Involvement of NK-1 and NK-2 tachykinin receptor mechanisms in jaw muscle activity reflexly evoked by inflammatory irritant application to the rat temporomandibular joint

The analysis of methaqualone (MTQ) in biological matrices by capillary electrophoresis (CE) is described. This methods uses liquid-liquid extraction and micellar electrokinetic capillary chromatography (MECC), an operation mode of CE. Separations are made using a 25 cm long capillary and a borate/phosphate buffer at pH 8.2. Using gas chromatography with mass spectrometry detection (GC-MS) as reference method, MTQ has been analyzed in urine, blood, gastric content and hair. For hair analysis, supercritical fluid extraction was compared with liquid-liquid extraction. Linearity was established in urine and blood between 0.25 and 10.0 micrograms/ml. MTQ recovery from blood was estimated at 60%. The limit of detection of this method in urine is about 0.10 microgram/ml. Drawbacks and advantages of MECC over GC-MS are discussed.     

In vitro and in vivo pharmacology of S 16474, a novel dual tachykinin NK1 and NK2 receptor antagonist

Since tachykinins released from lung sensory nerve endings are thought to play a role in inflammatory diseases of airways via NK1 and NK2 receptors, dual tachykinin NK1 and NK2 receptor antagonists may have a great therapeutic potential. In vitro, the cyclopeptide S 16474 (cyclo-[Abo-Asp(D-Trp(Suc0Na)-Phe-N-(Me)Bzl)]) bound to both human tachykinin NK1 and NK2 receptors expressed in two lines of transfected Chinese hamster ovary cells (IC50 values 85 nM and 129 nM, respectively), while showing a poor affinity for the rat tachykinin NK1 receptor. S 16474 inhibited the contractions induced by substance P in isolated rabbit vena cava (pA2 7.0) and by neurokinin A in rabbit pulmonary artery (pA2 5.6). In vivo in anaesthetized guinea-pigs, S 16474 was found to dose dependently inhibit the bronchoconstrictions induced by intravenously administered substance P, neurokinin A and capsaicin. Plasma extravasation evoked in bronchi by endogenously released tachykinins under vagus nerve stimulation was abolished by S 16474 (10 mu mol/kg i.v.). These results demonstrate clearly that S 16474 is a tachykinin receptor antagonist exhibiting, in vitro and in vivo, a dual inhibitory effect on NK1 and NK2 receptors.     

MEN 10,573 and MEN 10,612, novel cyclic pseudopeptides which are potent tachykinin NK-2 receptor antagonists

The activity and selectivity of MEN 10,573 and MEN 10,612, novel cyclic pseudopeptides which are selective tachykinin NK-2 receptor antagonists is described, as compared to that of previously characterized linear and cyclic compounds. For the NK-2 receptor, the activity of test compounds was investigated in the hamster isolated trachea (HT) and the endothelium-deprived rabbit isolated pulmonary artery (RPA), two preparations which are endowed with pharmacologically distinct forms of the NK-2 receptor. The novel cyclic pseudopeptides, MEN 10,573 and MEN 10,612 displayed very high affinity for the NK-2 receptor in the HT (pA2 8.66 and 9.06, respectively) which is higher than that observed in the RPA (pA2 7.31 and 7.41 for MEN 10,573 and MEN 10,612, respectively). The antagonism exerted by MEN 10,573 and MEN 10,612 was of competitive nature in both preparations. MEN 10,573 and MEN 10,612 also displayed competitive antagonism for NK-2 receptor-mediated responses in the rabbit bronchus (RB), rat vas deferens (RVD), circular muscle of the human colon (HUC) and ileum (HUI). In the RB, HUC and HUI, the potency of the novel cyclic pseudopeptides was comparable to that of MDL 29,913 and about 10-fold greater than that of L659,877. In the RVD however, the potency of MEN 10,573 MEN 10,612 or MDL 29,913 was similar to that of L659,877. In anaesthetized rats, i.v. injection of MEN 10,612 produced a selective and long-lasting blockade of the urinary bladder contraction produced by the i.v. injection of the NK-2 receptor selective agonist [beta Ala8]neurokinin A(4-10), without affecting the response to the NK-1 receptor selective agonist [Sar9]substance P sulfone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a tachykinin NK3 receptor antagonist, SR 142801, studied in isolated neonatal rat spinal cord

