| 9402049 |
MEN 11420, a potent and selective tachykinin NK2 receptor antagonist in the guinea-pig and human colon |
10.1007/pl00005105. |
Naunyn Schmiedebergs Arch Pharmacol |
MEN 11420, a potent and selective tachykinin NK2 receptor antagonist in the guinea-pig and human colon
Abstract
- We have characterized the action of the novel, water-soluble, tachykinin NK2 receptor antagonist MEN 11420 (Asn(2-AcNH-beta-D-Glc)-Asp-Trp-Phe-Dap-Leu c(2 beta-5 beta)) on the circular muscle of the guinea-pig and human colon in vitro and on the guinea-pig colon in vivo. In organ bath experiments on guinea-pig colon MEN 11420 produced a concentration-dependent rightward shift of the concentration-response curve to the NK2 receptor selective agonist, beta Ala8neurokinin A (NKA) (4-10) with a pKB value of 8.1. Up to 1 microM MEN 11420 had no effect on the concentration-response curve to methacholine, to the NK1 receptor selective agonist, Sar9substance P (SP) sulfone, to the NK3 receptor selective agonist, senktide, or on the response to exogenous SP. The response to exogenous NKA was inhibited, although the shift of the concentration-response curve to NKA produced by MEN 11420 at 1 microM (dose ratio 5.3) was much smaller than that produced against beta Ala8NKA (4-10) (dose ratio 102), presumably because NKA also stimulates NK1 receptors at relatively low concentrations. In sucrose gap, MEN 11420 concentration-dependently inhibited both depolarization (IC50 0.34 microM) and contraction (IC50 = 0.32 microM) produced by beta Ala8NKA (4-10) (0.3 microM for 10 s) in the guinea-pig colon without affecting the corresponding responses produced by Sar9SP sulfone. When similar experiments were performed in the circular muscle of the human colon MEN 11420 concentration-dependently inhibited both depolarization and contraction induced by beta Ala8NKA(4-10) with IC50s of 99 and 75 nM, respectively. MEN 11420 (1 microM) had no effect on the nonadrenergic noncholinergic (NANC) depolarization and contraction produced by a short period of electrical field stimulation (EFS, 10 Hz for 1 s) in the guinea-pig colon and selectively inhibited the sustained component of depolarization produced during a prolonged period of EFS (3 Hz for 3 min), without affecting the concomitant depolarization. Nifedipine (1 microM) eliminated the NANC contraction to a short period of EFS and the phasic contraction in response to a prolonged period of EFS. MEN 11420 (1 microM) abolished the nifedipine-resistant NANC contraction produced by prolonged period of electrical field stimulation (EFS, 3 Hz for 3 min). All electrical and mechanical NANC responses to EFS which were resistant to MEN 11420, either in the absence or presence of nifedipine, were abolished by the subsequent application of the NK1 receptor antagonist, SR 140333 (1 microM). Up to 3 microM, MEN 11420 had no significant effect on the cholinergic excitatory junction potential or the NANC inhibitory junction potential evoked by single pulse EFS, nor did it affect membrane conductance. In urethane-anaesthetized guinea-pigs MEN 11420 (10-100 nmol/kg i.v.) produced a dose-dependent and long lasting (> 3 h) inhibition of the contractile response (15 +/- 2 mmHg) of the proximal colon induced by beta Ala8NKA (4-10) (3 nmol/kg i.v.). MEN 11420 (300 nmol/kg i.v.) did not affect the contraction produced by Sar9SP sulfone. MEN 11420 (300 nmol/kg) produced a limited (Emax about 40% inhibition) and transient (recovery within 60 min) inhibition of the atropine- and hexamethonium-sensitive phasic contractions of the proximal colon induced by threshold distension of a colonic balloon. On the other hand, MEN 11420 (10-300 nmol/kg i.v.) produced a dose-dependent complete and prolonged (> 2 h from administration) inhibition of the atropine-resistant and hexamethonium-sensitive phasic contraction induced by suprathreshold distension of the colonic balloon. We conclude that MEN 11420 is a potent and selective tachykinin NK2 receptor antagonist devoid of significant inhibitory activity toward excitatory transmission mediated via tachykinin NK1 or muscarinic receptors. The present findings indicate that SP and NKA are likely involved in the preferential activation of NK1 and NK2 receptors during tachykininergi
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| 9414128 |
PPP1R6, a novel member of the family of glycogen-targeting subunits of protein phosphatase 1. |
10.1016/s0014-5793(97)01385-9 |
FEBS Lett. |
PPP1R6, a novel member of the family of glycogen-targeting subunits of protein phosphatase 1.
