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  • Biochemistry and Biotechnology  (2)
  • antibody  (2)
  • ATP-binding cassette (ABC) transporters  (1)
  • Chemotherapy  (1)
  • Human bladder carcinoma  (1)
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  • 1
    ISSN: 1573-6881
    Keywords: P-glycoprotein ; multidrug resistance ; MDR ; ATPase ; drug transport ; ATP-binding cassette (ABC) transporters
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Chemotherapy, though it remains one of the front-line weapons used to treat human cancer, is often ineffective due to drug resistance mechanisms manifest in tumor cells. One common pattern of drug resistance, characterized by simultaneous resistance to multiple amphipathic, but otherwise structurally dissimilar anticancer drugs, is termed multidrug resistance. Multidrug resistance in various model systems, covering the phylogenetic range from bacteria to man, can be conferred by mammalian P-glycoproteins (PGPs), often termed multidrug transporters. PGPs are 170-kD polytopic membrane proteins, predicted to consist of two homologous halves, each with six membrane spanning regions and one ATP binding site. They are members of the ATP-binding cassette (ABC) superfamily of transporters, and are known to function biochemically as energy-dependent drug efflux pumps. However, much remains to be learned about PGP structure-function relationships, membrane topology, posttranslational regulation, and bioenergetics of drug transport. Much of the recent progress in the study of the human and mouse PGPs has come from heterologous expression systems which offer the benefits of ease of genetic selection and manipulation, and/or short generation times of the organism in which PGPs are expressed, and/or high-level expression of recombinant PGP. Here we review recent studies of PGP inE. coli, baculovirus, and yeast systems and evaluate their utility for the study of PGPs, as well as other higher eukaryotic membrane proteins.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-6881
    Keywords: Chemotherapy ; ATP ; drug transport ; colchicine ; actinomycin D ; doxorubicin ; vinblastine ; vincristine ; introns ; evolution ; P-glycoprotein ; transmembrane domains ; MDR1 gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Multidrug resistance in animal cells is defined as the simultaneous resistance to a variety of compounds which appear to be structurally and mechanistically unrelated. One type of multidrug resistance is characterized by the decreased accumulation of hydrophobic natural product drugs, a phenotype which is mediated by an ATP-dependent integral membrane multidrug transporter termed P-glycoprotein or P170. The gene coding for P170 is calledMDR. The nucleotide-binding domain of P-glycoprotein shares sequence homology with a family of bacterial permease ATP-binding components. In addition, P170 as a whole is structurally very similar to a number of prokaryotic and eukaryotic proteins believed to be involved in transport activities. This review summarizes our current knowledge of the molecular biology and clinical significance ofMDR expression and P-glycoprotein transport activity, as well as some theories about the function of this protein in normal cells.
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  • 3
    ISSN: 0887-3585
    Keywords: immunoglobulin ; antibody ; mAb B3 ; protein engineering ; disulfide bond ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Fv fragments are the smallest units of antibodies that retain the specific antigen binding characteristics of the whole molecule and are being used for the diagnosis and therapy of human diseases. These are noncovalently associated heterodimers of the heavy (V H) and the light (VL) chain variable domains, which, without modification, tend to dissociate, unfold, and/or nonspecific ally aggregate. The fragment is usually stabilized by producing it as a single chain recombinant molecule in which the two chains are linked by means of a short polypeptide linker. An alternative strategy is to connect the two chains by means of an interchain disulfide bond. We used molecular graphics and other modeling tools to identify two possible interchain disulfide bond sites in the framework region of the Fv fragment of the monoclonal mouse antibody (mAb) B3. The mAb B3 binds to many human cancer cells and is being used in the development of a new anticancer agent. The two sites identified are VH44-VL105 and VH111-VL48. (VH44-VL100 and VH105-VL43 in the numbering scheme of Kabat et al., “Sequence of Proteins of Immunological Interest,” U.S. DHHS, NIH publication No. 91-3242, 1991.) This design was recently tested using the chimeric protein composed of a truncated form of Pseudomonas exotoxin and the Fv fragment of mAb B3 with the engineered disulfide bond at VH44-VL105 (Brinkmann et al., Proc. Natl. Acad. Sci. U.S.A. 90:7538, 1993). The chimeric toxin was found to be just as active as the corresponding single chain counterpart and considerably more stable. Because these disulfide bond sites are in the framework region, they can be located from sequence alignment alone. We expect that the disulfide bond at these sites will stabilize the Fv fragment of most antibodies and the antigen-specific portion of the T-cell receptors, which are homologous. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 0887-3585
