Biomedical subjects

R Banerjee

Publications and source records attributed to R Banerjee.

At least 37 records · Page 2Linked to original sources

PubMed2001

Nucleolin stimulates viral internal ribosome entry site-mediated translation.

Previous results from our laboratory have identified a small (60 nt) RNA from the yeast S. cerevisiae that specifically inhibits internal ribosome entry site (IRES)-mediated translation programmed by poliovirus (PV) and hepatitis C virus (HCV) 5'-untranslated region (5'UTR). The yeast inhibitor RNA (called IRNA) was found to efficiently compete with viral 5'UTR for binding of several cellular polypeptides that presumably play important roles in IRES-mediated translation. One such IRNA (and 5'UTR)-binding protein has previously been identified as the La autoantigen. In this report, we have identified a 110-kDa IRNA-binding protein (which also interacts with viral 5'UTR) as nucleolin, a nucleolar RNA binding protein that was previously shown to translocate into the cytoplasm following infection of cells with poliovirus. We demonstrate that nucleolin (called C23) stimulates viral IRES-mediated translation both in vitro and in vivo. We also show that nucleolin mutants containing the carboxy-terminal RNA binding domains but lacking the amino terminal domain inhibit IRES-mediated translation in vitro. The translation inhibitory activity of these mutants correlates with their ability to bind the 5'UTR sequence. These results suggest a role of nucleolin/C23 in viral IRES-mediated translation.

5' Untranslated Regions↗

PubMed2001

Aggressive phenotypic and genotypic features in pediatric and NF2-associated meningiomas: a clinicopathologic study of 53 cases.

Pediatric and NF2-associated meningiomas are uncommon and poorly characterized in comparison to sporadic adult cases. In order to elucidate their molecular features, we analyzed MIB-1, progesterone receptor (PR), NF2, merlin, DAL-1, DAL-1 protein, and chromosomal arms 1p and 14q in 53 meningiomas from 40 pediatric/NF2 patients using immunohistochemistry and dual-color fluorescence in situ hybridization (FISH). Fourteen pediatric (42%) patients, including 5 previously undiagnosed patients, had NF2. The remaining 19 (58%) did not qualify. All 7 of the adult patients had NF2. Meningioma grading revealed 21 benign (40%), 26 atypical (49%), and 6 anaplastic (11%) examples. Other aggressive findings included high mitotic index (32%), high MIB-1 LI (37%), aggressive variant histology (e.g. papillary, clear cell) (25%), brain invasion (17%), recurrence (39%), and patient death (17%). FISH analysis demonstrated deletions of NF2 in 82%, DAL-1 in 82%, 1p in 60%, and 14q in 66%. NF2-associated meningiomas did not differ from sporadic pediatric tumors except for a higher frequency of merlin loss in the former (p = 0.020) and a higher frequency of brain invasion in the latter (p = 0.007). Thus, although pediatric and NF2-associated meningiomas share the common molecular alterations of their adult, sporadic counterparts, a higher fraction are genotypically and phenotypically aggressive. Given the high frequency of undiagnosed NF2 in the pediatric cases, a careful search for other features of this disease is warranted in any child presenting with a meningioma.

Adolescent↗

PubMed2001

Chromosome 1p and 14q FISH analysis in clinicopathologic subsets of meningioma: diagnostic and prognostic implications.