Abstract
- A complementary DNA encoding a novel human protein phosphatase 1 (PP1) glycogen-targetting subunit of molecular mass 33 kDa has been sequenced. PPP1R6 is 31% identical to the glycogen-targetting subunit (G(L)) of PP1 from rat liver, 28% identical to the N-terminal region of the glycogen-targetting subunit (G(M)) from human skeletal muscle and 27% identical to glycogen-targetting subunit PPP1R5. Unlike human PPP1R5 and its murine homologue PTG, whose mRNAs are most abundant in skeletal muscle, heart and liver, PPP1R6 is present at similar levels in a wide variety of tissues. The PPP1R6 is associated with glycogen in muscle but is not subject to the same modes of covalent and allosteric regulation as G(M) and G(L).
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| 9416423 |
Conformational preference for segetalins G and H, cyclic peptides with estrogen-like activity from seeds of Vaccaria segetalis |
10.1016/s0968-0896(97)00135-1. |
Bioorg Med Chem |
Conformational preference for segetalins G and H, cyclic peptides with estrogen-like activity from seeds of Vaccaria segetalis
Abstract
- Three-dimensional structures in DMSO-d0 of segetalins G cyclo(-Gly-Val-Lys-Tyr-Ala-) and H cyclo(-Gly-Tyr-Arg-Phe-Ser-), cyclic pentapeptides from seeds of Vaccaria segetalis, showing estrogen-like activity, were determined by the distance geometry calculation and restrained energy minimization from NMR data. The backbone structure of segetalin G contains one beta-turn: a beta II-like turn at Tyr4-Ala5, and that of segetalin H one beta-turn: a beta II' turn at Gly1-Tyr2 and one gamma-turn at Arg3-Phe4-Ser5 sequence. The results of distance comparison analysis proposed a pharmacophore model of estrogen-like cyclic peptides, segetalins.
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| 9418016 |
Differential effects of somatostatin analogues on proliferation of murine colonic cancer cells in vitro |
None |
Cytobios |
Differential effects of somatostatin analogues on proliferation of murine colonic cancer cells in vitro
Abstract
- The effects of somatostatin analogues octreotide (SMS 201-995), ASS-51 and ASS-52 on 3H-thymidine incorporation into DNA of the murine colon 38 cancer cells in vitro were investigated. It was found that SMS 201-995 and ASS-51 inhibited the tritiated thymidine incorporation in a dose-dependent manner. In contrast, analogue ASS-52 in spite of a very similar structure to ASS-51, which differed from the latter only by one CH2OH group, was devoid of remarkable antiproliferative activity. These results indicate that slight modification of the molecule of somatostatin analogues may deeply influence their antiproliferative activity.
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| 9419975 |
Epiregulin binds to epidermal growth factor receptor and ErbB-4 and induces tyrosine phosphorylation of epidermal growth factor receptor, ErbB-2, ErbB-3 and ErbB-4 |
10.1038/sj.onc.1201458. |
Oncogene |
Epiregulin binds to epidermal growth factor receptor and ErbB-4 and induces tyrosine phosphorylation of epidermal growth factor receptor, ErbB-2, ErbB-3 and ErbB-4
Abstract
- Epiregulin is a member of the epidermal growth factor (EGF) family, and has certain characteristics that are different from that of EGF, including mitogenic responses and binding to EGF receptor (EGFR). Epiregulin may also have another cell surface receptor and/or induces different receptor heterodimerizations for intracellular signaling. We investigated the binding ability of epiregulin to four ErbB family receptors using four human breast carcinoma cell lines that expressed different subsets of receptors. Chemical cross-linking experiments showed that [125I]epiregulin directly bound to each of EGFR and ErbB-4 but not to ErbB-2 and ErbB-3. Furthermore, although epiregulin stimulated tyrosine phosphorylation of all four ErbB receptors, the main intracellular signal was mediated by ErbB-4 and/or EGFR. The pattern of activation of ErbB family receptors was different from that of other EGF-related ligands. Our findings indicate that ErbB-4 and EGFR are receptors for epiregulin, and suggest that EGF-related ligands transduce signals for different biological responses by the hierarchical mechanism.
