[IMPACTCHIMIO: Development and multicentric validation of a tool for collecting pharmaceutical interventions carried out within chemotherapy reconstitution units]. / IMPACTCHIMIO : élaboration et validation multicentrique d'un outil de recueil des interventions pharmaceutiques réalisées au sein des unités de reconstitution de chimiothérapies.

OBJECTIVES: To develop and validate prospectively a specific tool for pharmaceutical interventions performed in centralized cytotoxic preparation units. METHODS: A pharmaceutical intervention is defined as a type of intervention performed in relation to a problem encountered. ImpactChimio is derived from the Act-IP® (SFPC) tool. The initial version (version 1) was developed from the pharmaceutical interventions collected over 1 year by the pilot centre. Its validation was carried out by the Delphi method via a prospective multicentric collection to assess its robustness (real life pharmaceutical interventions) and reproducibility (50 pharmaceutical interventions classified by pharmacists naive or not to the tool and study of classification divergences). RESULTS: The development of the tool (version 1) was based on the analysis of 412 pharmaceutical interventions. For its validation, 196 pharmaceutical interventions were provided by 6 centers for 5 months. The changes have been incorporated into the new versions of the tool (version 2 and version 3). Six naive and six non-naive pharmacists then tested reproducibility by reclassifying 50 selected pharmaceutical interventions into version 3. A total of 136 discrepancies (11.3 %) were found out of 1200 responses: 66 related to the problem encountered and 70 to the type of intervention. No statistically significant differences were found between naive and non-naive pharmacists. CONCLUSIONS: ImpactChimio is the first pharmaceutical interventions' specific tool for centralized cytotoxic preparation units, developed and validated by a multicentric study using the Delphi method. It makes possible to enhance the value of the analysis activity and to identify training areas for the teams.

Risk evaluation of the use of complementary and alternative medicines in cancer.

INTRODUCTION: Cancer patients use complementary and alternative medicines (CAM) to improve their well-being. Little is known about real risks. OBJECTIVE: To highlight 3 different types of axes: 1/cancer patients' perceptions concerning CAM; 2/misinformation/miscommunication about CAM; 3/CAM toxicity (direct toxicity, CAM-anticancer drugs, CAM-cancer interactions). METHOD: A questionnaire was proposed to cancer patients for 2 months. The CAM toxicity was analyzed if patients documented their drugs and CAM. RESULTS: Eighty-five patients responded: 72/85 were taking≥1CAM. In total, 95% patients were satisfied. There was an increasing CAM intake after cancer diagnosis. One hundred and seventeen different CAM were identified (63 herbs, 24 essential oils, 28 food supplements, 2 homeopathic specialities). Only 30/85 were aware CAM could interact with anticancer drugs. No other type of risk was perceived. INFORMATION SOURCES: 43/85 Internet, 38/85 general practitioner, 38/85 community pharmacist, 32/85 entourage, 25/85 other patients, 22/85 oncologist. In total, 81.3% questioned healthcare professionals (HCP) about CAM. Twelve patients noticed HCP lacked knowledge regarding CAM. The toxicity analysis was carried out for 24 patients who consumed 1 to 24CAM. In total, 133CAM were reported, including 87 different CAM. For only 43CAM/87, studies were found. All patients presented≥1risk: 14 at risk of CAM-cancer interactions, 15 of CAM-anticancer drug interactions, 21 of CAM direct toxicities. CONCLUSION: Many CAM are used by patients. The diagnosis of cancer favors their use. The risks are manifold: low perception of risk that can be induced by CAM, diverse and insecure sources of information and many potential toxicities that are not scientifically documented.

Acral lentiginous melanoma: a single-centre retrospective review of four decades in East-Central Europe.

