypsin inhibitors: molecular modelling of cucurbitaceae family peptides using the X-ray structure of MCTI-II.

Several trypsin inhibitor peptides (with 28-32 amino acid residues) belonging to the Cucurbitaceae (LA-1, LA-2, MCTI-I, CMTI-I, CMTI-III, CMTI-IV), characterized by a distinctive tertiary fold with three conserved disulphide bonds and with mostly arginine at their active centre, were modelled using the high-resolution X-ray structure of a homologous inhibitor, MCTI-II, isolated from bitter gourd. All the inhibitors were modelled in both their native and complexed state with the trypsin molecule, keeping the active site the same as was observed in the trypsin-MCTI-II complex, by homology modelling using the InsightII program. The minimized energy profile supported the binding constants (binding behaviour) of the inhibitor-trypsin complexes in the solution state. A difference accessible surface area (DASA) study of the trypsin with and without inhibitors revealed the subsites of trypsin where the inhibitors bind. It revealed that the role of mutation of these peptides through evolution is to modulate their inhibitory function depending on the biological need rather than changing the overall structural folding characteristics which are highly conserved. The minor changes of amino acids in the non-conserved regions do not influence significantly the basic conformational and interactional sequences at the trypsin binding subsites during complex formation.

Amino Acid Sequence↗
PubMed2000

Protection against endotoxemia in rats by a novel tetrahydrobiopterin analogue.

We studied the effects of a novel pterin antagonist of NO synthase, the 4-amino analogue of tetrahydrobiopterin (4-ABH4), in a rat model of endotoxic shock and compared its properties with those of N(G)-monomethyl L-arginine (L-NMMA). Treatment with a bolus dose of 4-ABH4 at 2 h after LPS challenge significantly improved the 6-day survival rate, compared with the controls treated with saline. L-NMMA treatment did not significantly influence the survival rate. This bolus treatment, using either compound, had no effect on the plasma nitrite + nitrate or plasma IL-6 levels. The continuous infusion of 4-ABH4 efficiently suppressed the enhanced calcium-dependent/independent NO synthase activities induced by endotoxin in lung homogenates and completely suppressed the increase in plasma nitrite + nitrate caused by endotoxin at 5 h, with no significant difference compared with the L- NMMA treatment. Treatment of RAW264.7 murine macrophages with 4-ABH4 but not with L-NMMA suppressed endotoxin-induced tumor necrosis factor-alpha release by the cells, whereas nitrite in the supernatant decreased in a dose-dependent fashion in both assay systems. Our data show that 4-ABH4, an inhibitor of inducible NO synthase, significantly improves survival in a rat model of endotoxic shock when administered in a bolus dose that does not reduce plasma total nitrite + nitrate levels. Because we observed no overt signs of toxicity and no influence on organ-specific tetrahydrobiopterin levels, we conclude that the novel compound 4-ABH4 is a promising drug candidate for protection against endotoxin-related mortality.

Animals↗
PubMed2000

Bronchoscopic photodynamic diagnosis and therapy for lung cancer.

Photodynamic diagnosis and therapy have exciting potential in lung cancer management. Fluorescence bronchoscopy enhances the detection of preinvasive lesions and early invasive carcinomas involving the central airways, whereas photodynamic therapy (PDT) provides an effective method for treating these lesions with preservation of lung tissue. However, the natural history of preinvasive lesions is poorly understood and so the value of treatment remains unclear. Although treatment for early invasive carcinomas'is not open to question, the possibility of undetected nodal disease means that PDT is unlikely to replace surgical resection in patients who are fit for thoracotomy. PDT also provides an effective method of palliating patients with advanced obstructing endobronchial tumors. Although it is considered superior to simple tumor debulking, its usefulness is limited by transient but potentially severe skin photosensitivity. There is therefore a need to develop new photosensitizing drugs with less severe side effects.

Bronchoscopy↗
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

Bench to bedside: tumor necrosis factor-alpha: from inflammation to resuscitation.

Proinflammatory mediators such as tumor necrosis factor-alpha (TNF) have been implicated in the pathophysiology in a number of acute disease states. Tumor necrosis factor-alpha can contribute to cell death, apoptosis, and organ dysfunction. Tumor necrosis factor-alpha can be generated with sepsis or ischemia-reperfusion by activation of cell mitogen-activated protein kinases and nuclear factor kappa B, leading to TNF production. A number of strategies to modulate TNF have been recently explored, including factors directed toward mitogen-activated protein kinases, TNF transcription, anti-inflammatory ligands, heat shock proteins, and TNF-binding proteins. However, TNF may also play an important role in the adaptive response to injury and inflammation. Control of the deleterious effects of TNF and other proinflammatory cytokines represents a realistic goal for clinical emergency medicine. The purpose of this article is to provide a background of relevance to emergency medicine academicians on the production and regulation of TNF, the acute effects of TNF on pathophysiology, and the rationale for therapeutic interventions directed toward TNF and the clinical experience with these strategies.

Apoptosis↗