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| 9422736 |
Identification and characterization of a novel protein interacting with Ral-binding protein 1, a putative effector protein of Ral |
10.1074/jbc.273.2.814. |
J Biol Chem |
Identification and characterization of a novel protein interacting with Ral-binding protein 1, a putative effector protein of Ral
Abstract
- Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral and exhibits a GTPase activating activity for Rac and CDC42. To clarify the function of RalBP1, we isolated a novel protein that interacts with RalBP1 by yeast two-hybrid screening and designated it POB1 (partner of RalBP1). POB1 consists of 521 amino acids, shares a homology with Eps15, which has been identified as an epidermal growth factor (EGF) receptor substrate, and has two proline-rich motifs. The POB1 mRNA was expressed in cerebrum, cerebellum, lung, kidney, and testis. POB1 interacted with RalBP1 in COS cells and the C-terminal region of POB1 was responsible for this interaction. The binding domain of RalBP1 to POB1 was distinct from its binding domain to Ral. Ral and POB1 simultaneously interacted with RalBP1 in COS cells. The binding of POB1 to RalBP1 did not affect the GTPase activating activity of RalBP1. Furthermore, POB1 bound to Grb2 but not to Nck or Crk. POB1 was tyrosine-phosphorylated in COS cells upon stimulation with EGF and made a complex with EGF receptor. These results suggest that RalBP1 makes a complex with POB1 and that this complex may provide a link between tyrosine kinase, Src homology 3 (SH3)-containing protein, and Ral.
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| 9422808 |
Characterization of receptors mediating contraction induced by tachykinins in the guinea-pig isolated common bile duct |
10.1038/sj.bjp.0701560. |
Br J Pharmacol |
Characterization of receptors mediating contraction induced by tachykinins in the guinea-pig isolated common bile duct
Abstract
- 1. We studied the effect of the natural tachykinins and of synthetic agonists selective for the tachykinin NK1, NK2 and NK3 receptors, on the motility of guinea-pig isolated common bile duct longitudinally-oriented smooth muscle. 2. All the tachykinins tested (both natural and synthetic) produced a concentration-dependent contractile response of the guinea-pig isolated common bile duct: these effects underwent a marked tachyphylaxis, especially the responses elicited by NK1 and NK3 receptor-selective agonists. 3. Among the natural tachykinins neurokinin B (EC50 = 3.2 nM; 95% c.l. = 2.0-5.1; n = 4) was the most potent, being about 40 and 25 fold more potent than substance P (EC50 = 121.6 nM; 95% c.l. = 94-157; P < 0.01; n = 4) and neurokinin A (EC50 = 83.4 nM; 95% c.l. = 62-112; P < 0.01; n = 4), respectively. Among the synthetic analogues the NK3 receptor-selective agonist senktide (EC50 = 1.1 nM; 95% c.l. = 0.7-1.8; n = 8) was the most potent, being about 120, 110 and 20 fold more potent than Sar9substance P sulfone (NK1 receptor-selective) (EC50 = 130.4 nM; 95% c.l. = 99-172; P < 0.01; n = 8), beta Ala8NKA (4-10) (NK2 receptor-selective) (EC50 = 120.1 nM; 95% c.l. = 95-151; P < 0.01; n = 8) and septide (NK1 receptor-selective) (EC50 = 22.6 nM; 95% c.l. = 18-28; P < 0.01; n = 8), respectively. All tachykinins (natural or synthetic receptor agonists) produced a similar Emax, averaging about 50% of that produced by KCl (80 mM). 4. Atropine (1 microM) did not affect the responses to either NK1 or NK2 receptor-selective agonists, whereas it reduced the Emax of senktide by about 50%, without affecting its potency (EC50). Tetrodotoxin (1 microM) totally blocked senktide-induced contractions, as did the combined pretreatment with atropine plus the tachykinin NK1 and NK2 receptor-selective antagonists GR 82334 and MEN 11420 (1 microM each), respectively. 