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Background: Recent studies, including those by the National Board of Medical Examiners, have highlighted the remarkable capabilities of recent large language models (LLMs) such as ChatGPT in passing the United States Medical Licensing Examination (USMLE). However, there is a gap in detailed analysis of LLM performance in specific medical content areas, thus limiting an assessment of their potential utility in medical education., Objective: This study aimed to assess and compare the accuracy of successive ChatGPT versions (GPT-3.5, GPT-4, and GPT-4 Omni) in USMLE disciplines, clinical clerkships, and the clinical skills of diagnostics and management., Methods: This study used 750 clinical vignette-based multiple-choice questions to characterize the performance of successive ChatGPT versions (ChatGPT 3.5 [GPT-3.5], ChatGPT 4 [GPT-4], and ChatGPT 4 Omni [GPT-4o]) across USMLE disciplines, clinical clerkships, and in clinical skills (diagnostics and management). Accuracy was assessed using a standardized protocol, with statistical analyses conducted to compare the models' performances., Results: GPT-4o achieved the highest accuracy across 750 multiple-choice questions at 90.4%, outperforming GPT-4 and GPT-3.5, which scored 81.1% and 60.0%, respectively. GPT-4o's highest performances were in social sciences (95.5%), behavioral and neuroscience (94.2%), and pharmacology (93.2%). In clinical skills, GPT-4o's diagnostic accuracy was 92.7% and management accuracy was 88.8%, significantly higher than its predecessors. Notably, both GPT-4o and GPT-4 significantly outperformed the medical student average accuracy of 59.3% (95% CI 58.3-60.3)., Conclusions: GPT-4o's performance in USMLE disciplines, clinical clerkships, and clinical skills indicates substantial improvements over its predecessors, suggesting significant potential for the use of this technology as an educational aid for medical students. These findings underscore the need for careful consideration when integrating LLMs into medical education, emphasizing the importance of structured curricula to guide their appropriate use and the need for ongoing critical analyses to ensure their reliability and effectiveness., (© Brenton T Bicknell, Danner Butler, Sydney Whalen, James Ricks, Cory J Dixon, Abigail B Clark, Olivia Spaedy, Adam Skelton, Neel Edupuganti, Lance Dzubinski, Hudson Tate, Garrett Dyess, Brenessa Lindeman, Lisa Soleymani Lehmann. Originally published in JMIR Medical Education (https://mededu.jmir.org).)

Peripheral artery disease (PAD) compromises blood flow, often leading to the need for amputation. This case report details a 72-year-old male with a history of PAD and type 2 diabetes mellitus who underwent below-knee amputation (BKA) due to atherosclerotic limb disease. He subsequently faced complications such as wound dehiscence, prolonged wheelchair dependency, and an eventual contralateral BKA. Despite interventions including wound care and revision surgeries, persistent delayed healing underscores the complex interplay between atherosclerosis, diabetes, and wound healing. This patient's journey highlights the critical importance of tailored management strategies and patient education in mitigating the impact of PAD-related amputations, emphasizing the need for comprehensive care to address the multifaceted challenges posed by vascular diseases., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Longley et al.)

Pathogenic variants in KCNB1 are associated with a neurodevelopmental disorder spectrum that includes global developmental delays, cognitive impairment, abnormal electroencephalogram (EEG) patterns, and epilepsy with variable age of onset and severity. Additionally, there are prominent behavioral disturbances, including hyperactivity, aggression, and features of autism spectrum disorder. The most frequently identified recurrent variant is KCNB1-p.R306C, a missense variant located within the S4 voltage-sensing transmembrane domain. Individuals with the R306C variant exhibit mild to severe developmental delays, behavioral disorders, and a diverse spectrum of seizures. Previous in vitro characterization of R306C described altered sensitivity and cooperativity of the voltage sensor and impaired capacity for repetitive firing of neurons. Existing Kcnb1 mouse models include dominant negative missense variants, as well as knockout and frameshifts alleles. While all models recapitulate key features of KCNB1 encephalopathy, mice with dominant negative alleles were more severely affected. In contrast to existing loss-of-function and dominant-negative variants, KCNB1-p.R306C does not affect channel expression, but rather affects voltage-sensing. Thus, modeling R306C in mice provides a novel opportunity to explore impacts of a voltage-sensing mutation in Kcnb1. Using CRISPR/Cas9 genome editing, we generated the Kcnb1 R306C mouse model and characterized the molecular and phenotypic effects. Consistent with the in vitro studies, neurons from Kcnb1 R306C mice showed altered excitability. Heterozygous and homozygous R306C mice exhibited hyperactivity, altered susceptibility to chemoconvulsant-induced seizures, and frequent, long runs of slow spike wave discharges on EEG, reminiscent of the slow spike and wave activity characteristic of Lennox Gastaut syndrome. This novel model of channel dysfunction in Kcnb1 provides an additional, valuable tool to study KCNB1 encephalopathies. Furthermore, this allelic series of Kcnb1 mouse models will provide a unique platform to evaluate targeted therapies., Competing Interests: Declaration of competing interest The authors declare no competing interests related to this study., (Copyright © 2023. Published by Elsevier Inc.)