In the ovariectomized (ovx) rat, the nonsteroidal antiestrogens, clomiphene (CLO) and tamoxifen (TAM), at dose levels that prevent development of osteopenia to a degree approaching that of 17beta-estradiol are, in contrast to 17beta-estradiol, only weakly uterotrophic. Metabolites of CLO and TAM might contribute differentially to these effects. Thus, we have evaluated bone protective and uterine effects in ovx rats of two such metabolites: 4-hydroxy CLO, produced by p-hydroxylation of CLO; and 4HTA, produced from TAM by stepwise replacement of its dimethylaminoethyl side chain with an acetic acid moiety, accompanied by p-hydroxylation. Also reported are effects of D4HTA, the dihydrodesethyl derivative of 4HTA previously characterized as a full estrogen mimetic in vitro. Administration of 4-hydroxy CLO (2.5 mg/kg subcutaneously) 5 days/week for 5 weeks to 3-month-old ovx rats resulted in complete prevention of bone loss and suppression of bone turnover to levels comparable to those of intact controls and to those of ovx animals similarly receiving 17beta-estradiol (10 microg/kg). However, uterine weight in animals receiving 4-hydroxy CLO was 64% less than that in 17beta-estradiol-treated animals. Although 4HTA (3.7 mg/kg s.c.) had a modest uterotrophic effect, it did not prevent bone loss associated with ovariectomy. In contrast, D4HTA (3.6 mg/kg s.c.) partially reduced bone turnover indicators and cancellous bone loss in a manner similar in many ways to that observed in TAM-treated ovx animals, but it had no uterotrophic effect. These results suggest that, although 4HTA does not contribute to the bone-protective effect of TAM, 4-hydroxy CLO might augment that of CLO.     

Pharmacological profiles of a novel non-peptide angiotensin II type I receptor antagonist HR720 in vitro and in vivo

The relationship, in 539 individuals infected with the human immunodeficiency virus (HIV), between two prognostic markers, the CD4 count and beta-2-microglobulin (B2M), and the development of the acquired immunodeficiency syndrome (AIDS) and death was investigated. Cox proportional hazards models were used to determine the risk of AIDS or death. In a multivariate model which adjusted for demographic factors and treatment, the most recent measurements of B2M (relative hazard (RH) 1.37 per g/l higher) and CD4 count (RH 2.17 per log-unit lower) were both significantly associated with the development of AIDS. Similarly, in a multivariate model which additionally adjusted for the development of AIDS as a time dependent covariate, there was a strong relationship with risk of death for the most recent measurements of B2M (RH 1.34 per g/l higher), and CD4 lymphocyte count (RH 1.91 per log-unit lower). A difference in the level of B2M could be used among patients with similar CD4 counts as an indicator of increased risk of progression to AIDS or death. Using the most recent values of these markers provides a better estimate of the risk of AIDS or death, compared to the more common method of analysis, where baseline values of the markers are used.     

Binding of the non-peptide vasopressin V1a receptor antagonist SR-49059 in the rat brain: an in vitro and in vivo autoradiographic study

The role of the sympathetic limb of the autonomic nervous system (ANS) in the mediation of oscillations in consecutive beat-to-beat RR interval (RRI) and systolic blood pressure (SBP) values of lizards, Gallotia galloti, was investigated using spectral analysis and measuring effects of autonomic blockers. alpha-Adrenergic blockade decreased the power spectral density (PSD) of both RRI and SBP very low frequency (VLF: 0.007-0.055 Hz) and low frequency (LF: 0.055-0.150 Hz) bands, whereas beta-adrenergic blockade increased the PSD of both RRI- and SBP-VLF and RRI- and SBP-LF bands. These findings suggest that in lizards 1) the VLF and LF peaks of RRI and SBP power spectra are alpha-adrenergic mediated, and that 2) the beta-adrenergic activity of the sympathetic system may act buffering all RRI and SBP oscillations below 0.150 Hz. These results, when analyzed jointly with the ones obtained from a previous study (De Vera and González. 1997. Comp Biochem Physiol 85A:389-394) on the effects of parasympathetic blockade on lizards' RRI and SBP oscillations, demonstrate that these reptiles, like mammals, exhibit spontaneous short-term oscillations in their HR and SBP which are mediated by the ANS. However, unlike mammals, the RRI and ABP low-frequency oscillations in Gallotia seem to be similarly affected by the ANS and appear to be powered by alpha-adrenergic and parasympathetic activities and buffered by beta-adrenergic activity.     