5. GR 82334 (1 microM) blocked with apparent competitive kinetics septide- (apparent pKB = 7.46 +/- 0.10; n = 5) and Sar9substance P sulfone- (apparent pKB = 6.80 +/- 0.04; n = 4) induced contractions. MEN 11420 (30-300 nM), a novel potent NK2 receptor antagonist, potently antagonized beta Ala8NKA (4-10), with competitive kinetics (pKB = 8.25 +/- 0.08; n = 12: Schild plot slope = -0.90; 95% c.l. = -1.4; -0.35). The NK3 receptor-selective antagonist SR 142801 (30 nM) produced insurmountable antagonism of the senktide-induced contractions (Emax inhibited by 64%). of the above antagonists, tested at the highest concentrations employed against tachykinins, affected the concentration-response curve to methacholine (0.1-300 microM). 6. We conclude that tachykinins produce contraction of the guinea-pig isolated common bile duct by stimulating NK1, NK2 and NK3 receptors. The responses obtained by activating NK1 and NK2 receptors are atropine-resistant. The contraction obtained by stimulating NK3 receptors is totally neurogenic, being mediated by the release of endogenous acetylcholine and tachykinins; the latter act, in turn, on postjunctional tachykinin NK1/NK2 receptors. The role of the NK3 receptor as prejunctional mediator of the excitatory transmission operated by tachykinins is discussed.
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| 9428692 |
Identification of Stat 5B as a substrate of the insulin receptor |
10.1111/j.1432-1033.1997.0411a.x. |
Eur J Biochem |
Identification of Stat 5B as a substrate of the insulin receptor
Abstract
- We have screened a human placenta library using the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. Doing so, we trapped a cDNA clone which encodes the Stat 5B region comprising amino acids 469 to 786. We show that interaction between Stat 5B and the receptor requires a functional insulin-receptor kinase, Tyr960 of insulin receptor is implicated in the interaction with Stat 5B, whereas asparagine and proline forming the NPEY960-motif are not, and Stat 5B mutated at Thr684, a potential phosphorylation site of mitogen-activated protein kinase, loses its ability to interact with the insulin receptor. Further, we found that insulin promotes rapid tyrosine phosphorylation of endogenous Stat 5B in 293 EBNA cells overexpressing insulin receptor and in NHIR cells. Taken together, our findings suggest that Stat 5B corresponds to a substrate for the insulin-receptor kinase, and this widens the repertoire of insulin-signaling pathways.
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| 9430433 |
des-AA-1,2,5D-Trp8, IAmp9somatostatin-14 allows the identification of native rat somatostatin sst1 receptor subtype |
10.1016/s0014-2999(97)01282-x. |
Eur J Pharmacol |
des-AA-1,2,5D-Trp8, IAmp9somatostatin-14 allows the identification of native rat somatostatin sst1 receptor subtype
Abstract
- Somatostatin exerts multiple activities by interacting with at least five different receptor subtypes (sst1-5). The affinity of des-AA(1,2,5)-D-Trp8, IAmp9somatostatin-14 (CH-275) was studied by competition experiments using the non-selective radioligand 125ILeu8, D-Trp22, Tyr25somatostatin-28 in areas of the rat brain and pituitary known to express identified receptor subtypes. In the cerebellar nuclei and cerebral cortex, which possess the somatostatin sst1 receptor subtype, CH-275 exhibited a moderate affinity (IC50: 10-50 nM). Conversely, in the hippocampus, immature cerebellum and pituitary which contain different subsets of receptors mRNAs (sst2-5), the IC50 values were > 1 microM. These data indicate that CH-275 is an appropriate ligand for the identification of native rat somatostatin sst1 receptor subtype.