In developed countries, vitamin A deficiency (VAD) remains rare but is a leading cause of global blindness. We describe the case of a 10-year-old girl with autism spectrum disorder (ASD) initially presenting mild VAD symptoms, escalating to severe photophobia and reluctance to leave a darkened room due to a self-restricted diet of fast-food french fries. A timely examination revealed severe VAD and vitamin A supplementation resolved her symptoms in three weeks. This case highlights the challenge of obtaining accurate patient history in ASD, emphasizing the need for routine dietary discussions and micronutrient testing, especially at ages nine and 13 when the United States Department of Agriculture (USDA) recommends increased vitamin A intake. Early intervention can prevent micronutrient deficiencies in pediatric patients, particularly those with ASD., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Dixon et al.)

The clinical association between Coxsackievirus B (CVB) and methicillin-sensitive Staphylococcus aureus (MSSA) has not been well established in the current literature. Here, we report a case of a 29-year-old male who presented with fever and malaise 24 hours after noticing a pruritic lesion on the anterior foreleg that resembled a mosquito bite. After multiple ED visits, laboratory studies, and imaging tests, the patient was admitted for treatment of high fevers and pancytopenia. The final diagnosis was viral sepsis complicated by co-infection with MSSA., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Ethridge et al.)

Introduction: The aims of lens removal surgery are to re-establish or preserve both a clear visual axis and emmetropic vision. Trans-scleral intraocular lens (IOL) fixation has been described in cases where lens capsule instability precludes the insertion of a prosthetic intraocular lens into the lens capsule. Previous techniques have necessitated enlargement of the corneal incision to accommodate either a rigid polymethylmethacrylate IOL or an acrylic foldable IOL inserted using forceps. This paper reports the modification of an endocapsular IOL to be used as an injectable suture-fixated IOL introduced through a 2.8 mm corneal incision., Materials and Methods: All cases underwent lens extraction by phacoemulsification followed by removal of the unstable lens capsule. A PFI X4 IOL (Medicontur) was modified to create four open-loop haptics. The IOL was injected into the anterior chamber, each haptic was captured in a loop of suture introduced ab externo, and the lens was sutured with four-point fixation., Results: The results from 20 eyes in 17 dogs are reported. Over an average follow-up time of 14.5 months, vision was retained in 16/20 eyes. Vision was lost in four eyes due to corneal ulceration and ocular hypertension (1/20), retinal detachment (2/20), and Progressive Retinal Atrophy (1/20)., Conclusions: The modified PFI X4 proved suitable for injection and scleral fixation through a 2.8 mm corneal incision, with a success rate comparable to previously published techniques., (© 2023 American College of Veterinary Ophthalmologists.)

Pathogenic variants in KCNB1 are associated with a neurodevelopmental disorder spectrum that includes global developmental delays, cognitive impairment, abnormal electroencephalogram (EEG) patterns, and epilepsy with variable age of onset and severity. Additionally, there are prominent behavioral disturbances, including hyperactivity, aggression, and features of autism spectrum disorder. The most frequently identified recurrent variant is KCNB1 -p.R306C, a missense variant located within the S4 voltage-sensing transmembrane domain. Individuals with the R306C variant exhibit mild to severe developmental delays, behavioral disorders, and a diverse spectrum of seizures. Previous in vitro characterization of R306C described loss of voltage sensitivity and cooperativity of the sensor and inhibition of repetitive firing. Existing Kcnb1 mouse models include dominant negative missense variants, as well as knockout and frameshifts alleles. While all models recapitulate key features of KCNB1 encephalopathy, mice with dominant negative alleles were more severely affected. In contrast to existing loss-of-function and dominant-negative variants, KCNB1 -p.R306C does not affect channel expression, but rather affects voltage-sensing. Thus, modeling R306C in mice provides a novel opportunity to explore impacts of a voltage-sensing mutation in Kcnb1 . Using CRISPR/Cas9 genome editing, we generated the Kcnb1 R306C mouse model and characterized the molecular and phenotypic effects. Heterozygous and homozygous R306C mice exhibited pronounced hyperactivity, altered susceptibility to flurothyl and kainic acid induced-seizures, and frequent, long runs of spike wave discharges on EEG. This novel model of channel dysfunction in Kcnb1 provides an additional, valuable tool to study KCNB1 encephalopathies. Furthermore, this allelic series of Kcnb1 mouse models will provide a unique platform to evaluate targeted therapies., Competing Interests: Declaration of Competing Interest The authors declare no competing interests related to this study.