The second most frequently reported genetic abnormalities in meningiomas after 22q loss are deletions of 1p and 14q. To assess the potential diagnostic and prognostic utility of these chromosomal alterations, we studied 180 well-characterized meningiomas using dual-color fluorescence in situ hybridization (FISH) with DNA probes localized to 1p32, 1p36, 14q13, and 14q32. Our cohort consisted of 77 benign (grade I), 74 atypical (grade II), and 29 anaplastic (grade III) meningiomas. Benign and atypical meningiomas were further stratified into subsets of recurring (despite gross total resection) vs non-recurring (at least 10 yr of follow-up) and mitotically active vs brain invasive subsets, respectively. Losses of 1p and 14q losses were identified in 23% and 31% of benign, 56% and 57% of atypical, and 75% and 67% of anaplastic meningiomas, respectively (p < 0.001 for 1p; p = 0.004 for 14q). Combined 1p/14q deletions were encountered in 7% benign. 39% atypical, and 63% anaplastic meningiomas (p < 0.001). Benign non-recurring meningiomas were less likely to harbor 14q deletions than recurring examples (17% vs 50%, p = 0.013). There was a trend for anaplastic meningiomas with 14q deletions and atypical meningiomas with combined 1p/14q deletions to have poorer overall survivals, though neither reached statistical significance. We conclude that 1p and 14q deletions are highly associated with increasing histologic grade and play an important role in meningioma tumor progression. Furthermore, 14q FISH analysis may aid in assessing recurrence risk in histologically benign meningiomas.

Adolescent↗

PubMed2001

Ultrastructure of exogenous surfactants using cryogenic scanning electron microscopy.

Therapy with specialised biomaterials, exogenous surfactants, is known to significantly decrease the mortality rates in Respiratory Distress Syndrome (RDS). Surfactants available commercially vary widely in composition and biophysical properties. The present paper studies the ultrastructure of three exogenous surfactants used for the treatment of Respiratory Distress Syndrome, namely, Survanta, ALEC and Exosurf Neonatal with respect to their ability to form liposomes using cryogenic scanning electron microscopy. Liposomal organisation is more obvious in Exosurf than in Survanta and is most pronounced in ALEC. ALEC forms closed regular liposomes with an onion-ring-like internal bilayer arrangement. Survanta forms open membranous structures with wavy ribbon-like membranes. The complex membrane-like structures seen with Survanta may be due to the interaction of lipids with surfactant-specific proteins present in this surfactant which is derived from natural lung extracts and might indicate superior spreading at the lipid-water interface. Artificial protein-free surfactants (ALEC and Exosurf) did not appear to form these open membranous structures. Further study of the ultrastructure of possible biomaterials as surfactants could help in the development of new, improved artificial protein-free surfactants with open membranous structures that might facilitate spreading at the air-liquid interface of lungs.

Biocompatible Materials↗

PubMed2001

Liposomes: applications in medicine.

Liposomes are spherical lipid bilayers from 50 nm to 1000 nm in diameter that serve as convenient delivery vehicles for biologically active compounds. The field of liposome research has expanded considerably over the last 30 years. It is now possible to engineer a wide range of liposomes varying in size, phospholipid composition and surface characteristics to suit the specific application for which they are intended. This paper gives an overview of the main advances in liposome research from a point of view of their applications in medicine. Aqueous contrast enhancing agents entrapped in liposomal carriers can be targeted to the liver and spleen and distinctions can be made between normal and tumorous tissue using computed tomography. Topical application of liposomes has great potential in dermatology. Liposomes have been used to deliver anticancer agents in order to reduce the toxic effects of the drugs when given alone or to increase the circulation time and effectiveness of the drugs. From the original concept of encapsulating hemoglobin in an inert shell, liposome-encapsulated hemoglobin (LEH) has evolved into a fluid proven to carry oxygen, capable of surviving for reasonable periods in the circulation and amenable to large-scale production. Liposomes may be used to target specific cells by attaching amino acid fragments such as antibodies or proteins or appropriate fragments that target specific receptor sites. Liposomal DNA delivery vectors and further enhancements in the forms of LPDI and LPDII are some of the safest and potentially most versatile transfer vectors used to date. DNA vaccination and improved efficiency of gene therapy are just a few of the upcoming applications of liposomes.

Drug Carriers↗

PubMed2001

Specific interaction of hepatitis C virus protease/helicase NS3 with the 3'-terminal sequences of viral positive- and negative-strand RNA.