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| 9449257 |
Therapeutic effects of water-soluble echinocandin compounds on Pneumocystis pneumonia in mice |
10.1128/AAC.42.1.37. |
Antimicrob Agents Chemother |
Therapeutic effects of water-soluble echinocandin compounds on Pneumocystis pneumonia in mice
Abstract
- The therapeutic effectiveness of water-soluble echinocandin compounds obtained from Coleophoma empetri F-11899, which has a strong inhibitory effect on the growth of fungi, was examined in nude mice with experimental Pneumocystis pneumonia. The studies demonstrated the potential usefulness of the compounds.
|
| 9461419 |
Localization and genomic organization of sheep antimicrobial peptide genes |
10.1016/s0378-1119(97)00569-6. |
Gene |
Localization and genomic organization of sheep antimicrobial peptide genes
Abstract
- Antimicrobial peptides are an abundant and diverse component of animal innate immunity. Within mammalian species, defensins and cathelicidins are the two principal antimicrobial peptide families. We identified and sequenced ten new sheep genes which encode potential antimicrobial peptides including two beta-defensins and eight cathelicidins. We mapped the two-exon beta-defensin genes to sheep chromosome 26 and the four-exon cathelicidin genes to sheep chromosome 19 using sheep-hamster somatic cell hybrids in conjunction with flow-sorted sheep chromosomes. These assignments confirm homology between sheep, cattle, mouse, and human antimicrobial peptide gene families. Contig construction for the sheep cathelicidin gene family demonstrates that three genes, OaDodeA, OaDodeB, and OaMAP-34, are present head-to-tail in a 14.5 kb region, and that four proline/arginine-rich genes, OaBac5, OaBac7.5, OaBac11, and OaBac6, are arranged head-to-tail in a region covering 30.5 kb. This richly diverse family of sheep cathelicidin peptides is encoded in a gene array which may reflect the mechanism of its evolution.
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| 9484780 |
Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins |
10.1038/sj.onc.1201607. |
Oncogene |
Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins
Abstract
- Shb is a recently described Src homology 2 (SH2) domain-containing adaptor protein. Here we show that Shb is expressed in lymphoid tissues, and is recruited into signaling complexes upon activation of Jurkat T cells. Grb2 binds proline-rich motifs in Shb via its SH3 domains. As a result, a number of proteins detected in anti-Shb and anti-Grb2 immunoprecipitates are shared, including phosphoproteins of 22, 36/38, 55/57 and 70 kDa. Shb-association with p22, which represents the T cell receptor associated zeta chain, occurs through the Shb SH2 domain. The central region of Shb binds p36/38. Since this interaction was inhibited by phosphotyrosine, this region of Shb is likely to contain a non-SH2 PTB (phosphotyrosine binding) domain. The Shb PTB domain was found to preferentially bind the sequence Asp-Asp-X-pTyr when incubated with a phosphopeptide library. A peptide corresponding to a phosphorylation site in 34 kDa Lnk inhibited association between Shb and p36/38. Overexpression of Shb in Jurkat cells led to increased basal phosphorylation of Shb-associated p36/38 and p70 proteins. Inactivation of the Shb SH2 domain by an R522K mutation resulted in a reduced stimulation of tyrosine phosphorylation of several proteins in response to CD3 crosslinking when expressed in Jurkat cells. Together, our results show three distinct domains of Shb all participate in the formulation of multimeric signaling complexes in activated T cells. These results indicate that the Shb protein functions in T cell receptor signaling.
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| 9485377 |
Epitope insertion favors a six transmembrane domain model for the carboxy- terminal portion of the multidrug resistance-associated protein. |
10.1021/bi972332v |
Biochemistry |
Epitope insertion favors a six transmembrane domain model for the carboxy- terminal portion of the multidrug resistance-associated protein.