Objectives: To report the outcomes following the insertion of a rhexis-fixated prosthetic intraocular lens (IOL) in dogs undergoing lens removal., Materials and Methods: The results are from 30 eyes of 28 dogs, undergoing lendectomy, in which the lens capsule could not accommodate a conventional prosthetic endo-capsular IOL. The reported cases had sustained either spontaneous or traumatic lens capsule rupture, or accidental intra-operative iatrogenic lens capsule disruption, or had required a planned, large, anterior or posterior continuous curvilinear capsulorhexis, all of which precluded insertion of a prosthetic IOL within the lens capsule. An acrylic IOL (XVET; Medicontur) was modified and positioned across the anterior and/or posterior capsulorhexes., Results: Other than haptic luxation in three cases, no complications were seen that were directly attributable to the rhexis-fixated lens. Over a follow-up period from three to 76 months (mean 20.7 months) 26/30 eyes remained visual. Blindness developed in three eyes due to retinal detachment and one eye was enucleated due to regrowth of a ciliary body adenoma., Clinical Significance: Rhexis fixation provided an alternative method to implant a prosthetic IOL when the lens capsule was unable to accommodate a conventional endo-capsular IOL., (© 2021 British Small Animal Veterinary Association.)

Digital food ordering platforms are used by millions across the world and provide easy access to takeaway fast-food that is broadly, though not exclusively, characterised as energy dense and nutrient poor. Outlets are routinely rated for hygiene, but not for their healthiness. Nutritional information is mandatory in pre-packaged foods, with many companies voluntarily using traffic light labels to support making healthier choices. We wanted to identify a feasible universal method to objectively score takeaway fast-food outlets listed on Just Eat that could provide users with an accessible rating that can infer an outlet's 'healthiness' . Using a sample of takeaway outlets listed on Just Eat, we obtained four complete assessments by nutrition researchers of each outlet's healthiness to create a cumulative score that ranged from 4 to 12. We then identified and manually extracted nutritional attributes from each outlet's digital menu, e.g., number of vegetables that have the potential to be numerated. Using generalized linear modelling we identified which attributes were linear predictors of an outlet's healthiness assessment from nutritional researchers. The availability of water, salad, and the diversity of vegetables were positively associated with academic researchers' assessment of an outlet's healthiness, whereas the availability of chips, desserts, and multiple meal sizes were negatively associated. This study shows promise for the feasibility of an objective measure of healthiness that could be applied to all outlet listings on Just Eat and other digital food outlet aggregation platforms. However, further research is required to assess the metric's validity, its desirability and value to users, and ultimately its potential influence on food choice behaviour.

Neonatal and infant exposure to volatile anesthetics has been associated with long-term learning, memory, and behavioral deficits. Although early anesthesia exposure has been linked to a number of underlying structural abnormalities, functional changes associated with these impairments remain poorly understood. To investigate the relationship between functional alteration in neuronal circuits and learning deficiency, resting state functional MRI (rsfMRI) connectivity was examined in adolescent rabbits exposed to general anesthesia as neonates (1 MAC isoflurane for 2 h on postnatal days P8, P11, and P14) and unanesthetized controls before and after training with a trace eyeblink classical conditioning (ECC) paradigm. Long-range connectivity was measured between several key regions of interest (ROIs), including primary and secondary somatosensory cortices, thalamus, hippocampus, and cingulate. In addition, metrics of regional BOLD fluctuation amplitudes and coherence, amplitude of low-frequency fluctuation (ALFF), fractional ALFF (fALFF), and regional homogeneity (ReHo) were calculated. Our results showed that the trace ECC learning rate was significantly lower in the anesthesia-exposed group. No anesthesia-related changes in long-range connectivity, fALFF, or ReHo were found between any ROIs. However, ALFF was significantly higher in anesthesia-exposed rabbits in the primary and secondary somatosensory cortices, and ALFF in those areas was a significant predictor of the learning performance for trace ECC. The absence of anesthesia-related changes in long-range thalamocortical connectivity indicates that functional thalamocortical input is not affected. Higher ALFF in the somatosensory cortex may indicate the developmental disruption of cortical neuronal circuits after neonatal anesthesia exposure, including excessive neuronal synchronization that may underlie the observed cognitive deficits., (Copyright © 2020 Drobyshevsky, Miller, Li, Dixon, Venkatasubramanian, Wyrwicz and Aksenov.)