Hypersecretion of LH and FSH by a pituitary adenoma

A 51-year-old man who had a pituitary adenoma that appeared to be hypersecreting LH and FSH is described. Not only were serum LH and FSH concentrations above the normal ranges, but the serum concentrations of testosterone, free testosterone, and dihydrotestosterone were also above normal. Serum LH and FSH concentration increased in response to synthetic thyrotropin-releasing hormone as well as to synthetic gonadotropin-releasing hormone. The elevated hormone concentrations decreased following an initial partial hypophysectomy and decreased further following repeat hypophysectomy.     

SR 48692, a non-peptide neurotensin receptor antagonist differentially affects neurotensin-induced behaviour and changes in dopaminergic transmission

Unilateral microinjection of neurotensin in the ventral tegmental area of the rat (2.5 micrograms/0.5 microliter) produced behavioural excitation illustrated by contralateral circling. Given orally, SR 48692, a selective and potent non-peptide neurotensin receptor antagonist, significantly reduced these rotations with a triphasic dose-effect relationship. Inhibition occurred at 0.12 mg/kg; further increases in dose up to 2.5 mg/kg produced no significant antagonism, then at doses > or = 5 mg/kg, a second phase of antagonism was observed. Bilateral injection of neurotensin (0.5 microgram each side) into the nucleus accumbens antagonized the increase in locomotor activity following intraperitoneal injection of amphetamine. Given orally, SR 48692 reduced dose-dependently (0.1-1 mg/kg) these intra-accumbens neurotensin effects. Using high pressure liquid chromatography with electrochemical detection, we showed that microgram amounts of neurotensin injected into the ventral tegmental area increased dihydroxyphenylacetate/dopamine ratios in the nucleus accumbens. Using in vivo voltammetry techniques, we found that the injection of nanogram and picogram amounts of neurotensin in the ventral tegmental area stimulated dopamine efflux in the nucleus accumbens. None of these biochemical changes were affected by SR 48692 (0.1-10 mg/kg). These results indicate complex interactions between neurotensin and the mesolimbic dopamine system. More particularly, the differential ability of SR 48692 to affect neurotensin-evoked behavioural versus biochemical changes supports the concept of neurotensin receptor heterogeneity.     

Effects of SR 48692, a selective non-peptide neurotensin receptor antagonist, on two dopamine-dependent behavioural responses in mice and rats

In this study, two transformation vectors (pMG101 and pMG103) for Phanerochaete chrysosporium were constructed, based on the ble phleomycin-resistance-encoding gene and a homologous histone H4 promoter. Transformation frequencies were 6-10 per micrograms of DNA. Transformed vector DNA could either exist as an unstable replicating plasmid or could be stably integrated. Integrated vector DNA from pMG101, which also contains a histone-encoding H3 gene in the promoter fragment, becomes methylated, resulting in inactivation of ble-dependent resistance. Plasmid pMG103, which lacks the H3, does not show methylation.     

Thioperamide, a histamine H3 receptor antagonist, increases GABA release from the rat hypothalamus

Using a microdialysis method and a new high performance liquid chromatography (HPLC)-fluorometric method for the detection of gamma-aminobutyric acid (GABA), we investigated the effect of thioperamide, an H3 receptor antagonist, on the GABA content in the dialysate from the anterior hypothalamic area of rats anesthetized with urethane. The addition of thioperamide to the perfusion fluid increased the release of GABA and histamine. Depleting neuronal histamine with alpha-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase, and the administration of immepip, an H3 agonist, had no effect on basal- and thioperamide-induced GABA release. In addition, an infusion of clobenpropit, the most specific H3 receptor antagonist available, did not alter the basal release of GABA. On the other hand, histamine release was decreased by immepip and increased by thioperamide and clobenpropit. Removing Ca2+ from the perfusion fluid did not alter the effect of thioperamide on the GABA release, whereas that on histamine release was abrogated. These results suggest that the effect of thioperamide on GABA release is not mediated by histamine H3 receptors and that thioperamide acts on the transporter to cause an efflux of GABA from neurons and/or glia. Thioperamide is a popular H3 receptor antagonist which has been used applied to many studies. However, results using this compound should be interpreted in consideration of its effects on GABA release.     

