Biomedical subjects

E E Trimmer

Publications and source records attributed to E E Trimmer.

3 recordsLinked to original sources

PubMed1999

Cisplatin.

Cisplatin is a widely used anti-cancer drug that is exceptionally effective against testicular cancer. trans-DDP, the geometric isomer of cisplatin, is ineffective as a chemotherapeutic agent. The anti-tumour activity of cisplatin is generally attributed to its formation of DNA adducts, both intrastrand and interstrand crosslinks, which induce structural distortions in DNA. The DNA adducts of cisplatin are thought to mediate its cytotoxic effects by inhibiting DNA replication and transcription and, ultimately, by inducing programmed cell death, or apoptosis. The adducts of both cis- and trans-DDP are removed from DNA by the nucleotide-excision-repair pathway. Cellular proteins possessing certain DNA-binding motifs, including the HMG domain, bind selectively to DNA modified by cisplatin, but not to DNA adducts of trans-DDP; evidence suggests a possible role for these proteins in modulating cisplatin cytotoxicity. Both intrinsic and drug-induced resistance often limit the success of cisplatin; several specific mechanisms of cisplatin resistance have been identified.

Antineoplastic Agents↗

PubMed1999

Purification and properties of NADH-dependent 5, 10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli.

A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15 degreesC and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 microM, the Km for CH2-H4folate is 0.8 microM, and the turnover number under Vmax conditions estimated for the reaction is 1,800 mol of NADH oxidized min-1 (mol of enzyme-bound FAD)-1. NADPH also serves as a reductant, but exhibits a much higher Km. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗

PubMed1998

Human testis-determining factor SRY binds to the major DNA adduct of cisplatin and a putative target sequence with comparable affinities.

cis-Diamminedichloroplatinum(II) (cis-DDP or cisplatin) is a widely used anticancer drug that is most effective against tumors of the testis. Although cisplatin is believed to mediate its cytotoxicity through the formation of DNA adducts, the precise biochemical mechanisms underlying its antitumor activity and selectivity for testicular tumors remain elusive. Of significance are the high-mobility group (HMG) domain and other proteins that bind specifically to cisplatin-DNA adducts. The present study focuses on the testis-specific HMG domain protein human SRY (hSRY). The full-length hSRY protein and its HMG domain region alone were expressed in Escherichia coli and purified to homogeneity. The affinities and specificities of full-length hSRY and the hSRY-HMG domain for 20 bp DNAs containing a single cis-[Pt(NH3)2{d(GpG)-N7(1), -N7(2)}] intrastrand cross-link or a putative hSRY target site in the CD3epsilon gene enhancer (AACAAAG) were determined in electrophoretic mobility shift assays. Full-length hSRY bound to the major 1,2-d(GpG) cisplatin adduct with a Kd(app) of 120 +/- 10 nM and exhibited a 20-fold specificity over unmodified DNA. The HMG domain of hSRY was sufficient for this interaction. The hSRY-HMG domain recognized the 1,2-d(GpG) intrastrand cross-link with higher affinity [Kd(app) = 4 +/- 0.7 nM] but with lower specificity (5-fold) than the full-length protein. The affinities of full-length hSRY and the hSRY-HMG domain for a single cisplatin-DNA adduct were comparable to those for the putative target sequence AACAAAG. These data suggest that cisplatin-DNA adducts may compete with specific DNA sequences in vivo for the binding of human SRY. A possible role for this testis-specific protein in the cytotoxicity and organotropic specificity of cisplatin for testicular tumors is proposed.

Binding, Competitive↗