Search

Listed as ‘threatened’ under the US Endangered Species Act in 1993, the spectacled eider Somateria fischeri population in western Alaska, USA, has since rebounded, prompting an assessment of their suitability for delisting. This assessment, however, is limited by aerial-based population estimates that are incompletely corrected for unobserved eiders. Notably, aerial counts of eiders are corrected with a visibility correction factor (VCF), calculated as the ratio of ground-based nest counts to aerial pair counts, which disregards spatial variation in eider density. Accordingly, we (1) stratified our study area into zones of low, medium, and high eider density, (2) developed density-adjusted VCFs for each stratum, (3) evaluated the influence of several ecological factors on VCFs, and (4) estimated eider population size using our density-adjusted VCFs. For the low-density stratum, we estimated a VCF (± SE) of 1.35 ± 0.15, indicating that aerial counts of eider pairs closely matched ground counts of nests. In medium and high-density strata, VCFs increased to 2.46 ± 0.17 and 3.09 ± 0.19, respectively, suggesting that aerial detection decreased as eider densities increased. VCFs also increased for surveys that occurred late relative to nest initiation. Population estimates produced with our density-adjusted VCFs were 42% (5580 eiders) lower, on average, than those produced with the traditional VCF, which is currently used. Such large differences underscore the importance of accounting for density when correcting counts for incomplete detection, and, for threatened species such as spectacled eiders, may determine whether populations retain protected status.

Significance: A shortcoming of the routine clinical use of diffuse optics (DO) in the injured head has been that the results from commercial near-infrared spectroscopy-based devices are not reproducible, often give physiologically invalid values, and differ among systems. Besides the limitations due to the physics of continuous-wave light sources, one culprit is the head heterogeneity and the underlying morphological and functional abnormalities of the probed tissue., Aim: The aim is to investigate the effect that different tissue alterations in the damaged head have on DO signals and provide guidelines to avoid data misinterpretation., Approach: DO measurements and computed tomography scans were acquired on brain-injured patients. The relationship between the signals and the underlying tissue types was classified on a case-by-case basis., Results: Examples and suggestions to establish quality control routines were provided. The findings suggested guidelines for carrying out DO measurements and speculations toward improved devices., Conclusions: We advocate for the standardization of the DO measurements to secure a role for DO in neurocritical care. We suggest that blind measurements are unacceptably problematic due to confounding effects and care using a priori and a posteriori quality control routines that go beyond an assessment of the signal-to-noise ratio that is typically utilized., (© 2024 The Authors.)

The facultative intracellular bacterium Listeria (L.) monocytogenes may cause severe diseases in humans and animals. The control of listeriosis/L. monocytogenes requires the concerted action of cells of the innate and adaptive immune systems. In this regard, cell-intrinsic immunity of infected cells, activated by the immune responses, is crucial for the control and elimination intracellular L. monocytogenes. Both the immune response against L. monocytogenes and cell intrinsic pathogen control are critically regulated by post-translational modifications exerted by the host ubiquitin system and ubiquitin-like modifiers (Ubls). In this review, we discuss our current understanding of the role of the ubiquitin system and Ubls in listeriosis, as well as future directions of research., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Hyperventilation (HV) therapy uses vasoconstriction to reduce intracranial pressure (ICP) by reducing cerebral blood volume. However, as HV also lowers cerebral blood flow (CBF), it may provoke misery perfusion (MP), in which the decrease in CBF is coupled with increased oxygen extraction fraction (OEF). MP may rapidly lead to the exhaustion of brain energy metabolites, making the brain vulnerable to ischemia. MP is difficult to detect at the bedside, which is where transcranial hybrid, near-infrared spectroscopies are promising because they non-invasively measure OEF and CBF. We have tested this technology during HV (∼30 min) with bilateral, frontal lobe monitoring to assess MP in 27 sessions in 18 patients with traumatic brain injury. In this study, HV did not lead to MP at a group level ( p > 0.05). However, a statistical approach yielded 89 events with a high probability of MP in 19 sessions. We have characterized each statistically significant event in detail and its possible relationship to clinical and radiological status (decompressive craniectomy and presence of a cerebral lesion), without detecting any statistically significant difference ( p > 0.05). However, MP detection stresses the need for personalized, real-time assessment in future clinical trials with HV, in order to provide an optimal evaluation of the risk-benefit balance of HV. Our study provides pilot data demonstrating that bedside transcranial hybrid near-infrared spectroscopies could be utilized to assess potential MP.

Significance: Benign external hydrocephalus (BEH) is considered a self-limiting pathology with a good prognosis. However, some children present a pathological intracranial pressure (ICP) characterized by quantitative and qualitative alterations (the so-called B-waves) that can lead to neurological sequelae., Aim: Our purpose was to evaluate whether there were cerebral hemodynamic changes associated with ICP B-waves that could be evaluated with noninvasive neuromonitoring., Approach: We recruited eleven patients (median age 16 months, range 7 to 55 months) with BEH and an unfavorable evolution requiring ICP monitoring. Bedside, nocturnal monitoring using near-infrared time-resolved and diffuse correlation spectroscopies synchronized to the clinical monitoring was performed., Results: By focusing on the timing of different ICP patterns that were identified manually by clinicians, we detected significant tissue oxygen saturation ( StO 2 ) changes ( p = 0.002 ) and blood flow index (BFI) variability ( p = 0.005 ) between regular and high-amplitude B-wave patterns. A blinded analysis looking for analogs of ICP patterns in BFI time traces achieved 90% sensitivity in identifying B-waves and 76% specificity in detecting the regular patterns., Conclusions: We revealed the presence of StO 2 and BFI variations-detectable with optical techniques-during ICP B-waves in BEH children. Finally, the feasibility of detecting ICP B-waves in hemodynamic time traces obtained noninvasively was shown., (© 2022 The Authors.)

This report is the second part of a comprehensive two-part series aimed at reviewing an extensive and diverse toolkit of novel methods to explore brain health and function. While the first report focused on neurophotonic tools mostly applicable to animal studies, here, we highlight optical spectroscopy and imaging methods relevant to noninvasive human brain studies. We outline current state-of-the-art technologies and software advances, explore the most recent impact of these technologies on neuroscience and clinical applications, identify the areas where innovation is needed, and provide an outlook for the future directions., (© 2022 The Authors.)