Millions of children undergo general anesthesia each year, and animal and human studies have indicated that exposure to anesthesia at an early age can impact neuronal development, leading to behavioral and learning impairments that manifest later in childhood and adolescence. Here, we examined the effects of isoflurane, a commonly-used general anesthetic, which was delivered to newborn rabbits. Trace eyeblink classical conditioning was used to assess the impact of neonatal anesthesia exposure on behavioral learning in adolescent subjects, and a variety of MRI techniques including fMRI, MR volumetry, spectroscopy and DTI captured functional, metabolic, and structural changes in key regions of the learning and sensory systems associated with anesthesia-induced learning impairment. Our results demonstrated a wide array of changes that were specific to anesthesia-exposed subjects, which supports previous studies that have pointed to a link between early anesthesia exposure and the development of learning and behavioral deficiencies. These findings point to the need for caution in avoiding excessive use of general anesthesia in young children and neonates.

Each year, millions of children undergo anesthesia, and both human and animal studies have indicated that exposure to anesthesia at an early age can lead to neuronal damage and learning deficiency. However, disorders of sensory functions were not reported in children or animals exposed to anesthesia during infancy, which is surprising, given the significant amount of damage to brain tissue reported in many animal studies. In this review, we discuss the relationship between the systems in the brain that mediate sensory input, spatial learning, and classical conditioning, and how these systems could be affected during anesthesia exposure. Based on previous reports, we conclude that anesthesia can induce structural, functional, and compensatory changes in both sensory and learning systems. Changes in myelination following anesthesia exposure were observed as well as the neurodegeneration in the gray matter across variety of brain regions. Disproportionate cell death between excitatory and inhibitory cells induced by anesthesia exposure can lead to a long-term shift in the excitatory/inhibitory balance, which affects both learning-specific networks and sensory systems. Anesthesia may directly affect synaptic plasticity which is especially critical to learning acquisition. However, sensory systems appear to have better ability to compensate for damage than learning-specific networks., (© 2020 Wiley Periodicals, Inc.)

Volatile general anesthetics are used commonly in adults and children, yet their mechanisms of action are complex and the changes in single unit firing and synaptic activity that underlie the broad decreases in neuronal activity induced by these drugs have not been well characterized. Capturing such changes throughout the anesthesia process is important for comparing the effects of different anesthetics and gaining a better understanding of their mechanisms of action and their impact on different brain regions. Using chronically implanted electrodes in the rabbit somatosensory cortex, we compared the effects of two common general anesthetics, isoflurane, and sevoflurane, on cortical neurons. Single unit activity and local field potentials (LFP) were recorded continuously before and during anesthetic delivery at 1 MAC, as well as during recovery. Our findings show that although isoflurane and sevoflurane belong to the same class of volatile general anesthetics, their effects upon cortical single units and LFP were quite different. Overall, the suppression of neuronal firing was greater and more uniform under sevoflurane. Moreover, the changes in LFP frequency bands suggest that effect of anesthesia upon beta oscillations does not necessarily depend on the level of single unit activity, but rather on the changes in GABA/glutamate neurotransmission induced by each drug.

The anaphylactoid reaction described follows cessation of ranitidine in a 19-year-old female with the disease cluster: mast cell activation syndrome, hypermobile Ehlers-Danlos syndrome and postural tachycardia syndrome. Anaphylaxis can give wide-ranging symptoms from rhinorrhoea and urticaria to tachycardia and system-wide, life-threatening, anaphylactic shock. Individuals with a disorder of mast cell activation can experience many such symptoms. H 2 receptor antagonists, such as ranitidine, are commonly prescribed in this population. A mechanism for the reaction is proposed in the context of ranitidine, as an inverse agonist, causing upregulation of H 2 histamine receptors and raised histamine levels due to enzyme induction. This effect, following extended and/or high antihistamine dosing, may have implications for other individuals with a disorder of mast cell activation, such as mastocytosis or mast cell activation syndrome. There are potential policy and patient guidance implications for primary and secondary care with respect to cessation of H 2 antagonists., (© 2018 The British Pharmacological Society.)

