Urinary tract infections caused by anaerobic bacteria. Utility of anaerobic urine culture.

Urinary tract infections (UTIs) caused by anaerobic bacteria have scarcely been reported. Since anaerobic bacteria are commensals of the genitourinary tract, their presence in a urine sample adds ambiguity in making a definitive diagnosis of anaerobic UTI. It is well known that standard urine culture is the gold standard method for the detection, identification, and antimicrobial susceptibility testing of uropathogens. Nonetheless, both the difficulties in establishing them as pathogens and the scarcity of reported anaerobic UTI cases led to the discontinuation of routine urine culture under an anaerobic atmosphere (UCAA). On the other hand, it is important to emphasize that culture-independent methods, such as proteomics and molecular technics, may detect anaerobes directly on a urine sample. Anaerobes are not included in guidelines for the diagnosis and management of UTIs. At the same time, as fastidious uropathogens and antibiotic resistance become more common, accurate pathogen identification becomes even more important for effective UTI treatment. As a result, we conducted a review of the clinical context, pathogen antimicrobial susceptibility, and treatment of patients with anaerobic UTIs. Because UCAA is a contentious topic, we narrowed our search to cases with both negative standard urine culture and positive UCAA.

Peptostreptococcus anaerobius: Pathogenicity, identification, and antimicrobial susceptibility. Review of monobacterial infections and addition of a case of urinary tract infection directly identified from a urine sample by MALDI-TOF MS.

Peptostreptococcus anaerobius is a gram-positive anaerobic coccus (GPAC) found in the gastrointestinal and vaginal microbiota. The organism is mainly found in polymicrobial and scarcely in monobacterial infections such as prosthetic and native endocarditis. Anaerobic bacteria have rarely been reported as the cause of urinary tract infection (UTI). Although GPAC are susceptible to most antimicrobials used against anaerobic infections, P. anaerobius has shown to be more resistant. Herein, we report a case of UTI caused by P. anaerobius from a 62-year-old man with a history of urological disease. Surprisingly, the microorganism was directly identified by Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) from the urine sample. The isolate was successfully identified by phenotypic methods, MALDI-TOF MS, and 16S rRNA gene sequencing. P. anaerobius showed no ß-lactamase-producing activity, was resistant to penicillin, ampicillin, ciprofloxacin and levofloxacin, and displayed intermediate susceptibility to ampicillin-sulbactam and amoxicillin-clavulanic acid. Successful treatment was achieved with oral amoxicillin-clavulanic acid. Antimicrobial susceptibility testing (AST) should be performed on P. anaerobius isolates due to their unpredictable AST patterns and because empirically administered antimicrobial agents may not be active. This report shows that MALDI-TOF MS, directly used in urine specimens, may be a quick option to diagnose UTI caused by P. anaerobius or other anaerobic bacteria. This review is a compilation of monobacterial infections caused by P. anaerobius published in the literature, their pathogenicity, identification, and data about the antimicrobial susceptibility of P. anaerobius.

Intra-peritoneal abscess after an abdominal hysterectomy involving Cutibacterium avidum (former Propionibacterium avidum) highly resistant to clindamycin.

Cutibacterium avidum is a gram-positive anaerobic rod belonging to the cutaneous group of human bacteria with preferential colonization of sweat glands in moist areas. The microorganism rarely cause disease, generally delayed prosthetic joint infections (PJIs). We describe the second case of intraperitoneal abscess by C. avidum after an abdominal surgery in an obese female patient and the first case after a non-prosthetic abdominal surgery due to a highly clindamycin resistant strain in a patient with underling conditions. The patient was successfully treated with surgical drainage and beta-lactam antibiotics. Although rare and apparently non-pathogenic, C. avidum may be involved in infections, especially in some high-risk patients with obesity who have undergone surgical incision involving deep folder of the skin. The microorganism was identified by phenotypic methods, MALDI-TOF MS and 16S rRNA gene sequencing. Susceptibility test should be performed in C. avidum because high level resistance to clindamycin could be present. We present a literature review of C. avidum infections.

