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Biomedical subjects

G Mitra

Publications and source records attributed to G Mitra.

At least 19 recordsLinked to original sources
PubMed2000

Enhanced 3D PET OSEM reconstruction using inter-update Metz filtering.

We present an enhancement of the OSEM (ordered set expectation maximization) algorithm for 3D PET reconstruction, which we call the inter-update Metz filtered OSEM (IMF-OSEM). The IMF-OSEM algorithm incorporates filtering action into the image updating process in order to improve the quality of the reconstruction. With this technique, the multiplicative correction image--ordinarily used to update image estimates in plain OSEM--is applied to a Metz-filtered version of the image estimate at certain intervals. In addition, we present a software implementation that employs several high-speed features to accelerate reconstruction. These features include, firstly, forward and back projection functions which make full use of symmetry as well as a fast incremental computation technique. Secondly, the software has the capability of running in parallel mode on several processors. The parallelization approach employed yields a significant speed-up, which is nearly independent of the amount of data. Together, these features lead to reasonable reconstruction times even when using large image arrays and non-axially compressed projection data. The performance of IMF-OSEM was tested on phantom data acquired on the GE Advance scanner. Our results demonstrate that an appropriate choice of Metz filter parameters can improve the contrast-noise balance of certain regions of interest relative to both plain and post-filtered OSEM, and to the GE commercial reprojection algorithm software.

Algorithms↗
PubMed1997

A colorimetric estimation of polyethyleneglycol-conjugated phospholipid in stealth liposomes.

The paper describes a colorimetric method for estimation of polyethyleneglycol (PEG)-conjugated phospholipid in either form, free or bound to liposomes. It provides a rapid, highly reproducible, and sensitive tool to detect PEG-coupled phospholipid in amounts as low as 1 microg giving a linear response over a range of 1-100 microg. The method makes use of the biphasic system comprising aqueous ammonium ferrithiocyanate and chloroform, developed by Stewart for estimation of phospholipids. The same system was also applied for quantitation of PEG in PEG-protein conjugates in a recent report. The samples were digested with phospholipase-C, prior to analysis, in order to eliminate the contributions from liposomal phospholipids other than the PEG-conjugated phospholipid. The technique can give valuable information regarding the retention of PEG coating on the surface of the vesicles when employed in combination with markers for the aqueous compartment. In addition, it does not suffer from interference by proteins. This makes it particularly suitable for monitoring the pharmacokinetics of stealth liposomes, using PEG-phospholipid as a lipid probe. Since it does not involve handling hazardous radioisotopes, the suggested technique could even be utilized in clinical trials.

Animals↗
PubMed1997

Stimulated human leukocytes cause activating mutations in the K-ras protooncogene.

Human tissues which are chronically infiltrated with inflammatory leukocytes are more likely to develop malignancies than non-inflamed tissues, however the mechanism(s) by which leukocytes contribute to carcinogenesis is unknown. Stimulated human leukocytes release superoxide anion and hydrogen peroxide which, in the presence of iron, can be converted into the potent oxidant, hydroxyl radical (.OH). Previous studies have shown that leukocyte-derived .OH (or a .OH-like species) can cause DNA damage, however a relationship between leukocyte-induced DNA damage and carcinogenesis has not been established. The present report demonstrates that leukocyte-derived .OH-induced DNA damage can cause K-ras oncogene activation, and suggests that there may be a characteristic pattern of .OH-induced K-ras oncogene activation. Since activation of the K-ras oncogene is believed to play a crucial role in the pathogenesis of many human malignancies, .OH-induced K-ras oncogene activation could be an important mechanism by which human leukocytes contribute to carcinogenesis.

3T3 Cells↗
PubMed1997

Enhanced in vivo catalytic activity of PEG-modified cellulase complex from Trichoderma reesei.

The cellulase complex from Trichoderma reesei was polyethylene glycol (PEG)-modified with considerable retention of endo-beta-1,4-glucanase activity, as evaluated by the carboxymethylcellulase (CMCase) assay. While resistance towards heat denaturation was the same for either form, susceptibility towards proteolysis was slightly greater for the PEG-conjugate, in contrast to most reports using other enzymes. The circulatory life of endoglucanase activity associated with the complex was enhanced upon PEG-modification. This was confirmed by demonstrating degradation of chromogen-labelled substrate injected (i.v.) after 24hr of administration of the PEG-ylated enzymes; in contrast the substrate remained undegraded in mice pretreated with the native complex. The PEG-modified complex could be a good candidate for the treatment of pneumoconioses of textile workers exposed to cotton dust.

