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D Banerjee

Publications and source records attributed to D Banerjee.

At least 109 records · Page 6Linked to original sources
PubMed1996

Characterization of native falcipain, an enzyme involved in Plasmodium falciparum hemoglobin degradation.

In Plasmodium falciparum, a cysteine protease known as falcipain has been implicated in the essential metabolic process of hemoglobin degradation. Parallel lines of investigation, using native or recombinant enzyme, have led to differing conclusions about the specificity and role of this protease. We have now determined that (1) Native falcipain does not cleave hemoglobin unless this substrate has first been denatured by reducing agents, acid-acetone treatment or plasmepsin action. (2) Reducing agents such as glutathione cannot denature hemoglobin in the presence of catalase, which is accumulated in the digestive vacuole. (3) The purified native enzyme has kinetics similar to those obtained with trophozoite extract, but substantially different from those of recombinant enzyme. (4) Although there are numerous cysteine protease genes in the P. falciparum genome, the falcipain gene is the only one whose transcript can be detected in the early intraerythrocytic parasites. We conclude that falcipain likely works by degrading hemoglobin fragments after initial aspartic protease attack has denatured the substrate. We propose that falcipain inhibitors block the initial steps of degradation indirectly by promoting vacuolar accumulation of osmotically active hemoglobin peptides.

Animals↗
PubMed1996

Molecular identity and cellular distribution of advanced glycation endproduct receptors: relationship of p60 to OST-48 and p90 to 80K-H membrane proteins.

Advanced glycation endproducts (AGEs) are derivatives of nonenzymatic reactions between sugars and protein or lipids, and together with AGE-specific receptors are involved in numerous pathogenic processes associated with aging and hyperglycemia. Two of the known AGE-binding proteins isolated from rat liver membranes, p60 and p90, have been partially sequenced. We now report that the N-terminal sequence of p60 exhibits 95% identity to OST-48, a 48-kDa member of the oligosaccharyltransferase complex found in microsomal membranes, while sequence analysis of p90 revealed 73% and 85% identity to the N-terminal and internal sequences, respectively, of human 80K-H, a 80- to 87-kDa protein substrate for protein kinase C. AGE-ligand and Western analyses of purified oligosaccharyltransferase complex, enriched rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membranes from rat liver or RAW 264.7 macrophages yielded a single protein of approximately 50 kDa recognized by both anti-p60 and anti-OST-48 antibodies, and also exhibited AGE-specific binding. Immunoprecipitated OST-48 from rat rough endoplasmic reticulum fractions exhibited both AGE binding and immunoreactivity to an anti-p60 antibody. Immune IgG raised to recombinant OST-48 and 80K-H inhibited binding of AGE-bovine serum albumin to cell membranes in a dose-dependent manner. Immunostaining and flow cytometry demonstrated the surface expression of OST-48 and 80K-H on numerous cell types and tissues, including mononuclear, endothelial, renal, and brain neuronal and glial cells. We conclude that the AGE receptor components p60 and p90 are identical to OST-48, and 80K-H, respectively, and that they together contribute to the processing of AGEs from extra- and intracellular compartments and in the cellular responses associated with these pathogenic substances.

Amino Acid Sequence↗
PubMed1996

Effect of cyclin D1 overexpression on drug sensitivity in a human fibrosarcoma cell line.