Survey of Aspergillus section Flavi presence in agricultural soils and effect of glyphosate on nontoxigenic A. flavus growth on soil-based medium.

AIMS: To evaluate the cultivable mycobiota from agricultural soils exposed to pesticides, the aflatoxigenic capacity of Aspergillus section Flavi strains and the effect of glyphosate on lag phase and growth rates of native nontoxigenic Aspergillus flavus under different water potential (MPa) conditions on soil-based medium. METHODS AND RESULTS: Culturable mycobiota analysis from different agricultural soils was performed by the surface spread method. The effect of glyphosate (0-20 mmol l(-1)) on the growth of A. flavus strains was evaluated on a soil extract solid medium. Mycobiota analysis of crop soils showed the presence of twenty-one genera of filamentous fungi. Aspergillus flavus and Aspergillus niger aggregate strains were isolated from the three soil types. Ninety-two per cent of A. flavus strains were toxigenic. In vitro assay results showed that at -0·70 MPa, a significant increase in growth rate in all strains was recorded at 5 and 20 mmol l(-1) of glyphosate. At -2·78 MPa, this parameter remained constant at all glyphosate concentrations, except in GM4 strain where an increase in growth rate was recorded with increasing pesticide concentrations. At -7·06 MPa, a significant increase in growth rate has also been observed in GM 3 strain with 5 mmol l(-1) and in GM 4 strain with 10 and 20 mmol l(-1). CONCLUSIONS: This study showed that the imperfecti fungi Aspergillus spp., Penicillium spp., Trichoderma spp., Cladosporium spp. and Paecilomyces spp. are isolated as prevalent groups in agricultural soil exposed to pesticides, and the capacity of nontoxigenic A. flavus strains to tolerate different glyphosate concentrations under different water potential (MPa) conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript makes a contribution to the knowledge of cultivable fungal populations from agricultural soils exposed to pesticides and the glyphosate tolerance of A. flavus strains.

Evaluation of the Andromas matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of aerobically growing Gram-positive bacilli.

Matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

Food-grade antioxidants and antimicrobials to control growth and ochratoxin a production by Aspergillus section Nigri on peanut kernels.

Each year, a significant portion of the peanuts produced cannot be marketed because of fungal disease at the postharvest stage and mycotoxin contamination. Antioxidants could be used as an alternative to fungicides to control ochratoxigenic fungi in peanuts during storage. This study was carried out to determine the effect of the antioxidant butylated hydroxyanisole (BHA) and the antimicrobial propyl paraben (PP) on the lag phase before growth, growth rate, and ochratoxin A (OTA) production by Aspergillus section Nigri strains in peanut kernels under different conditions of water activity (aw) and temperature. At 20 mM/g BHA, 18 degrees C, and 0.93 aw, complete inhibition of growth occurred. For PP, there was no growth at 20 mM/g, 18 degrees C, and 0.93, 0.95, and 0.98 aw. BHA at 20 mM/g inhibited OTA production in peanuts by Aspergillus carbonarius and Aspergillus niger aggregate strains at 0.93 aw and 18 degrees C. PP at 20 mM/g completely inhibited OTA production at 18 degrees C. The results of this work suggest that PP is more appropriate than BHA for controlling growth and OTA production by Aspergillus section Nigri species in peanut kernels.

Clostridium ramosum rapidly identified by MALDI-TOF MS. A rare gram-variable agent of bacteraemia.

Clostridium ramosum is an enteric anaerobic, endospore-forming, gram-positive rod with a low GC content that is rarely associated with disease in humans. We present a case of C. ramosum bacteraemia. To the best of our knowledge, this is the second case of C. ramosum bacteraemia in an elderly patient presenting with fever, abdominal pain and bilious emesis. We highlight the Gram stain variability, the lack of visualization of spores and the atypical morphology of the colonies that showed C. ramosum in a polymicrobial presentation that initially appeared to show monomicrobial bacteraemia. The microorganism was rapidly identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We present a comprehensive literature review of 32 cases of clinical infections by C. ramosum in which we describe, if available, sex, age, clinical symptoms, predisposing conditions, other organisms present in the blood culture, other samples with C. ramosum , identification methodology, treatment and outcome.

Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist.

SR 121463A, a potent and selective, orally active, nonpeptide vasopressin V2 receptor antagonist, has been characterized in several in vitro and in vivo models. This compound displayed highly competitive and selective affinity for V2 receptors in rat, bovine and human kidney (0.6 < or = Ki [nM] < or = 4.1). In this latter preparation, SR 121463A potently antagonized arginine vasopressin (AVP)-stimulated adenylyl cyclase activity (Ki = 0.26+/-0.04 nM) without any intrinsic agonistic effect. In autoradiographic experiments performed in rat kidney sections, SR 121463A displaced [3H]AVP labeling especially in the medullo-papillary region and confirmed that it is a suitable tool for mapping V2 receptors. In comparison, the nonpeptide V2 antagonist, OPC-31260, showed much lower affinity for animal and human renal V2 receptors and lower efficacy to inhibit vasopressin-stimulated adenylyl cyclase (Ki in the 10 nanomolar range). Moreover, OPC-31260 exhibited a poor V2 selectivity profile and can be considered as a V2/V1a ligand. In normally hydrated conscious rats, SR 121463A induced powerful aquaresis after intravenous (0.003-0.3 mg/kg) or oral (0.03-10 mg/kg) administration. The effect was dose-dependent and lasted about 6 hours at the dose of 3 mg/kg p.o. OPC-31260 had a similar aquaretic profile but with markedly lower oral efficacy. The action of SR 121463A was purely aquaretic with no changes in urine Na+ and K+ excretions unlike that of known diuretic agents such as furosemide or hydrochlorothiazide. In addition, no antidiuretic properties have been detected with SR 121463A in vasopressin-deficient Brattleboro rats. Thus, SR 121463A is the most potent and selective, orally active V2 antagonist yet described and could be a powerful tool for exploring V2 receptors and the therapeutical usefulness of V2 blocker aquaretic agents in water-retaining diseases.

Ochratoxin A production in relation to ecophysiological factors by Aspergillus section Nigri strains isolated from different substrates in Argentina.

Contamination of foodstuff with mycotoxins such as ochratoxins is a major matter of concern for human and animal health. In Aspergillus species, ochratoxin synthesis depends on several environmental factors. The aims of this work were to evaluate the effect of water activity (0.995-0.85), temperature (15, 25 and 30 degrees C), incubation time (7, 14 and 21 days) and their interactions on OTA production on peanut, maize kernels, dried grapes and coffee beans meal extract agar medium by eight strains of Aspergillus section Nigri isolated from human food in Argentina. The optimum temperature for OTA production was 25 or 30 degrees C depending on the strains assayed, in most cases the highest OTA levels were achieved after 7 days of incubation, whereas this situation occurred at 15 degrees C after 14 days of incubation for only one strain. The maximum OTA level was obtained at earlier growth states when incubation temperature increased. In general, OTA concentration increased as water activity (a(W)) increased with no significant production at 0.85-0.91 a(W) under all temperature levels tested. Production occurred over a range of temperatures (15-30 degrees C) with optimum production at 30 degrees C depending on a(W) assayed. The knowledge of Aspergillus section Nigri ecophysiology is critical in the development and prediction of the risk models of raw material and final product contamination by these species under fluctuating and interacting environmental parameters.

Unusual presentations of Comamonas kerstersii infection.

The association of Comamonas kerstersii with peritonitis resulting from the presence of perforated appendix has previously been described by our research team. In the present study, we describe the isolation of this microorganism from two forms of unusual presentations of C. kerstersii infection not previously described in the literature: localized intra-abdominal infection (psoas abscess) and pelvic peritonitis.

[Phenotypic and genotypic characterization of resistance to third-generation cephalosporins in Enterobacter spp]. / Caracterización fenotípica y genotípica de la resistencia enzimática a las cefalosporinas de tercera generación en Enterobacter spp.