BACKGROUND: Acral lentiginous melanoma (ALM) occurs on the palms, soles and subungual surface and has poor prognosis. It is uncommon in the Caucasian population and has remained unreported in East-Central Europe. OBJECTIVES: Our aim was to collect data from East-Central Europe by analysing the demographic and clinicopathologic features of patients with ALM and comparing data with the reports in literature. METHODS: We conducted a single-centre, retrospective review between 1976 and 2016 at one of the largest melanoma referral centres in Hungary. RESULTS: We identified 176 patients with ALM (3.83%) from 4593 patients with melanoma (mean age: 66.2 years). The tumours were mainly located on the lower extremities (88.63%). The mean Breslow tumour thickness was 3.861 mm, 37.50% of the tumours were thicker than 4.00 mm, and 71.6% exhibited microscopic ulceration. Nearly one-third of the patients underwent sentinel lymph node (SLN) biopsy, and 60.3% of the biopsies were positive for metastasis. The positive SLN status was associated with significantly thick tumours and reduced survival. Patients with ALM had 5- and 10-year overall survival rates of 60.5% and 41.6%, respectively. The mean delay in diagnosis was 18 months after the discovery of skin tumours. In multivariate analyses, age, tumour thickness and distant metastasis were independent risk factors for poor survival (P < 0.001). CONCLUSIONS: Our study, which is the first single-centre report in East-Central Europe focusing on ALM, confirms that patient and tumour characteristics and prognostic factors are similar with previous literature data involving Caucasians; however, tumour thickness and survival suggest even worse prognosis.

Rapid adaptive evolution in novel environments acts as an architect of population range expansion.

Colonization and expansion into novel landscapes determine the distribution and abundance of species in our rapidly changing ecosystems worldwide. Colonization events are crucibles for rapid evolution, but it is not known whether evolutionary changes arise mainly after successful colonization has occurred, or if evolution plays an immediate role, governing the growth and expansion speed of colonizing populations. There is evidence that spatial evolutionary processes can speed range expansion within a few generations because dispersal tendencies may evolve upwards at range edges. Additionally, rapid adaptation to a novel environment can increase population growth rates, which also promotes spread. However, the role of adaptive evolution and the relative contributions of spatial evolution and adaptation to expansion are unclear. Using a model system, red flour beetles (Tribolium castaneum), we either allowed or constrained evolution of populations colonizing a novel environment and measured population growth and spread. At the end of the experiment we assessed the fitness and dispersal tendency of individuals originating either from the core or edge of evolving populations or from nonevolving populations in a common garden. Within six generations, evolving populations grew three times larger and spread 46% faster than populations in which evolution was constrained. Increased size and expansion speed were strongly driven by adaptation, whereas spatial evolutionary processes acting on edge subpopulations contributed less. This experimental evidence demonstrates that rapid evolution drives both population growth and expansion speed and is thus crucial to consider for managing biological invasions and successfully introducing or reintroducing species for management and conservation.

In vitro skin models in the optimization of skin formulations.

BACKGROUND: Transdermal drug delivery is assumed to have a growing importance in drug development recently, thus it is crucial to optimize the penetration properties of drug into through the skin. Most of the current developments rely on the use of appropriate ex vivo animal or artificial models. However, the limited availability of human skin and the increasing restrictions in connection with animal testing encouraged the searchfor suitable artificial skin models. METHOD: For the review, we have searched the databases of scientific and medical research to collect the available publications about the in vitro skin models. Furthermore, we overviewed the methods of the DataBase service on ALternative Methods to animal experimentation (DB-ALM) database and the guidelines of Organisation for Economic Co-operation and Development (OECD). RESULTS: In vitro skin models have advantages like reproducibility, relatively low cost, easy storage, uncomplicated handling, and they offer a possibility for rapid screening and faster optimization of skin formulations. Furthermore, their composition can be easily modified which allows studying the relationship between certain pathological conditions and barrier function. However, the limitations of these models are needed to be taken into account. CONCLUSION: This review attempts to provide an overview of the most frequently used models, focusing on their limitations and advantages. Accessibility, easiness of the application, cost and the respective limitations have to be considered in order to choose the most appropriate in vitro model for the particular objective.

Adequacy of hyaluronan binding assay and a new fertility index derived from it for measuring of male fertility potential and the efficacy of supplement therapy.

The aim of the study was to statistically prove that the HBA(®) test is an efficient practical method for andrologists to determine the fertility potential as well as to measure the efficiency of oral supplement therapy in case of male infertility. In the study, 175 patients were involved and it also included the follow-up studies of 39 patients after supplement therapy. Completing the 'classic' spermatological parameters with the results of HBA(®) test, the authors have also determined a new fertility index to be used for practical rating of the measure of fertility potential. After the supplement therapy, both sperm density and hyaluronan binding capacity increased significantly. The authors are convinced that the HBA(®) analysis is an objective, standardisable test, which provides a better approach to fertility potential. This analysis enables us to detect spermatozoa that were previously misjudged as normal by morphological assay and also makes the efficiency of the therapy more measurable.