BACKGROUND: Alterations in the expression of genes that control the cell cycle may be of critical importance in determining the sensitivity of cells and tumors to drugs (chemosensitivity) and radiation. Mutations and deletions of the p53 tumor suppressor gene in cell lines and tumors are associated with resistance to a variety of DNA-damaging agents. The effects of alterations in the cyclin genes and their products on drug action have not been studied. One of these genes, cyclin D1, is expressed in early G1 phase, and its protein product, together with the cyclin-dependent kinases CDK4 and CDK6, mediates the phosphorylation and functional inactivation of the retinoblastoma protein (pRb). Elevated levels of expression of cyclin D1 protein have been found in a variety of cancers, including breast cancer, head and neck cancer, non-small-cell lung cancer, and mantle cell lymphomas. PURPOSE: This study was conducted to investigate the effect of increased expression of cyclin D1 protein on the chemosensitivity profile of a human fibrosarcoma cell line. METHODS: Expression plasmids containing either the neomycin-resistance gene and the complementary DNA sequence encoding human cyclin D1 or the neomycin-resistance gene only (control) were transfected by lipofection into the human HT1080 fibrosarcoma cell line, and cell colonies resistant to the antibiotic neomycin (G418) were isolated. Cyclin D1 messenger RNA (mRNA) and protein levels were measured by ribonuclease protection and western blot analyses, respectively. Dihydrofolate reductase (DHFR) mRNA and protein levels were measured by northern blot and western blot analyses, respectively. The phosphorylation status of pRb was assessed by western blot analysis. Cell cycle analysis was performed by use of the technique of fluorescence-activated cell sorting. Cytotoxicity assays were carried out by use of the sulforhodamine blue assay. RESULTS: Of the 16 cyclin D1-transfected cell clones that were isolated, four were randomly selected for further study. Two cell clones expressed high levels of cyclin D1 mRNA and protein as compared with control cells transfected with plasmids containing the neomycin-resistance gene only. A relative increase in the phosphorylated form of pRb in cells expressing high versus low levels of cyclin D1 was also revealed by western blot analysis. There was an increased fraction of cells in the S and G2 phases of the cell cycle among cells expressing higher levels of cyclin D1. Transfectants with increased cyclin D1 expression also had increased DHFR mRNA and protein expression. Cytotoxicity assays revealed a statistically significant (P < .01) increase in resistance to methotrexate in cells expressing high levels of cyclin D1 compared with cells expressing lower levels. There was no difference in resistance to doxorubicin, paclitaxel (Taxol), and cytarabine. CONCLUSION: Alterations in the expression of cyclin D1 led to altered cell cycle distribution in a human sarcoma cell line. The associated increase in DHFR expression resulted in increased resistance to methotrexate but had no effect on other classes of anticancer agents. IMPLICATIONS: These results indicate that alterations in cell cycle genes may differ in their effects on cytotoxicity. It will be important to determine the effects of alterations of other cell cycle regulatory genes on the responses of cells to specific classes of drugs. Tumors with overexpression of cyclin D1 may be relatively refractory to methotrexate treatment.

Antimetabolites, Antineoplastic↗
PubMed1996

Active site-directed double mutants of dihydrofolate reductase.

Variants of dihydrofolate reductase (DHFR), which confer resistance to antifolates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy. To identify improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu22 and Phe31 (i.e., Phe22-Ser31, Tyr22-Ser31, Phe22-Gly31, and Tyr22-Gly31). Antifolate resistance has been observed previously when individual changes are made at these active-site residues. Substrate and antifolate binding properties of these "double" mutants revealed that each have greatly diminished affinity for antifolates (> 10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified for one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.

3T3 Cells↗
PubMed1996

Resistance mechanisms to methotrexate in tumors.

The mechanisms of intrinsic and acquired resistance to methotrexate (MTX) in human tumors are reviewed herein. In blasts from patients with acute lymphocytic leukemia, resistance mechanisms found are decreased uptake and increased dihydrofolate reductase (DHFR) activity. A major cause of intrinsic resistance to MTX in soft tissue sarcoma cells and in acute myelocytic leukemia appears to be a lack of drug retention, due mainly to low levels of polyglutamylation. A novel association between lack of the retinoblastoma protein and intrinsic MTX resistance has been found. This has been attributed to an increase in DHFR activity, due to an increased rate of transcription of this gene, stimulated by an increase in levels of free E2F, not sequestered by hypophosphorylated retinoblastoma protein.

Antimetabolites, Antineoplastic↗
PubMed1996

A study of hepatobiliary function in chronic heroin smokers.

The controversy over the relationship between heroin addiction and hepatocellular dysfunction was examined by assay of serum activities of aspartate and alanine aminotransferases, total protein, albumin and globulin in 48 chronic heroin smokers and 33 controls. Biliary secretion in the addicts was compared to controls by assay of serum bilirubin and its conjugated and unconjugated fractions. Serum aminotransferases, total protein, albumin and globulin did not differ significantly between the addict and the control groups (p < 0.05), whereas total bilirubin and its conjugated fraction were significantly higher in the addicts (p < 0.02). The results indicate that chronic abuse of heroin by smoking affects biliary secretion but exercises little influence over hepatocellular function.

Journal Article↗
PubMed1996

Resistance Mechanisms to Methotrexate in Tumors.