Approximately 422 million people have diabetes mellitus worldwide, with the majority diagnosed with type 2 diabetes mellitus (T2DM). The complications of diabetes mellitus include diabetic peripheral neuropathy (DPN) and retinopathy, both of which can lead to balance impairments. Balance assessment is therefore an integral component of the clinical assessment of a person with T2DM. Although there are a variety of balance measures available, it is uncertain which measures are the most appropriate for this population. Therefore, the aim of this study was to conduct a systematic review on clinical balance measures used with people with T2DM and DPN. Databases searched included: CINAHL plus, MEDLINE, SPORTDiscus, Dentistry and Oral Sciences source, and SCOPUS. Key terms, inclusion and exclusion criteria were used to identify appropriate studies. Identified studies were critiqued using the Downs and Black appraisal tool. Eight studies were included, these studies incorporated a total of ten different clinical balance measures. The balance measures identified included the Dynamic Balance Test, balance walk, tandem and unipedal stance, Functional Reach Test, Clinical Test of Sensory Interaction and Balance, Berg Balance Scale, Tinetti Performance-Oriented Mobility Assessment, Activity-Specific Balance Confidence Scale, Timed Up and Go test, and the Dynamic Gait Index. Numerous clinical balance measures were used for people with T2DM. However, the identified balance measures did not assess all of the systems of balance, and most had not been validated in a T2DM population. Therefore, future research is needed to identify the validity of a balance measure that assesses these systems in people with T2DM., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Androsace vitaliana (syn. Vitaliana primuliflora ; Primulaceae) has been subject to several taxonomic treatments, whose conclusions ranged from a single species with numerous infraspecific taxa to several species usually without infraspecific taxa. Here, following molecular investigation, several taxonomic changes are made. A single species with the following infraspecific taxa is recognized: subsp. vitaliana (Pyrenees), subsp. cinerea (south-western Alps), subsp. lepontina (Pennine Alps), subsp. sesleri (south-eastern Alps), subsp. praetutiana (Apennines) and subsp. assoana (Iberian Peninsula excluding the Pyrenees), the last of which is divided into the four allopatrically distributed varieties assoana , centriberica , flosjugorum and nevadensis . Contrary to some previous assertions, all taxa are allopatric and, especially for subspp. vitaliana , cinerea and lepontina , where clear diagnostic characters are lacking, they can best be determined by their geographic origin.

Achromatopsia was identified in three Labrador Retriever littermates. The dogs demonstrated day blindness, negotiating obstacles under low-light conditions, but apparently blind when outdoors. One of the dogs presented with immature bilateral diffuse posterior cortical cataracts and clinical signs of day blindness became apparent following cataract extraction surgery. Electroretinography demonstrated an absence of a cone photoreceptor response to a bright stimulus and a flicker response of 30 Hz in all three dogs. No fundic lesions have been apparent ophthalmoscopically in any of the dogs as the initial presentation of each case. No abnormalities were detected with DNA screening for known mutations of the CNGB3 gene in any of the dogs., (© 2015 American College of Veterinary Ophthalmologists.)

Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.

Moapa dace (Moapa coriacea) is a critically endangered thermophilic minnow native to the Muddy River ecosystem in southeastern Nevada, USA. Restricted to temperatures between 26.0 and 32.0 °C, these fish are constrained to the upper two km of the Muddy River and several small tributaries fed by warm springs. Habitat alterations, nonnative species invasion, and water withdrawals during the 20th century resulted in a drastic decline in the dace population and in 1979 the Moapa Valley National Wildlife Refuge (Refuge) was created to protect them. The goal of our study was to determine the potential effects of reduced surface flows that might result from groundwater pumping or water diversions on Moapa dace habitat inside the Refuge. We accomplished our goal in several steps. First, we conducted snorkel surveys to determine the locations of Moapa dace on three warm-spring tributaries of the Muddy River. Second, we conducted hydraulic simulations over a range of flows with a two-dimensional hydrodynamic model. Third, we developed a set of Moapa dace habitat models with logistic regression and a geographic information system. Fourth, we estimated Moapa dace habitat over a range of flows (plus or minus 30% of base flow). Our spatially explicit habitat models achieved classification accuracies between 85% and 91%, depending on the snorkel survey and creek. Water depth was the most significant covariate in our models, followed by substrate, Froude number, velocity, and water temperature. Hydraulic simulations showed 2-11% gains in dace habitat when flows were increased by 30%, and 8-32% losses when flows were reduced by 30%. To ensure the health and survival of Moapa dace and the Muddy River ecosystem, groundwater and surface-water withdrawals and diversions need to be carefully monitored, while fully implementing a proactive conservation strategy.