The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and Delta pNS3 (helicase domain only) and examined their abilities to interact with the 3'-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3' untranslated region (UTR). The results presented here demonstrate that NS3 (and Delta pNS3) interacts efficiently and specifically with the 3'-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5'-terminal RNA sequences. The interaction of NS3 with the 3'-terminal negative strand [called 3'(-) UTR(127)] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3' terminus (nucleotides 5 to 20 from the 3' end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and Delta pNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3'-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3'(-) UTR binding, specific interaction of NS3 (or Delta pNS3) with the 3'-terminal sequences of the positive-strand RNA [3'(+) UTR] appears to require the entire 3'(+) UTR of HCV. Deletion of either the 98-nucleotide 3'-terminal conserved region or the 5' half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3'-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.

3' Untranslated Regions↗

PubMed2001

Targeted disruption of the methionine synthase gene in mice.

Alterations in homocysteine, methionine, folate, and/or B12 homeostasis have been associated with neural tube defects, cardiovascular disease, and cancer. Methionine synthase, one of only two mammalian enzymes known to require vitamin B12 as a cofactor, lies at the intersection of these metabolic pathways. This enzyme catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. Human patients with methionine synthase deficiency exhibit homocysteinemia, homocysteinuria, and hypomethioninemia. They suffer from megaloblastic anemia with or without some degree of neural dysfunction and mental retardation. To better study the pathophysiology of methionine synthase deficiency, we utilized gene-targeting technology to inactivate the methionine synthase gene in mice. On average, heterozygous knockout mice from an outbred background have slightly elevated plasma homocysteine and methionine compared to wild-type mice but seem to be otherwise indistinguishable. Homozygous knockout embryos survive through implantation but die soon thereafter. Nutritional supplementation during pregnancy was unable to rescue embryos that were completely deficient in methionine synthase. Whether any human patients with methionine synthase deficiency have a complete absence of enzyme activity is unclear. These results demonstrate the importance of this enzyme for early development in mice and suggest either that methionine synthase-deficient patients have residual methionine synthase activity or that humans have a compensatory mechanism that is absent in mice.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2001

Scoring of surface parameters of physiological relevance to surfactant therapy in respiratory distress syndrome.

The Wilhelmy balance was used for in vitro testing of surface parameters of surfactants used for respiratory distress syndrome therapy. Two commercial protein-free surfactants, ALEC and Exosurf, were compared with pure forms of the three main phospholipids in natural surfactants, dipalmitoyl phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE), and their binary mixtures, PC with PE and PG each in the ratio 2:3. Surface excess films (15 A2/molecule) were compressed at 1.2 cycles/min past collapse to a compression ratio of 4:1. The maximum surface pressure, spreading time, compressibility, respreading ratio, recruitment index, and hysteresis area were compared. A consolidated list of criteria for selection of suitable surfactants was compiled from the literature. A relative scoring system was devised for comparison based on these criteria. PC/PG (2:3) performed the best as it fulfilled all the criteria and obtained the highest relative score. Exosurf also performed well, except on the respreading criterion. ALEC and PC/PE were equivalent in their performance and performed well, except on two criteria: hysteresis area and recruitment index. Thus the scoring system proposed here proved valuable to rate the overall efficacy as well as relative merits of surfactant formulations.

Drug Combinations↗

PubMed2001

Effect of calcium on the surface properties of phospholipid monolayers with respect to surfactant formulations in respiratory distress syndrome.