Objective: To evaluate the effect of ceramic thicknesses, bonding surface (enamel vs. dentin), and preparation design (box vs. no box) on the fatigue survival and failure load of minimally invasive full-veneer restorations., Materials and Methods: Human-premolars (n = 60) were divided into five test groups (n = 12). All teeth received full-veneer preparation with the following occlusal/labial thicknesses: standard: 1.5/0.8 mm; thin: 1.0/0.6 mm; ultrathin 0.5/0.4 mm. Preparations for each ceramic thickness were refined in enamel (E-1.0 and E-0.5) or dentin (D-1.5, D-1.0, and D-0.5). Control groups DB-1.5, EB-1.0, and EB-0.5 received box preparations. Monolithic lithium disilicate restorations (IPS-e.max-Press, Ivoclar Vivadent) were adhesively cemented (Syntac-Classic/Variolink-II, Ivoclar Vivadent) and subjected to cyclic mechanical loading (F = 49 N, 1.2 million cycles) with simultaneous thermocycling (5-55°C). All specimens were exposed to single load-to-failure. Pair-wise differences were calculated by using a linear regression model and Student-Newman-Keuls method (p < 0.05)., Results: All full-veneers of group D-1.5, E-1.0, E-0.5, DB-1.5, EB-1.0, and EB-0.5 survived fatigue. Two full-veneers (D-1.0 and D-0.5) revealed cracks during fatigue, resulting in an overall fatigue survival rate of 98.1%. Mean load-to-failure values (N) were as followed: 1005 (D-1.5); 866 (D-1.0); 816 (D-0.5); 1495 (E-1.0); 1279 (E-0.5); 1129 (DB-1.5); 1087 (EB-1.0); and 833 (EB-0.5). Irrespective of ceramic thicknesses, enamel-based full-veneers resulted in higher failure loads than dentin-based restorations. Box preparation reduced the failure loads of thin and ultrathin enamel-based restorations., Conclusion: All tested monolithic lithium disilicate full-veneer restorations exceeded physiological masticatory forces. Minimally invasive full-veneer restorations with enamel as a bonding surface and a non-retentive preparation design showed superior performance., Clinical Significance: Enamel-based non-retentive full-veneers made of monolithic lithium disilicate may serve as a reliable and esthetical minimally invasive treatment option for premolars., (© 2022 The Authors. Journal of Esthetic and Restorative Dentistry published by Wiley Periodicals LLC.)

In a study by Law and colleagues recently published in Neuroimage, the authors reported that wearing a surgical mask during an fMRI scan leads to a statistically significant subject-specific change (30%) in the baseline BOLD level in gray matter, although the response to a sensory-motor task was unaffected. An average increase in end-tidal CO 2 of 7.4% was found when wearing a mask, despite little support in the literature for major effects of mask wearing on blood gas levels. We comment on these findings, point out a several relevant limitations of the study design and provide alternative interpretations of these data., (Copyright © 2021. Published by Elsevier Inc.)

The COVID-19 pandemic led to widespread mandates requiring the wearing of face masks, which led to debates on their benefits and possible adverse effects. To that end, the physiological effects at the systemic and at the brain level are of interest. We have investigated the effect of commonly available face masks (FFP2 and surgical) on cerebral hemodynamics and oxygenation, particularly microvascular cerebral blood flow (CBF) and blood/tissue oxygen saturation (StO 2 ), measured by transcranial hybrid near-infrared spectroscopies and on systemic physiology in 13 healthy adults (ages: 23 to 33 y). The results indicate small but significant changes in cerebral hemodynamics while wearing a mask. However, these changes are comparable to those of daily life activities. This platform and the protocol provides the basis for large or targeted studies of the effects of mask wearing in different populations and while performing critical tasks., Competing Interests: Competing interest statement: T.D. and J.B.F. are inventors on relevant patents. Institut de Ciències Fotòniques (ICFO) has equity ownership in the spin-off company HemoPhotonics S.L. Potential financial conflicts of interest and objectivity of research have been monitored by ICFO’s Knowledge and Technology Transfer Department., (Copyright © 2021 the Author(s). Published by PNAS.)

Objectives: This study aimed to describe demographic, clinical, and laboratory values in febrile pediatric intestinal failure patients with indwelling central venous catheters presenting to the emergency department to determine if there are low-risk features predictive of negative blood cultures that could help identify a subset that do not need hospitalization and/or broad-spectrum antibiotics., Methods: We conducted a retrospective cohort study of febrile patients younger than 18 years with diagnosis of intestinal failure requiring a central venous catheter for home parenteral nutrition to identify factors associated with normal blood cultures to identify a low-risk subset., Results: Of 119 encounters, 46% had a bloodstream infection. Children with a bloodstream infection were older than those without. A normal lactate and heart rate were associated with negative blood cultures., Conclusion: Pediatric intestinal failure patients with a central venous catheter for home parenteral nutrition presenting to the emergency department with fever and found to have a normal lactate and heart rates had lower rates of bloodstream infection. Although we were unable to create a highly sensitive clinical decision-making rule to identify a low-risk cohort because of the low number of patients meeting both criteria, the promising candidate variables identified merit for future multicenter studies., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.)

Assessing species status and making classification decisions under the Endangered Species Act is a critical step towards effective species conservation. However, classification decisions are liable to two errors: i) failing to classify a species as threatened or endangered that should be classified (underprotection), or ii) classifying a species as threatened or endangered when it is not warranted (overprotection). Recent surveys indicate threatened spectacled eider populations are increasing in western Alaska, prompting the U.S. Fish and Wildlife Service to reconsider the federal listing status. There are multiple criteria set for assessing spectacled eider status, and here we focus on the abundance and decision analysis criteria. We estimated population metrics using state-space models for Alaskan breeding populations of spectacled eiders. We projected abundance over 50 years using posterior estimates of abundance and process variation to estimate the probability of quasi-extinction. The decision analysis maps the risk of quasi-extinction to the loss associated with making a misclassification error (i.e., underprotection) through a loss function. Our results indicate that the Yukon Kuskokwim Delta breeding population in western Alaska has met the recovery criteria but the Arctic Coastal Plain population in northern Alaska has not. The methods employed here provide an example of accounting for uncertainty and incorporating value judgements in such a way that the decision-makers may understand the risk of committing a misclassification error. Incorporating the abundance threshold and decision analysis in the reclassification criteria greatly increases the transparency and defensibility of the classification decision, a critical aspect for making effective decisions about species management and conservation., Competing Interests: The authors have declared that no competing interests exist.