STIP1 Tissue Expression Is Associated with Survival in Chemotherapy-Treated Bladder Cancer Patients.

To optimize treatment decisions in advanced bladder cancer (BC), we aimed to assess the therapy predictive value of STIP1 with regard to cisplatin therapy. Cisplatin-based chemotherapy represents the standard first-line systemic treatment of advanced bladder cancer. Since novel immunooncologic agents are already available for cisplatin-resistant or ineligible patients, biological markers are needed for the prediction of cisplatin resistance. STIP1 expression was analyzed in paraffin-embedded bladder cancer tissue samples of 98 patients who underwent adjuvant or salvage cisplatin-based chemotherapy by using immunohistochemistry. Furthermore, pre-chemotherapy serum STIP1 concentrations were determined in 48 BC patients by ELISA. Results were correlated with the clinicopathological and follow-up data. Stronger STIP1 nuclear staining was associated with worse OS in both the whole patient group (p = 0.034) and the subgroup of patients who received at least 2 cycles of chemotherapy (p = 0.043). These correlations remained significant also in the multivariable analyses (p = 0.035 and p = 0.040). Stronger STIP1 cytoplasmatic immunostaining correlated with shorter PFS both in the whole cohort (p = 0.045) and in the subgroup of patients who received at least 2 cycles of chemotherapy (p = 0.026). Elevated STIP1 serum levels were associated with older patient's age, but we found no correlation between STIP1 serum levels and patients' outcome. Our results suggest that tissue STIP1 analysis might be used for the prediction of cisplatin-resistance in BC. In contrast, pretreatment STIP1 serum levels showed no predictive value for chemotherapy response and survival.

Melphalan potently substitutes the N-terminal Tyr of D-Ala2-Leu5-enkephalin methyl ester.

In search of an affinity label of the opioid receptor, the nitrogen mustard melphalan, Mel, was built into the peptide chain of D-Ala2-Leu5-enkephalin (DALE) methyl ester in different positions. We report now that in contrast to the previous observations that an intact Tyr in position 1 is essential for opioid activity [(1980) Annu. Rev. Pharmacol. Toxicol. 20, 81-110], substitution of Tyr by Mel did not result in a loss of the binding affinity. Mel1, Leu5-enkephalin-OMe competed for the binding sites of [3H]naloxone as potently as DALE did; IC50 values for both compounds were 50 nM. Mel substitution has led to one order potency decrease in binding to the delta-sites. 0.5-1 microM of the compound irreversibly inactivates 50% of the binding sites of [3H]naloxone, and 5-10 microM of that of [3H]DALE. These results shed new light on the structural requirements established for opioid peptides. In addition, the new derivative can be used as an affinity label of the opioid receptor.

Separation of kappa-opioid receptor subtype from frog brain.

Complete separation of the [3H]ethylketocyclazocine [( 3H]EKC) specific binding (kappa subtype) from tritiated Tyr-D-Ala2-Me-Phe4-Gly-ol5 enkephalin (DAGO) and Tyr-D-Ala2-L-Leu5-enkephalin (DALA) binding (mu-and delta-subtypes, respectively) was achieved by Sepharose-6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (s20.w) for the kappa receptor-detergent complex was 13.1 S and the corresponding Stokes radius 64 A. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas mu- and delta-specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular entity from the mu and delta receptor sites.

A monoclonal anti-idiotypic antibody to mu and delta opioid receptors.