The effects of calcium, in the form of calcium chloride, at concentrations of 5 and 20 mM, were studied on the surface properties of physiologic relevance to specialised biomaterials which replace lung surfactant in Respiratory Distress Syndrome. The dynamic surface pressure, re-spreading ratio, compressibility, hysteresis area and recruitment index of pure films of the main phospholipids of pulmonary surfactant namely dipalmitoyl phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and of binary mixed films of these phospholipids in the ratio of 2:3 were studied both in the presence and absence of calcium by in vitro analysis using a Wilhelmy balance. Surface excess films, of each of the surfactant systems, with initial concentration 15 A2 molecule were compressed at the rate of 50 seconds/cycle past collapse till a compression ratio of 4:1. The presence of 5 mM calcium caused a significant decrease in compressibility (p < 0.05 Mann-Whitney U test) of all the surfactant monolayer films. No further benefit was obtained by adding 20 mM calcium over that of 5 mM calcium. A significant beneficial effect of calcium (p < 0.05 Mann-Whitney U test) on film stability was observed when analysing the materials in a pulsating bubble surfactometer, in which liposomal suspensions of 1% concentration in the presence of 5 mM calcium were pulsated at a high frequency of 40 cycles per minute, corresponding to the respiratory frequency of neonates. The ultrastructure of the liposomal suspensions were also studied using cryogenic scanning electron microscopy and longitudinal micro-tubular structures were found on addition of 5 mM calcium, which could have resulted in the improved performance of the exogenous surfactants with respect to compressibility and stability.

Biocompatible Materials↗

PubMed2001

Optimization of some additives to improve protease production under SSF.

In a locally isolated Rhizopus oryzae strain highest-production of protease (388.54/g wheat bran) was observed in presence of Tween-80 and dioctyl sodium sulfosuccinate individually at 40mg/g wheat bran concentration. Under solid state fermentation biotin (0.0025mg/g wheat bran); Ca2+ (0.05mg/g wheat bran) and 1-Naphthyl acetic acid (0.01mg/g wheat bran) also showed some inducing effect on the synthesis of the enzyme protease by solid state fermentation.

Biotin↗

PubMed2000

Neutrophil dysplasia caused by mycophenolate mofetil.

Two transplant recipients developed striking morphological abnormalities in their circulating neutrophils while receiving mycophenolate. The changes included nuclear hypolobulation and abnormal clumping of nuclear chromatin and were presumably related to inhibition of guanosine nucleoside synthesis. In both cases the neutrophil abnormalities preceded the development of neutropenia and were thus a sign of impending hematological toxicity. The hematological changes resolved after the drug was discontinued.

Adult↗

PubMed2000

Dynamics of compact denatured states of glutaminyl-tRNA synthetase probed by bis-ANS binding kinetics.

Bis-ANS binds to native glutaminyl-tRNA synthetase (GlnRS) with a fast and a slow phase. The rate constant of the slow phase is independent of bis-ANS concentration suggesting a slow conformational change in the pathway of bis-ANS binding. Aging of GlnRS causes a large decrease of the slow phase amplitude with concomitant increase of the fast phase amplitude. Several other large, multi-domain proteins show similar patterns upon aging. The near UV-CD spectra of the native and the aged GlnRS remain similar. Significant changes in far UV-CD, acrylamide quenching and sulfhydryl reactivity, are seen upon aging, suggesting disruptions in native interactions. Refolding of GlnRS from the urea-denatured state rapidly produces a state that is very similar to the equilibrium molten globule state. Bis-ANS binds to the molten globule state with kinetics similar to that of the aged state and unlike that of the native state. This suggests that the slow binding phase of bis-ANS, seen in native proteins, originate from relatively high energy barriers between the native and the more open states. Thus bis-ANS can be used as a powerful probe for large amplitude, low-frequency motions of proteins.

Amino Acyl-tRNA Synthetases↗

PubMed2000

The quantitatively important relationship between homocysteine metabolism and glutathione synthesis by the transsulfuration pathway and its regulation by redox changes.