Over the recent years, a typical implementation of diffuse correlation spectroscopy (DCS) instrumentation has been adapted widely. However, there are no detailed and accepted recipes for designing such instrumentation to meet pre-defined signal-to-noise ratio (SNR) and precision targets. These require specific attention due to the subtleties of the DCS signals. Here, DCS experiments have been performed using liquid tissue simulating phantoms to study the effect of the detected photon count-rate, the number of parallel detection channels and the measurement duration on the precision and SNR to suggest scaling relations to be utilized for device design., Competing Interests: The role in the project of all the companies and their employees involved has been defined by the project objectives, tasks, and work packages and has been reviewed by the European Commission (grant agreement No. 688303, LUCA-project). Turgut Durduran is an inventor of a relevant patent (Patent US8082015B2). ICFO has equity ownership in the spin-off company HemoPhotonics S.L.. Udo Weigel is the CEO, has equity ownership and is an employee of the company. The potential financial conflicts of interest and objectivity of research have been monitored by ICFO’s Knowledge & Technology Transfer Department and none were identified. Davide Contini and Alberto Dalla Mora are co-founders of pioNIRS s.r.l., spin-off company from Politecnico di Milano (POLIMI, Italy). Their potential financial conflicts of interest and the objectivity of research has been monitored by POLIMI and none were identified., (© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Intracranial pressure (ICP) is an important parameter to monitor in several neuropathologies. However, because current clinically accepted methods are invasive, its monitoring is limited to patients in critical conditions. On the other hand, there are other less critical conditions for which ICP monitoring could still be useful; therefore, there is a need to develop non-invasive methods. We propose a new method to estimate ICP based on the analysis of the non-invasive measurement of pulsatile, microvascular cerebral blood flow with diffuse correlation spectroscopy. This is achieved by training a recurrent neural network using only the cerebral blood flow as the input. The method is validated using a 50% split sample method using the data from a proof-of-concept study. The study involved a population of infants ( n  = 6) with external hydrocephalus (initially diagnosed as benign enlargement of subarachnoid spaces) as well as a population of adults ( n  = 6) with traumatic brain injury. The algorithm was applied to each cohort individually to obtain a model and an ICP estimate. In both diverse cohorts, the non-invasive estimation of ICP was achieved with an accuracy of <4 mm Hg and a negligible small bias. Further, we have achieved a good correlation (Pearson's correlation coefficient >0.9) and good concordance (Lin's concordance correlation coefficient >0.9) in comparison with standard clinical, invasive ICP monitoring. This preliminary work paves the way for further investigations of this tool for the non-invasive, bedside assessment of ICP.

Background: Pediatrics residents are expected by the Accreditation Council for Graduate Medical Education to competently perform 13 procedures. However, residents are graduating with poor self-perceived competency for these procedures., Objective: We developed a curriculum using simulation training at the beginning of internship and 'refresher' workshops throughout the year in order to increase procedure exposure and improve self-perceived competency., Design: Procedural workshops were taught during intern orientation and to all pediatrics residents throughout the academic year. Residents provided a quantitative competency self-assessment before and after each workshop; interns provided an additional self-assessment at the end of the intern year., Results: The curriculum was well-liked and led to more procedural experience. Mean competency self-assessment scores improved immediately after almost every procedure workshop. Mean scores were retained at the end of intern year for most procedures. However, end-of-year mean competency self-assessment and procedural experience on actual patients were similar to interns from a previous year that had not participated in the curriculum., Conclusions: A pre-internship procedure workshop coupled with longitudinal workshops is a feasible way to improve intern exposure to pediatric procedural training. However, it was not sufficient to improve mean competency self-assessments compared to a traditional model of bedside procedural training.

Background: Validated clinical scales, such as the Canadian Acute Respiratory Illness and Flu Scale (CARIFS), have not been used to differentiate influenza (FLU) from other respiratory viruses., Methods: Secondary analysis of a prospective cohort presenting to the emergency department (ED) with an influenza-like infection from 2008 to 2010. Subjects were children aged 0 to 19 years who had a venipuncture and respiratory virus polymerase chain reaction. Demographics and CARIFS items were assessed during the ED visit; comparisons were made between FLU and non-FLU subjects., Results: The 203 subjects had median age 30.5 months; 61.6% were male. Comorbid conditions (51.2%) were common. FLU was identified in 26.6%, and were older than non-FLU patients (69.7 vs 47.9 months, P = .02). Demographic, household factors, and mean CARIFS score did not differ between FLU (33.7), and non-FLU (32.0) (mean difference 1.6, 95% CI: -2.0 to 5.2) groups., Conclusions: CARIFS cannot discriminate between FLU and non-FLU infection in ED children with influenza-like infection., (© The Author(s) 2014.)

Objective: Data on complications from upper respiratory infection are limited. We examined development of severe complications in children presenting to the emergency department (ED) for moderate to severe influenza-like illness (ILI)., Methods: Prospective cohort study of children 0 to 19 years presenting to a tertiary care children's hospital ED during peak respiratory viral seasons from 2008 to 2010. Subjects included had moderate to severe ILI, defined by performance of venipuncture and nasopharyngeal multiplex polymerase chain reaction for respiratory viruses. Severe complications (respiratory failure, encephalopathy, seizures, pneumonia, bacteremia, death) were prospectively determined. Risk factors for severe complications were collected, including demographics, comorbidities, and household exposures., Results: There were 241 enrolled subjects with median age of 27.4 months (interquartile range 8.9-68.5); 59.3% were boys and 48.5% were black. High-risk conditions were present in 53.5%. Severe complications developed in 35.3% (95% confidence interval [CI] 29.3-41.3), most frequently pneumonia (26.1%). The risk for severe complications was increased in subjects with neurologic or neuromuscular conditions (relative risk 4.0; 95% CI 1.9-8.2). No specific respiratory virus was associated with development of severe complications. Among patients with influenza, severe complications were greater with subtype H1N1 infection (relative risk 1.45, 95% CI 0.99-2.13, P = .048), and were at highest risk for pneumonia (relative risk 4.2, 95% CI 1.2-15.9)., Conclusion: In children presenting to the ED for moderate to severe ILI, those with neurologic and neuromuscular disease are at increased risk for severe complications. Development of severe complications did not differ by infecting virus; however, risk of severe complications was greater with subtype H1N1 compared with other influenza., (Copyright © 2014 by the American Academy of Pediatrics.)