Enterobacter spp. are becoming increasingly frequent nosocomial pathogens with multiple resistance mechanism to beta-lactam antibiotics. We carried out the phenotypic and genotypic characterization of beta-lactamases in 27 Enterobacter spp. (25 Enterobacter cloacae y 2 Enterobacter aerogenes), as well as the ability of different extended spectrum-lactamase (ESBL) screening methods. Resistance to third generation cephalosporins was observed in 15/27 (63%) isolates. Twelve resistant isolates produced high level chromosomal encoded AmpC beta-lactamase; 6 of them were also producers of PER-2. Resistance to third generation cephalosporins in the remaining 3 isolates was due to the presence of ESBLs, PER-2 in 2 cases, and CTX-M-2 in the other. Only CTX-M-2 production was detected with all tested cephalosporins using difusion synergy tests, while cefepime improved ESBLs detection in 7/8 PER-2 producers, 4/8 in the inhibitor approximation test and 7/8 with double disk test using cefepime containing disk with and without clavulanic acid. Dilution method, including cephalosporins with and without the inhibitor detected 1/9 ESBLs producers.

Vasopressin stimulates steroid secretion in human adrenal glands: comparison with angiotensin-II effect.

Autoradiographic experiments using iodinated vasopressin analog revealed the presence of specific vasopressin-binding sites in the human adrenal cortex (zona glomerulosa and zona fasciculata). These receptors exhibited a good affinity for arginine vasopressin (3.3 nM), with classical V1a pharmacology and densities of 65 and 135 fmol/mg protein-enriched membranes from zona glomerulosa and fasciculata, respectively. Vasopressin receptors present in both glomerulosa and fasciculata cell-enriched primary cultures were coupled to phospholipase C (ED50, 0.9 and 1.8 nM; maximal stimulation, 4.3- and 5.8-fold, respectively). Vasopressin also stimulated an increase in intracellular calcium through at least two distinct mechanisms: the mobilization of intracellular pools via vasopressin-stimulated inositol phosphate accumulation and the activation of calcium influx. In glomerulosa cell-enriched primary cultures, vasopressin increased aldosterone secretion (ED50, 0.4 nM; maximal stimulation, 2.5-fold) and was found to be as potent as angiotensin-II in stimulating aldosterone secretion, phosphoinositide turnover, and calcium mobilization. In fasciculata cells, vasopressin and angiotensin-II were also able to stimulate cortisol secretion and inositol phosphate accumulation. Moreover, perifusion experiments demonstrated that vasopressin was released from the adrenal medulla. Together, these results indicate that vasopressin can be considered a potent paracrine modulator of adrenal steroid secretion in man.

[3H]vasopressin binding to rat hippocampal synaptic plasma membrane. Kinetic and pharmacological characterization.

Characterization of specific vasopressin binding sites to rat hippocampal membranes has been assayed using tritiated lysine-vasopressin labelled on the tyrosyl residue. At 30 degrees C specific [3H]vasopressin binding was saturable. The estimated equilibrium dissociation constant was 7.1 nM, the mean maximal binding capacity was 78 fmol/mg protein. Arginine-vasopressin has a high affinity (Kd = 2.8 nM) and dDAVP has a low affinity (Kd = 249 nM) for hippocampal synaptic membranes. (OH)AVP and Phe2Orn8VT are at least as active as AVP in inhibiting [3H]vasopressin binding. Adenylate cyclase was activated by VIP and inhibited by PIA, but not affected by lysine-vasopressin.

A radioiodinated linear vasopressin antagonist: a ligand with high affinity and specificity for V1a receptors.