Homocysteine is a key junction metabolite in methionine metabolism. It suffers two major metabolic fates: transmethylation catalyzed by methionine synthase or betaine homocysteine methyl transferase and transsulfuration catalyzed by cystathionine beta-synthase leading to cystathionine. The latter is subsequently converted to cysteine, a precursor of glutathione. Studies with purified mammalian methionine synthase and cystathionine beta-synthase have revealed the oxidative sensitivity of both junction enzymes, suggesting the hypothesis that redox regulation of this pathway may be physiologically significant. This hypothesis has been tested in a human hepatoma cell line in culture in which the flux of homocysteine through transsulfuration under normoxic and oxidative conditions has been examined. Addition of 100 microM H(2)O(2) or tertiary butyl hydroperoxide increased cystathionine production 1.6- and 2.1-fold from 82 +/- 7 micromol h(-)(1) (L of cells)(-)(1) to 136 +/- 15 and 172 +/- 23 micromol h(-)(1) (L of cells)(-)(1), respectively. The increase in homocysteine flux through the transsulfuration pathway exhibited a linear dose dependence on the concentrations of both oxidants (50-200 microM H(2)O(2) and 10-200 microM tertiary butyl hydroperoxide). Furthermore, our results reveal that approximately half of the intracellular glutathione pool in human liver cells is derived from homocysteine via the transsulfuration pathway. The redox sensitivity of the transsulfuration pathway can be rationalized as an autocorrective response that leads to an increased level of glutathione synthesis in cells challenged by oxidative stress. In summary, this study demonstrates the importance of the homocysteine-dependent transsulfuration pathway in the maintenance of the intracellular glutathione pool, and the regulation of this pathway under oxidative stress conditions. Aberrations in this pathway could compromise the redox buffering capacity of cells, which may in turn be related to the pathophysiology of the different homocysteine-related diseases.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2000

Comparison of global transcription responses allows identification of Vibrio cholerae genes differentially expressed following infection.

Comparison of global transcription profiles of Vibrio cholerae grown in vitro and in vivo revealed that 20% of the genome was repressed and about 5% was induced under in vivo conditions. Hybridization with the cloned genes revealed that the virulence genes ctx, toxR, toxT and tcpA were induced under in vivo conditions. Dissection of two in vivo induced cosmids identified another set of three genes homologous to che Y1 involved in motility and chemotaxis, pnuC encoding the major component of the nicotinamide mononucleotide transport system and icmF belonging to a cassette involved in multiplication inside host cells. These results demonstrate that the global transcription profile approach might be a powerful method for identification of differentially expressed transcripts under in vivo conditions.

Animals↗

PubMed2000

Characterization of the heme in human cystathionine beta-synthase by X-ray absorption and electron paramagnetic resonance spectroscopies.

Human cystathionine beta-synthase is one of two key enzymes involved in intracellular metabolism of homocysteine. It catalyzes a beta-replacement reaction in which the thiolate of homocysteine replaces the hydroxyl group of serine to give the product, cystathionine. The enzyme is unusual in its dependence on two cofactors: pyridoxal phosphate and heme. The requirement for pyridoxal phosphate is expected on the basis of the nature of the condensation reaction that is catalyzed; however the function of the heme in this protein is unknown. We have examined the spectroscopic properties of the heme in order to assign the axial ligands provided by the protein. The heme Soret peak of ferric cystathionine beta-synthase is at 428 nm and shifts to approximately 395 nm upon addition of the thiol chelator, mercuric chloride. This is indicative of 6-coordinate low-spin heme converting to a 5-coordinate high-spin heme. The enzyme as isolated exhibits a rhombic EPR signal with g values of 2.5, 2.3, and 1.86, which are similar to those of heme proteins and model complexes with imidazole/thiolate ligands. Mercuric chloride treatment of the enzyme results in conversion of the rhombic EPR signal to a g = 6 signal, consistent with formation of the high-spin ferric heme. The X-ray absorption data reveal that iron in ferric cystathionine beta-synthase is 6-coordinate, with 1 high-Z scatterer and 5 low-Z scatterers. This is consistent with the presence of 5 nitrogens and 1 sulfur ligand. Together, these data support assignment of the axial ligands as cysteinate and imidazole in ferric cystathionine beta-synthase.

Cystathionine beta-Synthase↗

PubMed2000

Thermodynamic and kinetic characterization of Co-C bond homolysis catalyzed by coenzyme B(12)-dependent methylmalonyl-CoA mutase.