To assess cardiorespiratory fitness (CF), usually a stress test is necessary. Our aims were to assess CF in a patient population with suspected or known coronary artery disease (CAD) based on a questionnaire (quest); to compare estimated CF with achieved workloads, and to evaluate its prediction of stress modality (physical/pharmacologic). Consecutive 612 patients undergoing myocardial perfusion SPECT (MPS) completed quest. They first chose one category which best described their daily physical activities. The second part contained patient characteristics (gender, age, BMI, and resting heart rate). An activity score was calculated and metabolic equivalents (METs) were estimated. Estimated and achieved results were compared. Patients with pharmacologic test (n = 208) provided a lower estimate of their performance than physically stressed patients (n = 404): 7.0 ± 2.1 and 8.2 ± 2.3 METs, respectively (P < 0.0001). The latter showed a good correlation between estimated and achieved METs (r = 0.63, P < 0.0001). Regarding prediction of the stress modality, area under the curve (ROC) was 0.65 (P < 0.0001). The quest can easily be applied in daily practice to assess CF in a patient population with CAD and for estimating whether an adequate physical stress test can be carried out.

Objective: To determine pharmacokinetic parameters and variables, firocoxib concentrations in urine and plasma, urine-to-plasma ratios, and the urine depletion profile of firocoxib and to evaluate whether the pharmacokinetic behavior of firocoxib was governed by linear processes after multiple doses of firocoxib were administered IV and orally., Animals: 6 healthy female horses (5 Paint horses and 1 Quarter Horse) in experiment 1 and 12 healthy male and female horses in experiment 2., Procedures: In experiment 1, 6 horses were orally administered firocoxib paste once daily for 12 consecutive days, and plasma and urine samples were obtained and analyzed. In a second experiment, 12 horses received IV injections of firocoxib solution once daily for 9 consecutive days, and plasma was obtained and analyzed., Results: Mean +/- SD clearance and steady-state volume of distribution of firocoxib were 40.5 +/- 14.7 mL/h/kg and 2.3 +/- 0.7 L/kg, respectively. Mean half-life was 44.2 +/- 21.6 hours and 36.5 +/- 9.5 hours for IV and oral administration, respectively. The urine concentration- time curve decreased in parallel with the plasma concentration-verus-time curve. Renal clearance (0.26 +/- 0.09 mL/kg/h) was low, compared with total body clearance, which indicated that the main route of elimination was hepatic clearance., Conclusions and Clinical Relevance: The pharmacokinetics of firocoxib during prolonged use were determined. Use of plasma or urine to ascertain drug concentrations in horses is scientifically valid because the plasma-to-urine ratio was consistent over time and among horses.

Background: The global spread of the highly pathogenic avian influenza H5N1 virus has stimulated interest in a better understanding of the mechanisms of H5N1 dispersal, including the potential role of migratory birds as carriers. Although wild birds have been found dead during H5N1 outbreaks, evidence suggests that others have survived natural infections, and recent studies have shown several species of ducks capable of surviving experimental inoculations of H5N1 and shedding virus. To investigate the possibility of migratory birds as a means of H5N1 dispersal into North America, we monitored for the virus in a surveillance program based on the risk that wild birds may carry the virus from Asia., Results: Of 16,797 birds sampled in Alaska between May 2006 and March 2007, low pathogenic avian influenza viruses were detected in 1.7% by rRT-PCR but no highly pathogenic viruses were found. Our data suggest that prevalence varied among sampling locations, species (highest in waterfowl, lowest in passerines), ages (juveniles higher than adults), sexes (males higher than females), date (highest in autumn), and analytical technique (rRT-PCR prevalence = 1.7%; virus isolation prevalence = 1.5%)., Conclusion: The prevalence of low pathogenic avian influenza viruses isolated from wild birds depends on biological, temporal, and geographical factors, as well as testing methods. Future studies should control for, or sample across, these sources of variation to allow direct comparison of prevalence rates.

The proposed phenyl-A region of sigma (sigma) receptors accommodates several structural features. In this study we explored the possibility that appropriate structural features located at the phenyl-A region of sigma receptor sites could lead to more potent and selective agents for the sigma receptor subtypes. By keeping the phenyl-B substituent as the optimum omega-phenylpentyl moiety, and varying substituents in the phenyl-A region, we have observed changes in binding potency and selectivity at the sigma receptor subtypes. SAR for the binding of these compounds at sigma-2 sites was also examined.

Solution-phase synthesis of various acylguanidine derivatives and the evaluation of a small library of compounds as potential sodium channel blockers are described.

The identification and characterization of a potentially ischemia-selective and orally-active sulfoxide based NMDA ion-channel blocker showing good neuroprotective activity, (R)-(+)-N-(2-chloro-5-methylthiophenyl)-N'-(3-methylsulfinylphenyl)-N'-methylguanidine (CNS 5788), is described.

A lingering question in sigma receptor ligand development is whether a nitrogen atom serves as an important pharmacophoric element in binding affinity. To address this question, we have synthesized several phenylalkylpiperidines and phenylalkylpiperazines and demonstrated that removal of the N atom from a typical phenylalkylpiperidine led to little or no binding to sigma receptors. In addition, where two N atoms occur in a compound, such as with phenylalkylpiperazines, the N atom on the longer alkyl chain appears to be more important. Thus, based on this study, the N atom is an important pharmacophoric element in the binding of phenylalkylpiperidines and phenylalkylpiperazines to sigma receptors.

Synthesis and structure-activity relationships (SAR) are described for a series of N,N'-diarylguanidines related to N-acenaphth-5-yl-N'-(4-methoxynaphth-1-yl)guanidine (3) as anticonvulsants through blockade of sodium channels. SAR studies on compound 3 led to several simpler diphenylguanidines with improved in vitro and in vivo activity. Compounds were screened for blockade of sodium channels in a veratridine-induced [14C]guanidinium influx assay (type IIA sodium channels) and for anticonvulsant activity in the audiogenic DBA/2 mouse model. Results indicated that N, N'-diphenylguanidines substituted with flexible and moderate size lipophilic groups were preferred over aryl and/or hydrophilic groups for biological activity. Among the compounds studied, n-butyl- and/or n-butoxy-containing guanidines showed superior biological activity. A possible relationship between in vitro and in vivo activity of this compound series and their measured/calculated lipophilicities was investigated. Compounds of this series showed only weak NMDA ion channel-blocking activity indicating that the anticonvulsant activity of these compounds is unlikely to be mediated by NMDA ion channels but, more likely, by acting at voltage-gated sodium channels.