Effect of a new CCK-A receptor antagonist, dexloxiglumide, on the exocrine pancreas in the rat

The effect of dexloxiglumide, a new potent cholecystokinin (CCK) antagonist, on pancreatic enzyme secretion and growth was studied in the rat. Pancreatic exocrine secretion was studied both in vitro (isolated and perfused pancreatic segments) and in vivo (anaesthetized animals with cannulation of the common bile duct) whereas the trophic effect was investigated after short-term (7 days) administration of the CCK-agonist, caerulein, or camostate (a potent trypsin inhibitor), with or without dexloxiglumide. CCK-8 stimulated amylase release from in vitro pancreatic segments in a concentration-dependent manner. Dexloxiglumide displaced the concentration response curves to CCK-8 to the right without affecting the maximum response, suggesting a competitive antagonism. The Schild plot analysis of data gave a straight line with a slope (0.90 +/- 0.36) not significantly different from unity. The calculated pA2 for dexloxiglumide was 6.41 +/- 0.38. In vivo experiments confirmed results from in vitro studies since intravenous dexloxiglumide reduced pancreatic exocrine secretion induced by submaximal CCK-8 stimulation (0.5 nmol/kg/h) in a dose-dependent manner, the ID50 being 0.64 mg/kg. Both exogenous and endogenous (released by camostate) CCK increased the weight of the pancreas, the total pancreatic protein and DNA, trypsin and amylase content. Dexloxiglumide (25 mg/kg), administered together with caerulein (1 microgram/kg), reduced the peptide-induced increase in pancreatic weight, protein and enzyme content. Similarly, when dexloxiglumide was given together with camostate (200 mg/kg), all the observed changes were reduced by concomitant administration of the antagonist. These results demonstrate the ability of dexloxiglumide to antagonize the effects of CCK on pancreatic secretion and growth, suggesting that this compound is a potent and selective antagonist of CCK-A-receptors in the pancreas.     

ZD1542, a potent thromboxane A2 synthase inhibitor and receptor antagonist in vitro

1. The thromboxane A2 synthase (TXS) inhibitory activity and the thromboxane A2 (TP)-receptor blocking action of ZD1542 (4(Z)-6-[2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]-4-(3- pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in vitro on platelets and whole blood from a range of species including man. Antagonist activity has also been investigated in vascular and pulmonary smooth muscle preparations in vitro. 2. ZD1542 caused concentration-dependent inhibition of human platelet microsomal thromboxane B2 (TXB2) production in vitro (IC50 = 0.016 microM); this inhibition was associated with an increase in prostaglandin E2 (PGE2) and PGF2 alpha formation. 3. ZD1542 also inhibited collagen-stimulated TXS in human, rat and dog whole blood giving IC50 values of 0.018, 0.009 and 0.049 microM respectively. The drug did not modify platelet cyclo-oxygenase activity as inhibition of TXB2 formation was associated with a concomitant increase in the levels of PGD2, PGE2 and PGF2 alpha. ZD1542 had little if any effect against cultured human umbilical vein endothelial cell (HUVEC) cyclo-oxygenase (IC50 > 100 microM) and prostacyclin (PGI2) synthase (IC50 = 18.0 +/- 8.6 microM). 4. ZD1542 caused concentration-dependent inhibition of U46619-induced aggregation responses of human, rat and dog platelets yielding apparent pA2 values of 8.3, 8.5 and 9.1 respectively. The drug was selective as, at concentrations up to 100 microM, it did not modify 5-hydroxytryptamine (5-HT) or the primary phases of adenosine diphosphate (ADP) and adrenaline-induced aggregation. Furthermore, ZD1542 (100 microM) modified only weakly the platelet effects of PGD2, PGE1 and PGI2. 5. ZD1542 also caused concentration-dependent inhibition of U46619-mediated contractions of rat thoracic aorta, guinea-pig trachea and lung parenchyma preparations giving apparent pA2 values of 8.6,8.3 and 8.5 respectively. At concentrations approaching three orders of magnitude greater than those required to block U46619-mediated contractions, the drug did not affect the actions of non-prostanoid agonists or exhibit agonist activity in any of the smooth muscle preparations employed; neither did it interact at EP- or FP-receptors.6. In conclusion, the present study demonstrates that ZD1542 is a drug that exhibits both potent,selective TXS inhibition and TXA2 receptor antagonism.     