Animals↗
PubMed1996

A colorimetric assay for estimation of polyethylene glycol and polyethylene glycolated protein using ammonium ferrothiocyanate.

A colorimetric method for quantitative assay of polyethylene glycol (PEG) described here is based on partitioning of a chromophore present in ammonium ferrothiocyanate reagent from an aqueous to a chloroform phase in the presence of PEG. The method is simple, reproducible, and can detect PEG in amounts as low as 5 microg. It gives a linear response over a range of 5-100 microg. The absence of any interference by proteins makes the assay equally suitable for the estimation of PEG in PEG-protein conjugates. The method was employed to monitor the separation profile of a mixture of free and PEG-5000 coupled to bovine serum albumin during purification through a gel filtration column. In this report we have also demonstrated for the first time an assay method which permits a critical evaluation of pharmacokinetic properties of any PEG-protein conjugate under in vivo conditions.

Animals↗
PubMed1994

Removal of DNA contaminants from therapeutic protein preparations.

Mammalian cell culture-derived biotechnical products for therapeutic use have risks associated with cell substrate DNA. Regulatory authorities in the USA and Europe have set stringent limits for this contaminant, requiring orders of magnitude reduction from fermenter harvests to purified products. This paper addresses the relevant unit processes (e.g., cell separation, affinity chromatography and ion-exchange chromatography) utilized for purification of mammalian cell culture-derived products as they pertain to removal of DNA contaminants.

Cell Separation↗
PubMed1994

Inactivation of hepatitis C virus in low pH intravenous immunoglobulin.

The safety from hepatitis C virus of intravenous immunoglobulin prepared by the cold ethanol method of Cohn-Oncley is demonstrated by clearance through the manufacturing process of 9 x 10(6) plaque-forming units of bovine viral diarrhea virus used as a surrogate for hepatitis C virus. Incubation of the intravenous immunoglobulin in its final formulation at pH 4.25 for 21 days at 21 degrees C caused a 10,000-fold decrease in bovine viral diarrhea virus intentionally added and complete inactivation of 1000 chimpanzee infectious doses per ml of hepatitis C virus.

Animals↗
PubMed1993

Epitope mapping and functional analysis of three murine IgG1 monoclonal antibodies to human tumor necrosis factor-alpha.

Immunologic and enzyme digest epitope mapping techniques were used to compare and contrast the interactions of three mouse mAb, designated A10G10, A6, and B6, with human TNF-alpha. These antibodies have previously been shown to protect mouse and human cells from TNF toxicity. ELISA showed that the antibodies recognize predominantly conformational epitopes, and that native TNF binds at least two molecules of the same mAb. Enzyme digestion of native TNF bound to each of the mAb in turn showed that each of the antibodies binds to or sterically masks a large fraction of the exposed surface of TNF. These epitope mapping data were compared with four putative TNF cell receptor binding sites presented in the literature. The results are consistent with the hypothesis that these antibodies prevent TNF-induced cell death by binding to or sterically masking the TNF receptor binding site.

Amino Acid Sequence↗
PubMed1993

Multiple pathways for activation of MAP kinases.

p42/Mitogen activated protein kinase (MAPK) and MAP Kinase Kinase (MAPKK) activities are constitutively elevated in v-raf transformed NIH3T3 cells, which correlates with increased tyrosine phosphorylation of p42mapk protein. These activities can be further enhanced to a moderate extent by treatment of raf-transformed cells with either serum, tetradecanoyl phorbol acetate (TPA), or aluminium fluoride. A similar activation of MAPK is observed in a cell line (M17raf) coexpressing a dominant inhibitory ras mutant (N-17 ras) along with v-raf. However, in this cell line, both the serum and TPA stimulated response of MAPK activity is reduced compared to similarly treated raf-transformed cells, while aluminium fluoride is equally potent in all the cell lines tested. These studies indicate that in addition to c-Raf-1, serine/threonine kinase, which is an upstream activator of MAPK, other c-ras dependent as well as c-ras independent pathways also can contribute to MAPK activation.