    Keywords: antibody ; antitumor ; single chain Fv ; variable domains ; X-ray crystallography ; protein structure ; protein stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A recombinant Fv construct of the B1 monoclonal antibody that recognizes the LewisY-related carbohydrate epitope on human carcinoma cells has been prepared. The Fv is composed of the polypeptide chains of the VH and VL domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amounts of guanidine chloride solubilized inclusion bodies. The Fv is stabilized by an engineered interchain disulfide bridge between residues VL100 and VH44. This construct has a similar binding affinity as that of the single-chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023-1029, 1995). The B1 disulfide-stabilized Fv (B1dsFv) crystallizes in space group P6122 with the unit cell parameters a = b = 80.1 Å, and c = 138.1 Å. The crystal structure of the B1dsFv has been determined at 2.1-Å resolution using the molecular replacement technique. The final structure has a crystallographic R-value of 0.187 with a root mean square deviation in bond distance of 0.014 Å and in bond angle of 2.74°. Comparisons of the B1dsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of B1dsFv is a deep depression 10-Å wide and 17-Å long with the walls of the depression composed of residues, many of which are tyrosines, from complementarity determining regions L1, L3, H1, H2, and H3. Model building studies indicate that the LewisY tetrasaccharide, Fuc-Gal-Nag-Fuc, can be accommodated in the antigen combining site in a manner consistent with the epitope predicted in earlier biochemical studies (Pastan, Lovelace, Gallo, Rutherford, Magnani, and Willingham, Cancer Res. 51:3781-3787, 1991). Thus, the engineered disulfide bridge appears to cause little, if any, distortion in the Fv structure, making it an effective substitute for the B1 Fab. Proteins 31:128-138, 1998. Published 1998 Wiley-Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.
    Additional Material: 10 Ill.
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  • 5
    ISSN: 1432-1335
    Keywords: Pseudomonas exotoxin ; TGFα ; Rat bladder carcinoma ; Human bladder carcinoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A protein formed by fusion of transforming growth factor α withPseudomonas exotoxin (TGFα-PE40) has been shown to have the ability to kill or inhibit the growth of several carcinoma cell lines. This study was designed to evaluate thein vitro cytotoxic effects of TGFα-PE40 on rat and human bladder carcinoma cell lines with different biological potential, and normal rat urothelial cells. The rat cell lines used were D44c, LMC19, and MYU3L, which were established in our laboratory. Human cell lines used were RT4, T24, and 253J. As a normal control, we used the first-passage culture of normal rat bladder urothelium (RU-P1). We examined the number and affinity of epidermal growth factor receptors (EGFR) in these cells, the ability of TGFα-PE40 to bind EGFR, and the cytotoxic effect of TGFα-PE40 and PE40. Rat cell lines, D44c, LMC19, and MYU3L (EGFR=4.9×103–11.4×103/cell) had ED50 values (the concentration of TGFα-PE40 needed to reduce the viable cell population by 50%) of 180 pM, 540 pM and 6000 pM respectively; forc 1 (the concentration required to achieve complete inhibition of growth under continuous serum stimulation) TGFα-PE40 concentrations of 104 pM, 104 pM and higher than 104 pM respectively were required. Human cell lines, RT4, T24, and 253J (EGFR=32×103–126×103/cell) had ED50 values of 20 pM, 66 pM, and 330 pM respectively and T24 showedc 1 values of 103 pM. RU-P1 (EGFR =92.6×103/cell) had the highest ED50 value of 8000 pM. These data indicate that the susceptibility to TGFα-PE40 does not always depend on the number of EGFR, that cells having a relatively small number of EGFR respond well to TGFα-PE40, and that normal urothelial cells are more resistant to TGFα-PE40 than are cancer cells. The differential effect of TGFα-PE40 on normal and neoplastic cells provides a rational basis for its use in vivo to control tumor growth.
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