In the present investigation, the rationale for the design, synthesis, and biological evaluation of potent inhibitors of neuronal Na+ channels is described. N,N'-diaryl- and N-aryl-N-aralkylguanidine templates were locked in conformations mimicking the permissible conformations of the flexible diarylguanidinium ion (AS+, AA+, SS+). The resulting set of constrained guanidines termed "lockamers" (cyclophane, quinazoline, aminopyrimidazolines, aminoimidazolines, azocino- and tetrahydroquinolinocarboximidamides) was examined for neuronal Na+ channel blockade properties. Inhibition of [14C]guanidinium ion influx in CHO cells expressing type IIA Na+ channels showed that the aminopyrimidazoline 9b and aminoimidazoline 9d, compounds proposed to lock the N,N'-diarylguanidinium in an SS+ conformation, were the most potent Na+ channel blockers with IC50's of 0.06 microM, a value 17 times lower than that of the parent flexible compound 18d. The rest of the restricted analogues with 4-p-alkyl substituents retained potency with IC50 values ranging between 0.46 and 2.9 microM. Evaluation in a synaptosomal 45Ca2+ influx assay showed that 9b did not exhibit high selectivity for neuronal Na+ vs Ca2+ channels. The retention of significant neuronal Na+ blockade in all types of semirigid conformers gives evidence for a multiple mode of binding in this class of compounds and can possibly be attributed to a poor structural specificity of the site(s) of action. Compound 9b was also found to be the most active compound in vivo based on the high level of inhibition of seizures exhibited in the DBA/2 mouse model. The pKa value of 9b indicates that 9b binds to the channel in its protonated form, and log D vs pH measurements suggest that ion-pair partitioning contributes to membrane transport. This compound stands out as an interesting lead for further development of neurotherapeutic agents.

In the mammalian central nervous system, the N-methyl-D-aspartate (NMDA) subclass of glutamate receptors may play an important role in brain diseases such as stroke, brain or spinal cord trauma, epilepsy, and certain neurodegenerative diseases. Compounds which specifically antagonize the actions of the neurotransmitter glutamate at the NMDA receptor ion-channel site offer a novel approach to treating these disorders. CERESTAT (4, aptiganel CNS 1102) is currently undergoing clinical trial for the treatment of traumatic brain injury and stroke. Previously, we reported that analogues of N-1-naphthyl-N'-(3-ethylphenyl)-N'-methylguanidine (4) bound to the NMDA receptor ion-channel site with high potency and selectivity. Recently, molecules active at both sigma receptors and NMDA receptor sites were investigated. A series of substituted diphenylguanidines 6 which are structurally related to N-1-naphthyl-N'-(3-ethylphenyl)-N'-methylguanidine was prepared. Compounds containing appropriate substitution pattern in one of the phenyl rings of diphenylguanidines displayed high affinity. For example, N-(2,5-dibromophenyl)-N'-(3-ethylphenyl)-N'- methylguanidine (27b, R2 = R5 = Br, R3 = C2H5) exhibited potency at both sigma receptors and NMDA receptor sites; 27b also showed high efficacy in vivo in a neonatal rat excitotoxicity model. Further studies indicated that substituent effects were important in this compound series, and 2,5-disubstituted phenyl was the preferred substitution pattern for high-affinity binding at NMDA receptor sites. Bromo and methylthio were the optimal substituents for the R2 and R5 positions of the 2,5-disubstituted phenyl group, respectively. N-(2-Bromo-5-(methylthio)phenyl)-N'- (3-ethylphenyl)-N'-methylguanidine (34b, R2 = Br, R5 = SMe, R3 = C2H5) was highly active at NMDA receptor sites. We found that the binding affinity of guanidines of type 6 could be further enhanced with the appropriate substitution at R3. Optimal activity in this series are afforded by 43b and 44b (R2 = Cl or Br, R5 = R3 = SCH3). Both 43b and 44b bound to NMDA receptor sites with high potency and selectivity (Ki vs [3H]MK-801: 1.87 and 1.65 nM, respectively); these compounds are active in vivo in various animal models of neuroprotection. The structure--activity relationships for these compounds at the NMDA receptor ion-channel site are discussed.

We investigated the psychosocial effects of bone marrow transplantation (BMT) on siblings of transplant recipients. We asked how donor siblings compared with nondonor siblings on quantitative measures of behavior, psychological distress, and sense of self. Participants included 44 siblings (21 donors and 23 nondonors, ages 6-18 yr) of surviving pediatric BMT patients. On self-report measures, donors reported significantly more anxiety and lower self-esteem than nondonors. On teacher-rated scales, donors showed significantly more adaptive skills in school. On these same scales, nondonors showed significantly more school problems than donors. One-third of the siblings in each group reported a moderate level of post-traumatic stress reaction. Exploratory multiple regression analyses point to factors that might influence sibling adjustment and suggest counseling strategies and avenues for future research.

We have originated a family of N,N'-disubstituted guanidines that block the voltage-activated Ca2+ and Na+ channels governing glutamate release. These compounds, CNS 1237 (N-acenaphthyl-N'-methoxynaphthyl guanidine) and its analogues, are "use dependent" in their ability to attenaute neurotransmitter release: they block glutamate release with greater efficacy under conditions of persistent or repetitive depolarization, as would be encountered under pathophysiological circumstances, relative to their ability to block glutamate release elicited by brief, transient depolarizations more characteristic of normal physiological release events in nonischemic brain. Using electrophysiological and rapid kinetic methods, we have differentiated the use-dependent block of the relevant Na+ and Ca2+ channels governing neurotransmitter release from the mechanism of channel antagonism exhibited by, respectively, the substituted guanidine Na+ channel blocker tetrodotoxin (TTX) and venom peptide Ca2+ antagonists. To characterize use-dependent Na+ channel block by CNS 1237, we have employed whole-cell voltage-clamp recordings from a Chinese hamster ovary (CHO) cell line expressing cloned mammalian type II Na+ channels. These experiments demonstrated that, in contrast to the actions of TTX under the same conditions, the potency of Na+ channel block by CNS 1237 is greatly enhanced by depolarizing stimuli in a frequency-dependent manner. Ca2+ channel-activated glutamate release from brain nerve terminal preparations was measured with approximately 300 msec time resolution over a 5-second period of high K(+)-depolarization, using a rapid superfusion technique. CNS 1237 and analogues, at 1-3 microM, accelerated the decay of glutamate release by 40-70%, reflecting depolarization-induced enhancement of block. In contrast, blockade of glutamate release by the Ca2+ channel antagonist peptide toxins omega-aga IV-A (from spider venom) and omega-conotoxin M-VII-C (from cone snail venom) exhibited "reverse-use-dependence:" at concentrations of 0.3 microM, which blocked the initial amplitude of glutamate release by 40-60%, the decay time constant for glutamate release was significantly increased, indicating depolarization-induced relief of block. These findings establish that CNS 1237 and other members of this compound series are use-dependent blockers of the voltage-activated ion channels governing glutamate release. Studies of CNS 1237 in the rat middle cerebral artery occlusion (MCAO) focal stroke model have indicated infarct size reduction comparable to that observed by the same investigators for the glutamate release blocker (BW 619C89 (Burroughs-Wellcome, now in clinical development). Maximal infarct size reduction is achieved with a 3-mg/kg bolus followed by a 4-hour infusion of 0.75 mg/kg/hr.(ABSTRACT TRUNCATED AT 400 WORDS)