A mouse monoclonal, anti-idiotypic, anti-opioid receptor antibody (Ab2-AOR) has been generated from monoclonal anti-morphine antibodies (Ab1). Hybridoma culture supernatants were screened by a solid phase radioimmunoassay (RIA), based on their competition with radiolabelled morphine for Ab1. One of the Ab2s that gave a positive RIA also competed at rat brain opioid receptors with tritiated opioid ligands dihydromorphine (DHM), naloxone, etorphine, Tyr-D-Ala-Gly-Phe-D-Leu (DADLE), Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE). SDS-PAGE revealed Ab2-AOR to be highly purified after successive affinity and protein A-Sepharose chromatography. Ab2-AOR at concentrations of 10-100 nM competed with both mu- and delta-selective specific ligands for brain opioid receptors. Less than 13 micrograms/ml Ab2-AOR completely inhibited specific opioid radioligand binding to both soluble and membrane-bound opioid receptors. To demonstrate its anti-delta receptor activity further, a double-antibody ELISA procedure was developed that is based on the binding of Ab2-AOR to immobilized NG 108-15 cells (which contain only delta opioid receptors). Dose-dependent, opioid peptide- and opiate alkaloid-competitive binding of Ab2-AOR-containing ascites fluid to NG 108-15 cells was observed. A mu opioid agonist effect was demonstrated for Ab2-AOR, in that it decreased by 70% [3H]thymidine incorporation into DNA of fetal brain cell aggregates. This agonist-like action of Ab2-AOR was blocked by naltrexone. The antibody bound specifically to brain tissue sections and the presence of diprenorphine blocked this interaction. Hence, an Ab2 with mu and delta specificity has been characterized.

Expression of G-protein subtypes in cultured cerebral endothelial cells.

This paper describes Western-blotting evidence for the presence of various guanine nucleotide binding proteins, G-proteins in cultured rat cerebral endothelial cells (CECs) and two immortalized cerebral endothelial cell lines, RBE4 and GP8. By using specific antibodies raised against known sequences of appropriate G-protein types that were previously characterized, we demonstrated the presence of Gsalpha, Gi2alpha, Gi3alpha, Gq/11alpha, Goalpha and Gbeta in cell lysates of primary cultures of CECs, and plasma membranes of RBE4 and GP8 cells. The appearance of Goalpha proteins in CECs might be of special importance, since they were not detected in peripheral endothelial cells in previous studies. Isoproterenol and bradykinin displayed significant, dose-dependent stimulation of [35S]GTPgammaS binding above basal values. This assay, reflecting the GDP-GTP exchange reaction on Galpha-subunits by receptor agonists, suggested that there were functional, G-protein coupled beta-adrenergic and bradykinin receptors in these systems. No significant stimulation of [35S]GTP7gammaS binding was noted with serotonin under our experimental conditions. Since stimulation of [35S]GTPgammaS binding by isoproterenol and bradykinin was additive, it was concluded that different Galpha proteins were activated by these two ligands. In analogy to other systems, activation of Gs is most likely by isoproterenol, while Gi and/or Gq/11 proteins might be activated by bradykinin receptors. The possible significance of the receptors and G-proteins detected is being discussed in the functioning of cerebral endothelium, and thus the blood-brain barrier.

Opiate receptor binding affinities of some D-amino acid substituted beta-casomorphin analogs.

beta-Casomorphin-(5) and some analogs modified by the introduction of some D-amino acids and D-pipecolic acid as well as by C-terminal amidation were tested for their affinities to mu- and delta-binding sites in rat brain membranes. The binding affinities of these compounds are compared with the known activities in the guinea pig ileum (GPI) and mouse vas deferens (MVD) test and their antinociceptive potencies in rats. The substitution of D-proline for proline in position 4 in beta-casomorphin-(5) and beta-casomorphin-(4)amide (morphiceptin) results in derivatives with very high mu-binding affinity and mu-selectivity. These affinities correspond to the respective analgesic potencies. Both binding to mu-receptors and analgesic potency are also enhanced by the introduction of D-Phe in position 3. Testing D-Ala2 substituted derivatives with respect to their ability to compete for 3H-naloxone, we observed apparent differences between the pentapeptide amides (biphasic displacement curves) and the tetrapeptide amides (monophasic displacement curves). The substitution of L-Pro2 by D-pipecolic acid yields an analog with preferential delta-receptor affinity in the organ preparations (MVD) but preferential mu-receptor affinity in brain membranes. This finding suggests a possible difference between peripheral and central mu-binding sites.

Long-lasting antinociceptive effect of DAMGO chloromethyl ketone in rats.