Abstract
- The overexpression of the multidrug resistance protein, MRP, in mammalian cells is associated with pleiotropic resistance to cytotoxic drugs. MRP is an integral membrane protein which belongs to the family of ATP-binding cassette transporters. Secondary structure predictions combined with biochemical analyses suggest that MRP encodes 11 transmembrane (TM) domains in the amino-terminal half of the protein and four or six transmembrane domains in the carboxy-terminal half of the protein. To gain insight into the membrane topology of the carboxy-terminal half of MRP, small, antigenic hemagglutinin (HA) epitopes (YPYDVPDYAS) were inserted within six predicted hydrophilic subfragments of this region (938, 1001, 1084, 1175, 1222, 1295). These epitope-tagged MRP variants were expressed in HeLa cells to evaluate their ability to confer resistance to the drug etoposide (VP-16). Insertion of the HA epitopes at positions 938, 1001, and 1222 resulted in functional proteins, while epitope insertion at positions 1084, 1175, and 1295 abrogated MRP function. The intracellular versus extracellular location of the HA epitopes present in biologically active MRP variants was then established in intact and permeabilized cells by immunofluorescence using an anti-HA antibody. Epitopes inserted at positions 1001 and 1222 were located on the extracellular side of the plasma membrane, while the epitope inserted at position 938 was located intracellularly. These
Results are consistent with a six TM rather than a four TM domain model for the membrane portion of the carboxy-terminal half of MRP.
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| 9488479 |
Epidermal growth factor receptor and the adaptor protein p52Shc are specific substrates of T-cell protein tyrosine phosphatase |
10.1128/MCB.18.3.1622. |
Mol Cell Biol |
Epidermal growth factor receptor and the adaptor protein p52Shc are specific substrates of T-cell protein tyrosine phosphatase
Abstract
- T-cell protein tyrosine phosphatase (TCPTP) exists as two forms generated by alternative splicing: a 48-kDa endoplasmic reticulum (ER)-associated form (TC48) and a 45-kDa nuclear form (TC45). To identify TCPTP substrates, we have generated substrate-trapping mutants, in which the invariant catalytic acid of TCPTP (D182) is mutated to alanine. The TCPTP D182A substrate-trapping mutants were transiently overexpressed in COS cells, and their ability to form complexes with tyrosine-phosphorylated (pTyr) proteins was assessed. No pTyr proteins formed complexes with wild-type TCPTP. In contrast, TC48-D182A formed a complex in the ER with pTyr epidermal growth factor receptor (EGFR). In response to EGF, TC45-D182A exited the nucleus and accumulated in the cytoplasm, where it bound pTyr proteins of approximately 50, 57, 64, and 180 kDa. Complex formation was disrupted by vanadate, highlighting the importance of the PTP active site in the interaction and supporting the characterization of these proteins as substrates. Of these TC45 substrates, the approximately 57- and 180-kDa proteins were identified as p52Shc and EGFR, respectively. We examined the effects of TC45 on EGFR signaling and observed that it did not modulate EGF-induced activation of p42Erk2. However, TC45 inhibited the EGF-induced association of p52Shc with Grb2, which was attributed to the ability of the PTP to recognize specifically p52Shc phosphorylated on Y239. These results indicate that TC45 recognizes not only selected substrates in a cellular context but also specific sites within substrates and thus may regulate discrete signaling events.
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| 9488644 |
Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA |
10.1126/science.279.5356.1504. |
Science |
Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA
Abstract
- Topoisomerases I promote the relaxation of DNA superhelical tension by introducing a transient single-stranded break in duplex DNA and are vital for the processes of replication, transcription, and recombination. The crystal structures at 2.1 and 2.5 angstrom resolution of reconstituted human topoisomerase I comprising the core and carboxyl-terminal domains in covalent and noncovalent complexes with 22-base pair DNA duplexes reveal an enzyme that "clamps" around essentially B-form DNA. The core domain and the first eight residues of the carboxyl-terminal domain of the enzyme, including the active-site nucleophile tyrosine-723, share significant structural similarity with the bacteriophage family of DNA integrases. A binding mode for the anticancer drug camptothecin is proposed on the basis of chemical and biochemical information combined with these three-dimensional structures of topoisomerase I-DNA complexes.
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