3T3 Cells↗
PubMed1992

Anti-tumor necrosis factor receptor and tumor necrosis factor agonist activity by an anti-idiotypic antibody.

Tumor necrosis factor (TNF) is a cytokine which, among other properties, is a principle mediator of inflammation and septic shock. It acts upon target cells by binding to specific cell surface receptors. A10G10 is a murine monoclonal antibody which recognizes human TNF and neutralizes its activity. A rabbit polyclonal antibody directed at the antigen-binding site of A10G10 was raised and affinity purified over an A10G10 column. The resultant anti-idiotypic antibody recognized not only A10G10 but also both TNF receptors. It showed TNF agonist activity in two different TNF bioassays, and competed with several anti-TNF receptor monoclonal antibodies and TNF itself for binding to cells. These results represent an example of a method for obtaining antibodies to a ligand-specific receptor in the absence of the receptor itself.

Animals↗
PubMed1991

Monoclonal anti-tumor necrosis factor (TNF) antibodies protect mouse and human cells from TNF cytotoxicity.

Tumor necrosis factor (TNF) is a cytokine produced by macrophages which mediates septic shock. TNF is also associated with anti-tumor activity and is cytotoxic to many cell lines in vitro. While the clinical efficacy of anti-TNF antibodies has yet to be established, such antibodies may prevent TNF action. We have evaluated three murine monoclonal anti-TNF antibodies for their capacity to protect mouse and human cells from the cytotoxic effects of human TNF. Mouse WEHI 164 cells were tested because of their known high sensitivity to TNF. Several human cells lines were also examined: U937, HeLa, MRC5, and ME180. Two antibodies protected mouse and human cells from the cytotoxic action of TNF. The neutralizing activity was also present in the Fab fragment and insensitive to the presence of carbohydrate on the antibody. The third antibody had substantially less TNF-neutralizing activity. The relative ability of these monoclonal antibodies to protect both mouse and human cell lines from TNF-induced cell death may be an effective indicator of their in vivo efficacy.

Animals↗
PubMed1991

Mutational analysis of conserved residues in the tyrosine kinase domain of the human trk oncogene.

The human trk oncogene (originally identified in a colon carcinoma) was activated by a genetic rearrangement which resulted in replacement of the extracellular ligand-binding domain of the proto-trk transmembrane receptor by non-muscle tropomyosin sequences. The product of the trk oncogene, a protein of 70 kDa (p70trk), possesses tyrosine-specific protein kinase activity, is autophosphorylated in vitro on tyrosine and is phosphorylated on serine, threonine and tyrosine residues in trk-transformed cells. By site-directed mutagenesis of trk oncogene cDNA, the codon for lysine (367) at the putative ATP-binding site was changed to that for methionine and the codons for tyrosines (503 and 504) at the putative autophosphorylation sites were changed to those for phenylalanine. Replacement of Lys-367 by methionine results in a biologically inactive, kinase-negative mutant. Phe-ala mutants of trk showed drastically reduced ability to induce morphologic transformation, anchorage-independent growth and tumorigenicity in mouse NIH3T3 cells and showed reduced in vitro tyrosine kinase activity when assayed by autophosphorylation and phosphorylation of histone as exogenous substrate. The present study indicates the role of these specific conserved residues in regulating the biochemical and biological properties of p70trk oncoprotein.

3T3 Cells↗
PubMed1990

Characterisation and virus safety of alpha 1-proteinase inhibitor.

Alpha 1-proteinase inhibitor (alpha 1-PI) concentrate is prepared from Cohn fraction IV-1 by precipitation and ion-exchange chromatography. A pasteurisation step (60 degrees, 10 hours) is introduced in this process with the aim at lowering the risk of transmitting infectious agents. Characterisation studies of alpha 1-PI with electrophoretic, immunologic and chemical methods demonstrated that the final product behaved the same as alpha 1-PI in human plasma. In particular, neo-antigens were not detected and the association rate constant of the final product was shown to be very similar to the constant published in the literature. The inactivation kinetic studies of eight different viruses showed that the heat treatment had a profound effect on their inactivation, the retroviruses such as HIV and Visna becoming undetectable after less than one hour of heat treatment.