Methylmalonyl-CoA mutase is a member of the family of coenzyme B(12)-dependent isomerases and catalyzes the 1,2-rearrangement of methylmalonyl-CoA to succinyl-CoA. A common first step in the reactions catalyzed by coenzyme B(12)-dependent enzymes is cleavage of the cobalt-carbon bond of the cofactor, leading to radical-based rearrangement reactions. Comparison of the homolysis rate for the free and enzyme-bound cofactors reveals an enormous rate enhancement which is on the order of a trillion-fold. To address how this large rate acceleration is achieved, we have examined the kinetic and thermodynamic parameters associated with the homolysis reaction catalyzed by methylmalonyl-CoA mutase. Both the rate and the amount of cob(II)alamin formation have been analyzed as a function of temperature with the protiated substrate. These studies yield the following activation parameters for the homolytic reaction at 37 degrees C: DeltaH(f)() = 18.8 +/- 0.8 kcal/mol, DeltaS(f)() = 18.2 +/- 0.8 cal/(mol.K), and DeltaG(f)() = 13.1 +/- 0.6 kcal/mol. Our results reveal that the enzyme lowers the transition state barrier by 17 kcal/mol, corresponding to a rate acceleration of 0.9 x 10(12)-fold. Both entropic and enthalpic factors contribute to the observed rate acceleration, with the latter predominating. The substrate binding step is exothermic, with a DeltaG of -5.2 kcal/mol at 37 degrees C, and is favored by both entropic and enthalpic factors. We have employed the available kinetic and spectroscopic data to construct a qualitative free energy profile for the methylmalonyl-CoA mutase-catalyzed reaction.

Catalysis↗

PubMed2000

A substrate switch: a new mode of regulation in the methionine metabolic pathway.

We propose a simple mathematical model of liver S -adenosylmethionine (AdoMet) metabolism. Analysis of the model has shown that AdoMet metabolism can operate under two different modes. The first, with low metabolic rate and low AdoMet concentration, serves predominantly to supply the cell with AdoMet, the substrate for various cellular methylation reactions. The second, with high metabolic rate and high AdoMet concentration, provides an avenue for cleavage of excess methionine and can serve as a source of cysteine when its increased synthesis is necessary. The switch that triggers interconversion between the "low" and "high" modes is methionine concentration. Under a certain set of parameters both modes may coexist. This behavior results from the kinetic properties of (i) the two isoenzymes of AdoMet synthetase, MATI and MATIII, that catalyse AdoMet production; one is inhibited by AdoMet, whereas the other is activated by it, and (ii) glycine- N -methyltransferase that displays highly cooperative kinetics that is different from that of other AdoMet-dependent methyltransferases. Thus, the model provides an explanation for how different cellular needs are met by regulation of this pathway. The model also correctly identifies a critical role for glycine N -methyltransferase in depleting excess methionine in the high mode, thus avoiding the toxicity associated with elevated levels of this essential amino acid.

Animals↗

PubMed2000

Cutting edge: infection by the agent of human granulocytic ehrlichiosis prevents the respiratory burst by down-regulating gp91phox.

The agent of human granulocytic ehrlichiosis (HGE) is an emerging tick-borne pathogen that resides in neutrophils and can be cultured in a promyelocytic (HL-60) cell line. In response to microbes, polymorphonuclear leukocytes normally activate the NADPH oxidase enzyme complex and generate superoxide anion (O2-). However, HL-60 cells infected with HGE bacteria did not produce O2- upon activation with PMA. RT-PCR demonstrated that HGE organisms inhibited mRNA expression of a single component of NADPH oxidase, gp91phox, and FACS analysis showed that plasma membrane-associated gp91phox protein was reduced on the infected cells. Infection with HGE organisms also decreased gp91phox mRNA levels in splenic neutrophils in a murine model of HGE, demonstrating this phenomenon in vivo. Therefore, HGE bacteria repress the respiratory burst by down-regulating gp91phox, the first direct inhibition of NADPH oxidase by a pathogen.

Animals↗