Antinociceptive activity of the tachykinin NK1 receptor antagonist, CP-99,994, in conscious gerbils

1. The ability of CP-99,994, and its less active enantiomer, CP-100,263, to inhibit spontaneous behaviours and hyperalgesia induced by central infusion of the NK1 receptor agonist, GR73632 or intraplantar injection of formalin was investigated in rats and gerbils. 2. GR73632 (3 pmol, i.c.v.)-induced foot tapping in gerbils was dose-dependently inhibited by CP-99,994 (0.1-1 mg kg-1, s.c.), but not by CP-100,263 (10 mg kg-1, s.c.) using pretreatment times up to 60 min. The centrally active dose-range for CP-99,994 was increased to 1-10 mg kg-1 s.c. with a higher challenge dose of GR73632 (30 pmol, i.c.v.). 3. In gerbils, intrathecal (i.t.) injection of GR73632 (30 pmol) elicited behaviours (licking, foot tapping or flinching and face washing) which closely resembled, but which was less specifically localized than, behaviours seen in animals injected with formalin (0.1-5%) into one hindpaw. 4. In rats, CP-100,263, but not CP-99,994 (up to 30 mg kg-1), inhibited the early phase response to intraplantar injection of 5% formalin (ID50 = 13.9 mg kg-1). The late phase was inhibited by both compounds (ID50 values 36.3 and 20.9 mg kg-1, respectively). In gerbils, there was marginal evidence for enantioselective inhibition of the early phase induced by formalin (2%). The ID50 values were 6.2 mg kg-1 for CP-99,994 and 13.4 mg kg-1 for CP-100,263. 5. Intrathecal injection of GR73632 (30 pmol) caused thermal hyperalgesia in igerbils which was inhibited enantioselectively by s.c. administration of CP-99,994 (ID50= 2.46 mg kg-1), but not by CP-100,263 (30 mg kg-1).6. In gerbils, intraplantar injection of formalin (0.1%) caused thermal hyperalgesia which was inhibited by CP-99,994 (ID50= 1.1 mg kg-1, s.c.). There was a nonsignificant trend for an anti-algesic effect of CP-100,236 (estimated ID50 = 8.2 mg kg-1, s.c.).7 These findings support the proposal that NK1 receptor antagonists may be useful in the clinical management of pain and reinforce the need to dissociate specific and nonspecific antinociceptive effects of available compounds.     

Effect of 17beta-estradiol on somatostatin receptor expression and inhibitory effects on growth hormone and prolactin release in rat pituitary cell cultures

Predictions of the minimal size an organism must have to swim along stimulus gradients were used to compare the relative advantages of sensory systems employing spatial (simultaneous) and temporal (sequential) gradient detection mechanisms for small free-swimming bacteria, leading to the following conclusions: 1) there are environmental conditions where spatial detection mechanisms can function for smaller organisms than can temporal mechanisms, 2) temporal mechanisms are superior (have a smaller size limit) for the difficult conditions of low concentration and shallow gradients, but 3) observed bacterial chemotaxis occurs mostly under conditions where spatial mechanisms have a smaller size limit, and 4) relevant conditions in the natural environment favor temporal mechanisms in some cases and spatial mechanisms in others. Thus, sensory ecology considerations do not preclude free-swimming bacteria from employing spatial detection mechanisms, as has been thought, and microbiologists should be on the lookout for them. If spatial mechanisms do not occur, the explanation should be sought elsewhere.     

LH-RH-dependent synthesis of protein necessary for LH release from rat pituitary glands in vitro

Anterior pituitary glands of intact diestrous and gonadectomized female rats were incubated with a supramaximally active dose of LH-RH. Puromycin (P) and cycloheximide (C) did not consistently affect the LH release from glands of ovariectomized rats. In intact rats, LH release induced by LH-RH occurred in two phases, an initial one (relatively slow rate of release) and a secondary one (high rate of release). P and C had no effect on the initial phase, but prevented the secondary one. Pre-incubation with LH-RH prevented the effect of P and C added after 4 hours. Since P and C did not affect the total amount of LH present in media and hypophyses, the LH-released during the P- and C-sensitive phase is not newly formed LH. It is concluded that LH-RH induces the synthesis of protein which is necessary for LH-RH-induced LH release. The initial phase of LH release is made possible by protein already present in the glands at the beginning of the experiment.     