Radioligand binding techniques were used to determine the affinity of a series of substituted guanidine derivatives for 1) the binding site within the ion channel of the N-methyl-D-aspartate (NMDA) receptor, as defined by displacement of MK-801 ([3H]dizocilpine) and 2) sigma sites as defined by displacement of [3H]N,N'-di-(o-tolyl)guanidine. The goal was to find ligands with high affinity and selectivity for the NMDA receptor ion-channel site. The neuroprotective activity of these compounds was assessed by their ability to protect cortical neurons from injury caused by a 5-min exposure to 500 microM glutamate in vitro. Release of lactate dehydrogenase into the culture medium by damaged neurons was used as an index of neuronal injury. The 14 compounds tested had IC50 values ranging from 37.3 nM to 12.7 microM for the NMDA receptor ion-channel site and from 8.3 nM to 7.25 microM for sigma sites. Affinity for the ion-channel site was improved by unsymmetrical substitutions on the guanidine moiety. All compounds in the series protected cortical neurons against glutamate toxicity, with EC50 values (concentration affording 50% protection) ranging from 0.38 to 28.25 microM. The neuroprotective effect of each compound was positively correlated with its ion-channel site affinity (r = 0.94); no correlation between neuroprotective efficacy and sigma site binding affinity was found (r V -0.13) establishing clearly that neuroprotection in this assay was linked to NMDA antagonist properties.

Two problems that have hampered sigma receptor research are (i) a lack of high-affinity agents and (ii) the recent identification of multiple populations of sigma receptors (i.e., sigma 1 and sigma 2 sites). Recently, several high-affinity sigma ligands have been identified, and the term superpotent sigma ligands has been coined to describe agents with Ki values of < 1 nM. We have previously shown that appropriately N-substituted phenylalkylamines bind at sigma receptors with high affinity. In the present investigation, we examine the structure-affinity relationships of these phenylalkylamine derivatives for sigma 1 binding and describe some of the first superpotent sigma 1 ligands. A binding model was developed to account for the structural features of the phenylalkylamines that appear to be important for the interaction of these agents with sigma 1 sites.

Diarylguanidines, acting as NMDA receptor ion channel site ligands, represent a new class of potential neuroprotective drugs. Several diarylguanidines structurally related to N,N'-di-o-tolylguanidine (DTG), a known selective sigma receptor ligand, were synthesized and evaluated in in vitro radioligand displacement assays, with rat or guinea pig brain membrane homogenates, using the NMDA receptor ion channel site specific radioligand [3H]-(+)-5(S)-methyl-10(R),11-dihydro-5H-dibenzo[a,d]cyclohepten-5 ,10- imine (MK-801, 3), and the sigma receptor-specific radioligand [3H]-di-o-tolylguanidine (DTG, 5). This paper presents the structure-activity relationships leading to novel tri- and tetrasubstituted guanidines, which exhibit high selectivity for NMDA receptor ion channel sites and weak or negligible affinity for sigma receptors. The in vitro binding results from symmetrically substituted diphenylguanidines indicated that compounds having ortho or meta substituents (with respect to the position of the guanidine nitrogen) on the phenyl rings showed greater affinity for the NMDA receptor ion channel site compared with para-substituted derivatives. Among the group of ring substituents studied for symmetrical diarylguanidines, an isopropyl group was preferred at the ortho position and an ethyl group was preferred at the meta position. Several unsymmetrical guanidines containing a naphthalene ring on one nitrogen atom and an ortho- or a meta-substituted phenyl ring on the second nitrogen atom, e.g., N-1-naphthyl-N'-(3-ethylphenyl)guanidine (36), showed a 3-5-fold increase in affinity for the NMDA receptor ion channel site and no change in sigma receptor affinity compared to the respective symmetrical counterparts. Additional small substituents on the guanidine nitrogen atoms bearing the aryl rings resulted in tri- and tetrasubstituted guanidine derivatives which retained affinity for NMDA receptor ion channel sites but exhibited a significant reduction in their affinities for sigma receptors. For example, N-1-naphthyl-N'-(3-ethylphenyl)-N'-methylguanidine (40) showed high affinity for the NMDA receptor ion channel site (IC50 = 36 nM vs [3H]-3) and low affinity for sigma receptors (IC50 = 2540 nM vs [3H]-5). Selectivity for the NMDA receptor ion channel sites over sigma receptors appears to be dependent upon the structure of the additional substituents on the guanidine nitrogen atoms bearing the aryl groups. Methyl and ethyl substituents are most preferred in the tri- and tetrasubstituted diarylguanidines. The trisubstituted guanidine, N-1-naphthyl-N'-(3-ethylphenyl)-N'-methylguanidine (40) and its close analogues showed good in vivo neuroprotection and are potential neuroprotective drug candidates for the treatment of stroke and other neurodegenerative disorders.