A linear vasopressin antagonist, Phaa-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (Linear AVP Antag) (Phaa = Phenylacetyl), was monoiodinated at the phenyl moiety of the tyrosylamide residue at position 9. This antagonist appeared to be a highly potent anti-vasopressor peptide with a pA2 value in vivo of 8.94. It was demonstrated to bind to rat liver membrane preparations with a very high affinity (Kd = 0.06 nM). The affinity for the rat uterus oxytocin receptor was lower (Ki = 2.1 nM), and affinities for the rat kidney- and adenohypophysis-vasopressin receptors were much lower (Ki = 47 nM and 92 nM, respectively), resulting in a highly specific vasopressin V1a receptor ligand. Autoradiographical studies using rat brain slices showed that this ligand is a good tool for studies on vasopressin receptor localization and characterization.

The D136A mutation of the V2 vasopressin receptor induces a constitutive activity which permits discrimination between antagonists with partial agonist and inverse agonist activities.

The substitution, in the human V2 vasopressin receptor, of the aspartate at position 136 by alanine leads to agonist-independent activation of this mutant V2 receptor. Pharmacological studies of the D136A V2 receptor helped us in characterizing different V2 receptor antagonists. SR-121463A and OPC-31260, two non-peptide antagonists, behaved as inverse agonists, while two cyclic peptides d(CH2)5[D-Tyr(Et)2,-Val4,Tyr-NH(2)9]AVP and d(CH2)5[D-Ile2,Ile4,Tyr-NH(2)9]AVP known to be V2 antagonists, demonstrated clear partial agonist properties. The finding of a constitutively activated human V2 receptor represents a useful tool in characterizing V2 receptor antagonist ligands.

Two aromatic residues regulate the response of the human oxytocin receptor to the partial agonist arginine vasopressin.

We investigated the mechanisms that regulate the efficacy of agonists in the arginine-vasopressin (AVP)/oxytocin (OT) receptor system. In this paper, we present evidence that AVP, a full agonist of the vasopressin receptors, acts as a partial agonist on the oxytocin receptor. We also found that AVP becomes a full agonist when two aromatic residues of the oxytocin receptor are replaced by the residues present at equivalent positions in the vasopressin receptor subtypes. Our results indicate that these two residues modulate the response of the oxytocin receptor to the partial agonist AVP.

125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9]AVP: iodination and binding characteristics of a vasopressin receptor ligand.

A radioiodinated vasopressin antagonist, d(CH2)5[Tyr(NH2)9]AVP has been prepared. Iodination was carried out at the phenyl moiety of the tyrosylamide residue at position 9, followed by HPLC purification. Non-radiolabelled monoiodinated antagonist was used as a reference for identification. 125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9]AVP binding appeared to take place with a dissociation constant of 0.28 +/- 0.09 nM (Kd +/- SD) to V1 vasopressin receptors on rat liver membranes.

Vasopressin binding in the cerebral cortex of the Mongolian gerbil is reduced by transient cerebral ischemia.

In Mongolian gerbils, the content of vasopressin in the cerebral cortex, the striatum, and the hypothalamus is increased after induction of acute cerebral ischemia. We used an iodinated vasopressin analogue and light microscopic autoradiography to study the distribution of vasopressin V1 receptors in the brain of adult male gerbils and to evaluate the effects of a transient bilateral cerebral ischemia (6 minutes) on the density of this receptor population. The animals were killed immediately or 10, 30, or 100 hours after transient bilateral occlusion of the common carotid arteries. In control animals, specific [125I]-VPA binding sites were present in various structures of the brain (olfactory bulb, anterior olfactory nucleus, lateral septum, bed nucleus of the stria terminalis, median preoptic area, ventral pallidum, substantia innominata, amygdala, thalamus, hypothalamic mammillary nuclei, superior colliculus, subiculum, central gray, nucleus of the solitary tract, hypoglossal nucleus). The strongest labeling was detected in the cerebral cortex, layers 5-6. After 30-100 hours of survival time following ischemia there was a marked decrease in [125I]-VPA binding site density in these cerebral cortex layers. To a lesser degree, a decrease was also detected in the lateral septal nucleus. In contrast, labeling in other noncortical structures remained unchanged. All animals with 100 hours recovery showed a loss of cells in hippocampus (CA1 layer) and striatum. In addition, ischemia induced concomitant and proliferative changes in cortical and hippocampal astrocytes assessed by glial fibrillary acid protein immunoreactivity. These observations indicate a role for vasopressin in the cerebral cortex either on neurons or on glial cells and the modulation of vasopressin receptor expression by transient cerebral ischemia.