Effect of GnRH antagonists on phorbol ester-induced LH release from rat pituitary gonadotrophs

We previously reported that a blockade of GnRH receptor activation inhibited the already-initiated C-kinase pathway(s). We tried to investigate whether this finding is a general phenomenon or not. In this study, we employed three GnRH antagonists, [D-Phe2,Pro3,D-Phe6]-GnRH, [Ac-D-Nal-Ala1,D-pCl-Phe2,D-Ser(Rha)6]-GnRH, and [Ac-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]-GnRH (referred to as #1-, #2-, #3-GnRH antag., respectively). Each antagonist was examined for its potency against GnRH by analyzing its inhibitory effect on LH release from pituitary gonadotrophs as well as on the increase in the cytosolic Ca2+ concentration. As a result, the #1-GnRH antag. was found to be weaker than the other two compounds. Consistent with a previous study, the #3-GnRH antag. inhibited the action of TPA on LH release. However, independently of their potency as GnRH-antagonists, the two other antagonists had no inhibitory effect on TPA-induced LH release. While it is generally accepted that the C kinase pathway plays a major role in the GnRH-induced LH release, not all GnRH antagonists can inhibit LH release by blocking the already-activated C kinase system.     

L-733,060, a novel tachykinin NK1 receptor antagonist; effects in [Ca2+]i mobilisation, cardiovascular and dural extravasation assays

Short RNA species that encompass the psi domain of the retroviral genome spontaneously form dimers in vitro, and the retroviral nucleocapsid protein activates this dimerization in vitro. Addition of gag RNA sequences downstream of the 3' end of the psi domain decreases the level of spontaneous dimerization. Here, we report the effects of RNA length on dimerization in vitro, studied with RNA fragments from Moloney murine leukaemia virus that contain the psi domain and all or part of the gag sequence. Extension of the RNA leads to progressive inhibition of the in vitro dimerization process. Sequences located downstream of the 3' end of the psi domain seem to stabilize the monomeric structures. This stabilization participates in dimerization of the RNA sequences involved in the recognition of two RNA molecules. We studied the ability of nucleocapsid protein 10 to promote dimerization of such long RNA fragments, and found that the protein greatly enhances their dimerization in vitro. We propose that nucleocapsid protein 10 stimulates the overall dimerization process by reduction of the energy barrier that must be overcome to allow dimer formation. Our results show that dimerization of RNA form Moloney murine leukaemia virus in vitro is enhanced by nucleocapsid protein 10. This finding is in agreement with the involvement of the nucleocapsid protein in RNA dimerization in vivo.     

Effects of FR173657, a non-peptide B2 antagonist, on kinin-induced hypotension, visceral and peripheral oedema formation and bronchoconstriction

The mutagenic and lethal effects of abasic sites in DNA are averted by repair initiated by 'class II' apurinic (AP) endonucleases, which cleave immediately 5'to abasic sites. We examined substrate binding by the human AP endonuclease, Ape protein (also called Hap1, Apex or Ref-1). In electrophoretic mobility-shift experiments, Ape bound synthetic DNA substrates containing single AP sites or tetrahydrofuran (F) residues. No complexes were detected with single-stranded substrates or unmodified duplex DNA. In EDTA, the concentration of Ape required to shift 50% of duplex F-DNA was approximately 50 nM, while the addition of 10 mM MgCl2 nearly eliminated detectable F-DNA@Ape complexes. Filter-binding studies demonstrated a half-life of approximately 50 s at 0 degrees C for F-DNA@Ape complexes in the presence of EDTA, and <15 s after the addition of Mg2+. The DNA recovered from F-DNA@Ape complexes was intact but was rapidly cleaved upon addition of Mg2+, which suggests that these protein-DNA complexes are on the catalytic pathway for incision. Methylation and ethylation interference experiments identified DNA contacts critical for Ape binding, and Cu-1, 10-phenanthroline footprinting suggested an Ape-induced structural distortion at the abasic site prior to cleavage.