Ischemic insults to the brain in stroke or traumatic brain injury produce excessive release of glutamate from depolarized nerve terminals. This excessive glutamate release in turn stimulates massive calcium entry into nerve cells, activating a biochemical cascade that results in cell death. A major pathway of calcium entry into depolarized nerve cells is through voltage-sensitive, high threshold calcium channels. A large fraction of this calcium entry is mediated through "R-type" calcium channels, channels resistant to blockage by dihydropyridine calcium antagonists such as nimodipine. A newly discovered compound derived from spider venom, CNS 2103, antagonizes both R-type channels and dihydropyridine-sensitive ("L-type") calcium channels. This broad spectrum of action, coupled with selectivity for calcium channels over other classes of voltage-sensitive and ligand-gated ion channels, makes CNS 2103 an interesting lead for development of drugs to treat ischemic brain injury. Activation of presynaptic ("N-type") calcium channels in nerve terminals is a primary cause of excessive neurotransmitter release in brain ischemia. Prevention of glutamate release by blockade of N-type channels in glutamatergic nerve terminals may, at an early stage in the pathophysiological cascade, abort the process leading to nerve cell death. Cambridge NeuroScience has developed a novel rapid kinetic approach for monitoring glutamate release from brain nerve terminals in vitro, and this has led to CNS 1145, a substituted guanidine that selectively blocks a kinetic component of calcium-dependent glutamate release mediated by persistent depolarization. Additional evidence suggests that CNS 1145 antagonizes presynaptic N-type calcium channels, and this may account at least in part for its ability to block glutamate release.

sigma receptors may represent an exciting new approach for the development of novel psychotherapeutic agents. Unfortunately, many of the commonly used sigma ligands lack selectivity (e.g., many bind at phencyclidine or dopamine receptors) or suffer from other serious drawbacks. Recently, we described a series of 2-phenylaminoethanes that bind at sigma receptors with high affinity and selectivity. Because there is evidence that 1-phenylpiperazines can structurally mimic the 2-phenylaminoethane moiety, we prepared a series of 1-phenylpiperazines and related analogues and incorporated structural features already shown to enhance the sigma binding of the 2-phenylaminoethanes. Several of these derivatives bind at sigma receptors with high affinity (Ki = 1-10 nM) and lack appreciable affinity for phencyclidine and dopamine receptors. In as much as certain of these agents structurally resemble the high-affinity, but nonselective, sigma ligand haloperidol, and because they bind with 10 times the affinity of haloperidol, we have apparently identified what appears to be the primary sigma pharmacophore of that agent.

Certain benzomorphans, such as N-allylnormetazocine, are classical "sigma-opiates" that bind both at sigma and phencyclidine (PCP) binding sites with modest affinity. Recently, we identified N-substituted 2-phenylaminoethane as being the primary sigma-pharmacophore of the benzomorphans and demonstrated that 1-phenyl-2-aminopropane (2) derivatives, depending upon their terminal amine substituents, constitute a novel class of high-affinity sigma-selective agents. With this pharmacophore, it is shown in the present investigation that the aromatic hydroxyl group (a prime feature of all the sigma-opiates) contributes little to the binding of 2 at sigma-sites. It is also demonstrated that an N-substituted aminotetralin moiety (such as 17, a conformationally restricted analogue of 2) may also be considered a sigma-opiate pharmacophore. Unlike the sigma-opiates, derivatives of 2 and 17 display no affinity for PCP sites and must consequently lack those structural features important for the binding of benzomorphans at PCP sites. Because 3-phenylpiperidines and related sigma-ligands also possess a phenylalkylamine imbedded within their structures, we propose that the 2-phenylaminoethane moiety is a common sigma-pharmacophore for derivatives of 2, the 3-phenylpiperidines, and the sigma-opiates.

Certain benzomorphan "sigma-opiates" such as N-allylnormetazocine (NANM) bind at sigma receptors with modest affinity and with little selectivity (i.e., they also bind at phencyclidine or PCP sites). In order to identify the primary pharmacophore of the benzomorphans, we prepared several amine-substituted derivatives of 1-phenyl-2-aminopropane. Several simple alkyl-substituted analogues were shown to bind at sigma sites with affinities comparable to that of NANM itself; among these was the N-benzyl derivative 9 (Ki = 117 nM). Lengthening the spacer between the terminal amine and the phenyl group from one to five methylene units resulted in a significant increase in affinity (e.g. 15, Ki = 6.3 nM). In addition, unlike the benzomorphans, these phenalkylamines do not bind at PCP sites. The results of the present study reveal that (a) the 1-phenyl-2-aminopropane nucleus of the benzomorphans is sufficient for binding at sigma sites provided that the terminal amine is not a primary amine and that (b) introduction of (phenylalkyl)amine substituents affords compounds that represent a new class of high-affinity sigma-selective agents.

With an eye toward the development of novel atypical antipsychotic agents, we have studied the structure-affinity relationships of N,N'-di-o-tolylguanidine (DTG, 3) and its congeners at the haloperidol-sensitive sigma receptor. A number of DTG analogues were synthesized and evaluated in in vitro radioligand displacement experiments with guinea pig brain membrane homogenates, using the highly sigma-specific radioligands [3H]-3 and [3H]-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and the phencyclidine (PCP) receptor specific compounds [3H]-N-[1-(2-thienyl)-cyclohexyl]piperidine and [3H]-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine. The affinity of N,N'-diarylguanidines for the sigma receptor decreases with increasing steric bulk of ortho substituents larger than C2H5. Hydrophobic substituents are generally preferred over similarly positioned hydrophilic ones. Furthermore, electroneutral substituents are preferred over strongly electron donating or withdrawing groups. Significant binding to the sigma receptor is usually retained as long as at least one side of the guanidine bears a preferred group (e.g. 2-CH3C6H5). Replacement of one or both aryl rings with certain saturated carbocycles (e.g. cyclohexyl, norbornyl, or adamantyl) leads to a significant increase in affinity. By combining the best aromatic and best saturated carbocyclic substituents in the same molecule, we arrived at some of the most potent sigma ligands described to date (e.g. N-exo-2-norbornyl-N'-(2-iodophenyl)guanidine, IC50 = 3 nM vs [3H]-3). All of the compounds tested were several orders of magnitude more potent at the sigma receptor than at the PCP receptor, with a few notable exceptions. This series of disubstituted guanidines may be of value in the development of potential antipsychotics and in the further pharmacological and biochemical characterization of the sigma receptor.