Previously, the opioid peptide Tyr-D-Ala-Gly-(NMe)Phe-CH2Cl (DAMCK) has been shown to bind irreversibly to mu opioid receptors in vitro. In the present work, the antinociceptive effect of DAMCK has been evaluated. Rats treated systemically with DAMCK (1-100 pg/kg) displayed a dose-dependent increase in tail-flick analgesia that peaked by 15 min, then stayed about the same until 60 min, followed by some decrease over time. Higher doses of DAMCK (10 ng/kg-100 microg/kg) produced a near-maximal antinociceptive effect that remained stable for 4 h. Significant antinociception was still detected 8 h, but not 24 h postinjection. In comparison, the parent peptide DAMGO (Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol) reached maximal effect by about 30 min, followed by a rapid cessation of its antinociceptive response. Naloxone administered before DAMCK antagonized the antinociceptive response of DAMCK, indicating that it was mediated via opioid receptors. Naloxone administered 45 min after DAMCK attenuated the tail-flick response to some extent, but a substantial part (40-60% depending on the peptide concentration and evaluation time) remained unaffected. Central administration of DAMCK also elicited time- and concentration-dependent, profound, opioid receptor mediated, apparently irreversible antinociception.

Synthesis and opioid binding activity of dermorphin analogues containing cyclic beta-amino acids.

In the present work, eight conformationally constrained analogues of the mu specific opioid peptide dermorphin were synthesized by replacing D-Ala2 with stereoisomers of beta-amino-cycloalkane or cycloalkene carboxylic acids. The resulting peptides were tested for their potency to mu and delta opioid binding sites of rat brain membranes labelled with [3H]Tyr1-D-Ala2-MePhe4-Gly-ol, [3H]DAMGO and [3H]Ile5,6deltorphin, respectively. All of the new derivatives displayed highly attenuated binding to both receptor types, albeit the decrease in their potency seemed to be less in the case of delta binding. Trans position of the beta-amino groups resulted in higher binding affinities than that of the corresponding cis isomers, the latter being more flexible than the former. It is concluded that conformational constraints caused either by a rigid ring structure or cis isomers instead of D-Ala2 in dermorphin-derived peptides are unfavourable for binding activity to either opioid receptors. We propose that interaction of the larger heptapeptide derivatives of dermorphins with the mu receptor is distinct from that of the tetrapeptide morphiceptin.

Enkephalin photoaffinity probes: synthesis and binding properties.

The diazomethyl ketone derivative of D-Ala2-Leu-enkephalin and Leu-enkephalin were synthesized. Replacement of the C-terminal carboxyl group with CO-CHN2 resulted in a potency decrease, the new compounds display micromolar affinities to 3H-naloxone and 3H-DALE binding sites. Photolysis of the ligands bound to rat brain membranes resulted in an approximately 30% irreversible loss of the receptors. Photoinactivation was prevented by the opiate antagonist, naloxone, thus providing that the ligands are specific photoaffinity probes of the opioid receptors.

Purification of a kappa-opioid receptor subtype from frog brain.

A kappa-opioid receptor subtype was purified from a digitonin solubilized preparation of frog brain membranes using affinity chromatography. The affinity resin was prepared by coupling D-Ala2-Leu5-enkephalin to Sepharose-6B matrix. After elution of the receptor by 50 mumol naloxone, the kappa-subtype was separated from the mu- and delta-subtypes by gel permeation chromatography on Sepharose-6B. The purified receptor binds 3,900 pmol [3H]-ethylketocyclazocine per mg protein (a 4,300-fold purification over the membrane-bound receptor) with a KD of 8.3 nM. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows two bands (Mr 65,000 and 58,000) in sodium dodecyl sulfate gel electrophoresis.

Irreversible labelling of rat brain opioid receptors by enkephalin chloromethyl ketones.

Chloromethyl ketone derivatives of leucine enkephalin (LE), D-Ala2-Leu5-enkephalin (DALE) and D-Ala2-D-Leu5-enkephalin (DADLE) were synthesized. They all show high affinity for rat brain opioid binding sites. Preincubation of the membrane fraction with enkephalin chloromethyl ketones causes a significant inhibition of /3H/-naloxone binding which cannot be reversed by extensive washing. It was found that the irreversible inhibition is selective for the high affinity (KD less than 1 nM) /3H/-naloxone binding site (putative mu-1 site). The irreversible blockade of opioid binding was partially protected by opiate alkaloids and opioid peptides, suggesting that non-specific labelling also occurs. Affinity of enkephalin chloromethyl ketones toward the mu sites is greater than that of the parent compounds. It was also found that the covalent inhibition of mu sites (/3H/-dihydromorphine and /3H/-DAGO binding) is more effective than that of delta sites (/3H/-DALE binding). We conclude that these chloromethyl ketone derivatives can be used as affinity labels for the opioid receptors, allowing us to study the structure of the mu receptor subtype.