Drug Contamination↗
PubMed1989

Detection and reduction of protein A contamination in immobilized protein A purified monoclonal antibody preparations.

Protein A chromatography is an excellent technique for the purification of monoclonal antibodies. However, the presence of Protein A in therapeutic monoclonal antibody preparations due to leaching has been linked with toxicity in animals and humans. Two sandwich ELISAs were developed to monitor Protein A column leaching: (1) rabbit anti-Protein A for capture and anti-Protein A F(ab')2-HRP for detection; and (2) rabbit anti-Protein A for capture and anti-Protein A-biotin for detection. The biotin ELISA is sensitive to the subnanogram range. In addition, these assays were used to develop DEAE and gel filtration chromatography techniques that substantially reduce Protein A levels in monoclonal antibodies purified by Protein A chromatography.

Animals↗
PubMed1989

Molecular analysis of O6-substituted guanine-induced mutagenesis of ras oncogenes.

We have designed an Ha-ras/thymidine kinase (TK) cassette that permits the incorporation of chemically synthesized adducts within specific domains of the rat Ha-ras protooncogene. This cassette has been used to evaluate the mutagenicity of O6-substituted guanine residues, including O6-methylguanine and O6-benzylguanine, incorporated within the 12th codon of this locus. Mutations were monitored by the ability of these modified Ha-ras DNAs to transform Rat4 TK-cells. Our results indicate that both types of O6-substituted guanines are substantially mutagenic, although the methyl substituent induced a 2-fold higher percentage of transformed Rat4 TK+ colonies than its bulkier benzyl analogue. Interestingly, the mutagenicity of both O6-substituted guanines was found to be independent of their relative position within codon 12, therefore suggesting that the specific activation of Ha-ras oncogenes by GGA----GAA mutations in tumors induced by methylating carcinogens might be due to differences in the accessibility of these guanine residues to the carcinogen rather than to a differential rate of repair. Molecular analysis of the mutations induced by these O6-substituted guanines indicated that O6-methylguanine exclusively induced G----A transitions. In contrast, O6-benzylguanine produced G----C and G----T transversions in addition to G----A transitions. These results suggest that O6-methylguanine and its bulkier analogue O6-benzylguanine may induce mutagenesis by different mechanisms.

Alkylation↗
PubMed1989

Molecular and biochemical characterization of the human trk proto-oncogene.

Molecular analysis of the human trk oncogene, a transforming gene isolated from a colon carcinoma biopsy, revealed the existence of a novel member of the tyrosine kinase gene family. This locus, which we now designate the trk proto-oncogene, codes for a protein of 790 amino acid residues that has several features characteristic of cell surface receptors. They include (i) a 32-amino-acid-long putative signal peptide, (ii) an amino-terminal moiety (residues 33 to 407) rich in consensus sites for N-glycosylation, (iii) a transmembrane domain, (iv) a kinase catalytic region highly related to that of other tyrosine kinases, and (v) a very short (15 residue) carboxy-terminal tail. Residues 1 to 392 were absent in the trk oncogene, as they were replaced by tropomyosin sequences. However, no other differences were found between the transforming and nontransforming trk alleles (residues 392 to 790), suggesting that no additional mutations are required to activate the transforming potential of this gene. The human trk proto-oncogene codes for a 140,000-dalton glycoprotein, designated gp140proto-trk. However, its primary translational product is a 110,000-dalton glycoprotein which becomes immediately glycosylated, presumably during its translocation into the endoplasmic reticulum. This molecule, designated gp110proto-trk, is further glycosylated to yield the mature form, gp140proto-trk. Both gp110proto-trk and gp140proto-trk proteins possess in vitro kinase activity specific for tyrosine residues. Finally, iodination of intact NIH 3T3 cells expressing trk proto-oncogene products indicated that only the mature form, gp140proto-trk, cross the plasma membrane, becoming exposed to the outside of the cell. These results indicate that the product of the human trk locus is a novel tyrosine kinase cell surface receptor for an as yet unknown ligand.

Amino Acid Sequence↗