The mechanisms of intrinsic and acquired resistance to methotrexate (MTX) in human tumors are reviewed herein. In blasts from patients with acute lymphocytic leukemia, resistance mechanisms found are decreased uptake and increased dihydrofolate reductase (DHFR) activity. A major cause of intrinsic resistance to MTX in soft tissue sarcoma cells and in acute myelocytic leukemia appears to be a lack of drug retention, due mainly to low levels of polyglutamylation. A novel association between lack of the retinoblastoma protein and intrinsic MTX resistance has been found. This has been attributed to an increase in DHFR activity, due to an increased rate of transcription of this gene, stimulated by an increase in levels of free E2F, not sequestered by hypophosphorylated retinoblastoma protein.

Journal Article↗
PubMed1996

Variants of human dihydrofolate reductase with substitutions at leucine-22: effect on catalytic and inhibitor binding properties.

We investigated the enzyme kinetic and antifolate inhibitory properties of human dihydrofolate reductase enzyme with mutations at position 22. Leu-22 was changed to isoleucine, methionine, phenylalanine, and tyrosine to generate the various mutant enzymes. The overall catalytic efficiency (kcat/Km) for methionine and phenylalanine mutants was reduced approximately 3-fold and >6-fold for isoleucine and tyrosine mutants. An arginine mutant (L22R) was also expressed but had a dramatically reduced catalytic potential (kcat>250-fold lower than wild-type) and therefore was not studied in detail. The Ki for antifolates, methotrexate, aminopterin, and trimetrexate are more dramatically affected (increased) than the Km for dihydrofolate, particularly for phenylalanine and tyrosine mutants. One remarkable feature is that the phenylalanine mutant is as potently inhibited by piritrexim as is the wild-type human enzyme, although the Ki values for methotrexate and aminopterin were increased 88- and 118-fold, respectively. This is likely related to different positioning of the methoxyphenyl side chain of piritrexim relative to the side chains of other compounds tested. A Chinese hamster cell line harboring the L22F mutant also demonstrated an increased sensitivity of piritrexim relative to antifolates.

Animals↗
PubMed1996

Concurrent papillary and squamous carcinoma in a thyroglossal duct cyst: a case report.

Carcinomas of thyroglossal duct cysts are rare. Most are papillary carcinomas; only about 5% are squamous cell carcinomas. Only one case of mixed papillary and squamous cell carcinoma of a thyroglossal duct cyst has been reported so far. The authors present a second case, that of a 38-year-old man who was first seen with a midline neck lump. It was diagnosed clinically as a thyroglossal duct cyst and was locally excised. Pathological examination showed both a concurrent papillary carcinoma and a squamous cell carcinoma. Treatment consisted of a near-total thyroidectomy, ablative radioactive iodine and adjuvant external radiation therapy. The authors review the literature and explain the rationale behind their choice of treatment.

Adult↗
PubMed1995

Identification of mutations by RNA conformational polymorphism "bar code" analysis.

DNA single-strand conformational polymorphism (SSCP) analysis is widely used for detection of point mutations in clinical specimens. Performing SSCP analysis with cRNA instead of DNA has been shown to improve mutation detection frequency. RNA can exist in numerous meta-stable conformations, which appear as patterns of bands on nondenaturing electrophoresis gels. Single base mutations can cause not only mobility shifts of major bands, but also loss of some conformations and appearance of new conformations. Unique RNA SSCP patterns associated with specific base sequences in many cases allow visual identification of point mutations. However, in some cases, the RNA SSCP pattern of a single base change in a sequence is not sufficiently different for a positive identification of the mutation. Improvement in the detection capability of RNA SSCP was obtained by adding 3'-deoxynucleotides to the transcription reaction. The presence of chain-terminating nucleotides in the transcription reaction formed numerous new RNA fragments, thereby generating complex band patterns ("bar codes") unique to each RNA sequence. This method was applied to analyzing p53 mutations in patients with colon cancer.

Base Sequence↗
PubMed1995

Lack of functional retinoblastoma protein mediates increased resistance to antimetabolites in human sarcoma cell lines.