After preloading with [3H]norepinephrine (NE), organ-cultured superior cervical ganglia released increased amounts of [3H]NE when incubated with depolarizing K+ concentrations, tyramine or amphetamine. K+-induced release was Ca++-dependent, whereas tyramine- and amphetamine-induced release were not. Analysis of the released radioactivity by high-pressure liquid chromatography showed that these releasing stimuli caused primarily an increase in NE release, with little increase in the release of NE metabolites. Incubation with 10 microM xylamine, an irreversible inhibitor of NE uptake, caused a small increase in [3H]NE efflux, but no reduction in the endogenous NE and dopamine levels in superior cervical ganglia. After xylamine treatment, tyramine-induced release was greatly inhibited, whereas release by amphetamine and K+ was not. The neuronal uptake inhibitor desipramine (1 microM), affected K+-, tyramine- and amphetamine-induced release in a manner similar to xylamine. It is concluded that xylamine is a very weak releasing agent in this tissue and that its effects on other release processes are consistent with its action as a NE uptake inhibitor. Amphetamine-induced release appears not to require the NE uptake system for either the uptake of amphetamine, as shown by the accumulation of [3H]amphetamine, or the efflux of NE.

Four diarylguanidine derivatives were synthesized. These compounds were found to displace, at submicromolar concentrations, 3H-labeled 1-[1-(2-thienyl)cyclohexyl]piperidine and (+)-[3H]MK-801 from phencyclidine receptors in brain membrane preparations. In electrophysiological experiments the diarylguanidines blocked N-methyl-D-aspartate (NMDA)-activated ion channels. These diarylguanidines also protected rat hippocampal neurons in vitro from glutamate-induced cell death. Our results show that some diarylguanidines are noncompetitive antagonists of NMDA receptor-mediated responses and have the neuroprotective property that is commonly associated with blockers of the NMDA receptor-gated cation channel. Diarylguanidines are structurally unrelated to known blockers of NMDA channels and, therefore, represent a new compound series for the development of neuroprotective agents with therapeutic value in patients suffering from stroke, from brain or spinal cord trauma, from hypoglycemia, and possibly from brain ischemia due to heart attack.

A study of the influence of various concentrations of NaC1 on 21 species of dermatophytes and other keratolytic fungi was made. Based on the sensitivity of the species to various concentrations of NaC1, it was possible to divide them into five groups. Microsporum ferrugineum and Trichophyton concentricum and T. tonsurans were the most sensitive to NaC1 and were inhibited by 5%. The greatest number of species were inhibited by 12% NaC1. The fungi most tolerant to NaC1 were M. cookei and M. nanum, and T. mentagrophytes, T. schoenleinii, and T. terrestre. These species were inhibited by 15%. NaC1 prevented any variant change in Epidermophyton floccosum, T. mentagrophytes, and M. gypseum, but promoted a change in phenotype in M. audouinii and M. cookei, and T. gallinae. It is suggested that the word "pleomorphism" be replaced by the term sterile albinism. This term refers to that kind of cultural change when there is no evidence of sporulation and the white fluffy mycelium consists of fine sterile hyphae. Sterile albino strains of E. floccosum were induced to form a macroconidia on Sabouraud cycloheximide chloramphenicol gentamicin agar (SCCGA) containing 3-5% NaC1. Also, M. audouinii formed microconidia and macroconidia in velvety growth cultured on SCCGA containing NaC1.

The accumulation of tritium-labeled xylamine ([3H]XYL), an irreversible inhibitor of neuronal norepinephrine (NE) uptake, was studied in organ-cultured rat superior cervical ganglia (SCG). At concentrations below those which significantly inhibit NE uptake, [3H]XYL accumulation was linear with time for up to 2 hr; at higher concentrations, accumulation slowed within the first 10 min. [3H]XYL accumulation was inhibited by desipramine, cocaine, bretylium and by NE at concentrations similar to those required to inhibit NE uptake. Replacing Na+ with Li+ in the bathing medium also inhibited [3H]XYL accumulation by about 70%. After 2 days of organ culture, SCG [3H]XYL accumulation was increased in parallel with an increase in NE uptake. Reserpine pretreatment of SCG did not affect [3H]XYL accumulation. The hydrolysis product of XYL, xylaminol, was also accumulated by SCG, but its accumulation was not inhibited by desipramine or the absence at Na+. By using trichloroacetic acid precipitation followed by ethanol extractions, about 40% of the accumulated tritium appeared to be irreversibly bound to protein, whereas about 30% appeared to be bound to lipids. We conclude that XYL is accumulated in sympathetic neurons by the NE uptake system and that this accumulation may be necessary in order for XYL to produce its irreversible inhibition of NE uptake.

The neuropeptide bombesin acts on a variety of target cells to stimulate the processes of secretion and cell proliferation. In this study we determined whether bombesin receptors interact with known guanine nucleotide-binding proteins in four different cell types: GH4C1 pituitary cells, HIT pancreatic islet cells, Swiss 3T3 fibroblasts, and rat brain tissue. Maximal concentrations of nonhydrolyzable GTP analogs decreased agonist binding to bombesin receptors in membranes from all four sources. In GH4C1 and HIT cell membranes GTP analogs inhibited bombesin receptor binding with IC50 values of about 0.1 microM, whereas GDP analogs were approximately 10-fold less potent. In contrast, GMP and the nonhydrolyzable ATP analog adenylyl-imidodiphosphate had no effect at 100 microM. Equilibrium binding experiments in GH4C1 and HIT cell membranes indicated a single class of binding sites with a dissociation constant (Kd) for [125I-Tyr4]bombesin of 24.4 +/- 7.0 pM and a binding capacity of 176 +/- 15 fmol/mg protein. Guanine nucleotides decreased the apparent affinity of the receptors without significantly changing receptor number. Consistent with this observation, guanine nucleotides also increased the rate of ligand dissociation. Pretreatment of GH4C1 or HIT cells with either pertussis toxin (100 ng/ml) or cholera toxin (500 ng/ml) for 18 h did not affect agonist binding to membrane bombesin receptors, its regulation by guanine nucleotides, or bombesin stimulation of hormone release. Although pertussis toxin pretreatment has been reported to block bombesin stimulation of DNA synthesis in Swiss 3T3 cells, it did not alter the binding properties of bombesin receptors in Swiss 3T3 membranes or inhibit the rapid increase in intracellular [Ca2+] produced by bombesin in these cells. In summary, our results indicate that the bombesin receptor interacts with a guanine nucleotide-binding protein which exhibits a different toxin sensitivity from those which regulate adenylate cyclase as well as those which couple some receptors to phospholipases.