A new series of photoactivatable and iodinatable linear vasopressin antagonists.

A series of new linear photoactivatable and iodinatable antagonists of the neuropeptidic hormone vasopressin was designed and synthesized by a combination of PyBOP-mediated Boc/solid-phase peptide synthesis and solution synthesis approaches. These were based on modifications of a previously reported potent and selective antagonist of the vasopressor response (V1a receptor) to [arginine]vasopressin, phenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2. (Azidophenyl)alkyl substitutions, of the general structure N3-C6H4(CH2)nCO (n = 0, 1, 2, or 3), were employed in position 1. The seven new analogues are 4-N3-C6H4CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (3), 3-N3-C6H4CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (12), 4-N3-C6H4CH2-CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (13), 3-N3-C6H4CH2CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (14), 4-N3-C6H4(CH2)2CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (15), 3-N3-C6H4(CH2)2CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (16), 4-N3-C6H4-(CH2)3CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (17). All analogues were tested for their affinity of the rat hepatic V1a receptor. Analogues 3 and 12 have a low affinity (Ki approximately 20 nM) and analogues 13-17 show a high affinity (Ki between 0.04 and 0.3 nM). The affinity values appear to be mainly a function of the alkyl chain length and to a lesser extent of the meta or para position of the azido group on the aromatic ring. Analogues 13-17 were iodinated on the Tyr-9 residue, giving compounds 18-22. All these five iodinated derivatives exhibited Ki values of 0.2-1 nM for rat liver membranes. Their affinities for oxytocin and renal V2 vasopressin receptors were much lower. Moreover, all analogues completely antagonized the vasopressin-stimulated inositol phosphates production in WRK1 cells and were devoided of any agonistic potency. Preliminary covalent binding studies showed improved covalent yields as compared to any previously reported results. They are very promising candidates as potential high-affinity, highly selective, photosensitive ligands for the V1a receptor. They could serve as a useful pharmacological tools for studies on the vasopressin binding site.

Design, synthesis and pharmacological characterization of a potent radioiodinated and photoactivatable peptidic oxytocin antagonist.

Using a segment strategy, we have synthesized four iodinated photoactivatable cyclic peptidic ligands of oxytocin, bearing a beta-mercapto-betabeta-cyclopentamethylene propionic group (Pmp) on their N-terminus. All the syntheses were RP-HPLC monitored, and the compounds were HPLC purified. They were characterized by 1H NMR, MALDI-TOF, or FAB mass spectrometries. The affinities of Pmp-Tyr(Me)-Ile-Thr-Asn-Cys-Gly-Orn-Phe(3I,4N3)-NH2 (20), Pmp-Tyr-Ile-Thr-Asn-Cys-Gly-Orn-Phe(3I,4N3)-NH2 (21), Pmp-Tyr(Me)-Ile-Thr-Asn-Cys-Pro-Orn-Phe(3I,4N3)-NH2 (22), and Pmp-Tyr-Ile-Thr-Asn-Cys-Pro-Orn-Phe(3I,4N3)-NH2 (23) were evaluated as inhibition constants (K(i), in nM) for the human oxytocin receptor expressed in Chinese hamster ovary cells by displacement of a radioiodinated disulfide-cyclized antagonist (Elands et al. Eur. J. Pharmacol. 1987, 147, 197-207). The most potent of them, compound 22, was synthesized by another method in order to allow its radiolabeling by 125I. Its dissociation constant (K(d)) for the human oxytocin receptor, directly measured in saturation studies, was 0.25 +/- 0.04 nM, and its antagonist properties were determined by inactivation of phospholipase C, thus obtaining an inactivation constant (K(inact)) of 0.18 +/- 0.02 nM, evaluated by inositol phosphate accumulation. This compound is a very good tool for the mapping of peptidic antagonist binding sites in the human oxytocin receptor.