Growth inhibition assays indicated that the IC50 values for methotrexate (MTX) and 5-fluorodeoxyuridine (FdUrd) in HS-18, a liposarcoma cell line lacking retinoblastoma protein (pRB), and SaOS-2, an osteosarcoma cell line with a truncated and nonfunctional pRB, were 10- to 12-fold and 4- to 11-fold higher, respectively, than for the HT-1080 (fibrosarcoma) cell line, which has wild-type pRB. These Rb-/- cell lines exhibited a 2- to 4-fold increase in both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) enzyme activities as well as a 3- to 4-fold increase in mRNA levels for these enzymes compared to the HT-1080 (Rb+/+) cells. This increase in expression was not due to amplification of the DHFR and TS genes. Growth inhibition by MTX and FdUrd was increased and DHFR and TS activities and expression were correspondingly decreased in Rb transfectants of SaOS-2 cells. In contrast, there was no significant difference in growth inhibition among these cell lines for the nonantimetabolites VP-16, cisplatin, and doxorubicin. A gel mobility-shift assay showed that parental SaOS-2 cells had increased levels of free E2F compared to the Rb-reconstituted SaOS-2 cells. These results indicate that pRB defective cells may have decreased sensitivity to growth inhibition by target enzymes encoded by genes whose transcription is enhanced by E2F proteins and suggest mechanisms of interaction between cytotoxic agents and genes involved in cell cycle progression.

Antimetabolites, Antineoplastic↗
PubMed1995

Proliferation measurements with flow cytometry Tpot in cancer of the uterine cervix: correlation between two laboratories and preliminary clinical results.

PURPOSE: To assess the prognostic value of the pretreatment potential doubling time (Tpot) in carcinoma of the uterine cervix, relative to other established clinical factors. METHODS AND MATERIALS: Fifty-two patients with cervical cancer were studied prospectively from March 1991 to October 1993. Pretreatment evaluation included examination under anesthesia and tumor biopsy 6 h following the intravenous administration of bromodeoxyuridine (200 mg). Tpot was determined by deriving the labeling index (LI) and S-phase synthesis time (Ts) using flow cytometry. Six patients were not evaluable and excluded. The remaining 46 patients (average age 55 years) were treated uniformly with radical radiation therapy. There were 39 squamous carcinomas and 7 adenocarcinomas. Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) stages were: Ib and IIa, 12 patients; IIb, 18 patients; III and IV, 16 patients. The median external beam dose was 50 Gy (range, 45-52.8 Gy) delivered in 25 fractions. The median intracavitary dose was 40 Gy (range. 25.5-40 Gy) delivered with a single line source to a point 2 cm lateral of the midline, with a mean dose rate of 0.71 Gy/h. The median overall treatment time was 45 days (range, 34-73 days). As of July 31, 1994, 12 patients had died of disease, and the average follow-up for alive patients was 1.4 years (range, 0.5-3.3 years). RESULTS: There were 27 tumors with diploid deoxyribonucleic acid (DNA) content and 19 tumors were aneuploid. The median and mean Tpot for the 46 patients were 5.5 and 6.6 days, respectively [range, 2.0-25.6 days; coefficient of variation (CV), 74%]. For 25 patients where Tpot measurements were performed at two separate laboratories, there was a fair correlation (r = 0.74), but systematic differences were detected suggesting that the lack of agreement was not simply due to intratumoral variation. To date, 30 patients remained disease free, while 8 patients had pelvic failure and 9 patients developed distant metastases as the first failure site (1 patient developed both at the same time). In univariate analysis, the only significant prognostic factor for disease-free survival was tumor size (p = 0.004). A short Tpot (or high LI) and long overall treatment time (OTT) were weakly associated with poorer disease-free survival, although not statistically significant (1/Tpot, p = 0.14; LI, p = 0.23; OTT, p = 0.04). Age, FIGO stage, hemoglobin level, S-phase fraction, DNA ploidy, and Ts were not associated with disease-free survival. Multiple regression analysis was not performed because of the relatively small number of patients and short follow-up. CONCLUSIONS: Tpot values determined with current techniques by different laboratories cannot be used interchangeably for the purpose of therapy decisions. Vigorous quality assurance and standardization of the laboratory procedures and analysis methods are important to reduce interlaboratory variation. In this uniformly treated group of patients with cancer of the uterine cervix, traditional clinical prognostic factors remain the most important. Preliminary data suggest that the flow cytometry-determined Tpot and labeling index predict for disease-free survival, although a larger number of patients with longer follow-up is required to assess the true prognostic significance of these assays and to determine if their effect is independent of other clinical factors.

Adenocarcinoma↗