Differential ontogeny of divalent cation effects on rat brain delta-, mu-, and kappa-opioid receptor binding.

The effect of the divalent cations, Mn2+, Mg2+ and Ca2+ on rat forebrain delta-, mu- and kappa-receptor binding was examined during postnatal development. It was found that delta-receptor binding, assessed with [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) (+ 10 nM D-Ala2- MePhe4-Gly-ol5-enkephalin (DAMGE)), was stimulated by the 3 cations in a dose- and developmental time-dependent manner. delta-Binding was most sensitive to the cations during the first week postnatal, prior to the appearance of high-affinity delta-binding. In contrast, inhibition of mu-receptor binding ([3H]DAMGE) by divalent cations appeared early in development and remained constant throughout the postnatal period. Divalent cation inhibition of kappa-binding ([3H]ethylketocyclazocine ([3H]EKC) + 100 nM DAMGE and 100 nM DADLE) appeared after the second week postnatal. These results demonstrate that the characteristics and postnatal development of divalent cation modulation of mu-, delta- and kappa-binding is distinctly different. Thus, the neonate may be a good model system to examine the binding properties and functions of delta- and kappa-receptor subtypes.

Exogenous cholecystokinin (CCK) reduces neonatal rat brain opioid receptor density and CCK levels.

Newborn rats were given saline or cholecystokinin8 (CCK8) (5 micrograms/kg, twice daily) i.p. for 3 weeks. On day 21, effects on brain development were assessed. CCK-like immunoreactivity was measured in 7 brain regions; a small (12-18%) but significant decrease in endogenous levels of this peptide was detected in cerebral cortex, medulla and pons of the CCK-treated rats. Morphometric measurements revealed a slight reduction in thickness of most cerebral cortical sections within the CCK-treated group. The area of a midsagittal section of the cerebellum was unchanged except for the Purkinje/granule cell layer, which was smaller in CCK-treated animals. Levels of mu-, delta- and kappa-opioid receptors were estimated by homologous displacement binding assays using selective radioligands. The CCK treatment resulted in a significant decrease in levels of mu- (11%) and delta- (13%)-sites in the cerebral cortex. Neither binding affinities nor kappa-receptor densities were altered. Other animals received the same treatment regimens for 21 days and were maintained for an additional 29 days without treatment; these rats had reductions only in cortical mu-sites (15%). Chronic intraventricular administration of CCK (0.1 microgram/h) to adult rats did not elicit a similar down-regulation of cortical mu or delta receptors, suggesting that the effects observed in neonates reflected developmental processes.

Receptor-mediated activation of G-proteins by kappa opioid agonists in frog (Rana esculenta) brain membranes.

This study delineates the heterotrimeric guanine nucleotide binding regulatory protein (G-protein) types in frog (Rana esculenta) brain membranes and their activation by kappa opioid agonists. Ethylketocyclazocine (EKC), trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)b enzeneacetamide (U-50,488) and bremazocine displayed dose-dependent, norbinaltorphimine-reversible stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding in crude membrane preparations. G-proteins were identified by Western-blotting using previously characterized specific antisera that were generated against mammalian G-protein alpha-subunits and beta-subunits. A photoreactive guanosine 5'-triphosphate (GTP) analog, [alpha-32P]GTP azidoanilide ([alpha-32P]AA-GTP) irreversibly labeled four proteins in the molecular weight range of 39-43 kDa. Ethylketocyclazocine and U-50,488 stimulated photolabelling of these proteins among which the 39 kDa band comigrated with the protein specifically labelled with the alpha(i2) antibody and the 40 kDa band was identified as alpha(o1). The other two bands were also stained with the alpha(common) antibody, but were not further identified. These results suggest that the endogenously expressed kappa opioid receptors that are present in frog brain interact with multiple G-proteins in situ. Furthermore, the structure of most G-proteins seems to be well preserved during phylogenesis.