Agonist-induced internalisation of receptors may lead to the formation of signalling endosomes. There is little evidence relating to whether this occurs to native receptors in non-transformed cells, and no previous studies asking whether this endosomal signalling can promote cell cycle progression in non-transformed cells. We investigated the hypothesis that in primary hepatocytes clathrin-dependent epidermal growth factor (EGF)-induced internalisation of the EGF receptor leads to signalling from endosomal EGF-EGF receptor complexes which may support EGF-stimulated cell cycle progression. We used EGF-stimulation of rat hepatocytes followed by confocal microscopy, and Western blots for phosphoproteins. [(3)H]thymidine incorporation into DNA was used as a indicator of progression to S-phase. Confocal microscopy demonstrated co-internalisation of EGF, EGF receptors and transferrin into endosomes. Internalisation of EGF/EGF receptor/transferrin was blocked by expression of dominant-negative dynamin, but not by the tyrosine kinase inhibitor AG 1478. Dominant-negative dynamin expression reduced EGF-stimulated extracellular signal-related kinase and Akt signalling, but increased tyrosine phosphorylated EGF receptor. EGF-stimulated cell cycle progression requires stimulation of EGF receptors during an initial period (e.g. 1h) and also later during a 24h incubation. EGF receptor internalisation in the presence of AG 1478 followed by removal of the inhibitor resulted in signalling from internalised EGF receptors that is sufficient for the initial stimulation to provide progression to S-phase of the cell cycle. These observations on hepatocytes characterise, for the first time in non-transformed cells, endosomal signalling from internalised EGF receptors, and provide evidence that this endosomal signalling may support the early phase of EGF-stimulated cell cycle progression., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Island radiations are often regarded as natural laboratories that allow us to study evolution in action. The genus Schiedea (Caryophyllaceae) is one of the largest radiations of angiosperms in the Hawaiian Islands, and Schiedea globosa is one of the few species in the genus to be found on more than one of the main islands. DNA sequences from nineteen nuclear and three chloroplast regions show a pattern of colonization from older to younger islands (west to east), with a concomitant decrease in genetic diversity eastwards (π=0.53% for O'ahu, 0.43% for Moloka'i and 0.36% for Maui). While polymorphisms in the maternally inherited chloroplast have become fixed on different islands (F(ST)=0.804), significant gene flow between islands is inferred for the nuclear genome (F(ST)=0.270). This gene flow appears to be uneven, with most gene flow outwards from the central island. The extent of inter-island gene flow through wind pollination was assessed in an isolation-migration framework; the inferred rate, c. 1 migrant per generation, may be sufficient to prevent divergence of S. globosa populations and ensure cohesion of the species following the colonization of new islands., (© 2010 The Authors. Journal of Evolutionary Biology © 2010 European Society For Evolutionary Biology.)

The relative contribution of advantageous and neutral mutations to the evolutionary process is a central problem in evolutionary biology. Current estimates suggest that whereas Drosophila, mice, and bacteria have undergone extensive adaptive evolution, hominids show little or no evidence of adaptive evolution in protein-coding sequences. This may be a consequence of differences in effective population size. To study the matter further, we have investigated whether plants show evidence of adaptive evolution using an extension of the McDonald-Kreitman test that explicitly models slightly deleterious mutations by estimating the distribution of fitness effects of new mutations. We apply this method to data from nine pairs of species. Altogether more than 2,400 loci with an average length of approximately 280 nucleotides were analyzed. We observe very similar results in all species; we find little evidence of adaptive amino acid substitution in any comparison except sunflowers. This may be because many plant species have modest effective population sizes.

Different protein secondary structure elements have different physicochemical properties and roles in the protein, which may determine their evolutionary flexibility. However, it is not clear to what extent protein structure affects the way Darwinian selection acts at the amino acid level. Using phylogeny-based likelihood tests for positive selection, we have examined the relationship between protein secondary structure and selection across six species of Drosophila. We find that amino acids that form disordered regions, such as random coils, are far more likely to be under positive selection than expected from their proportion in the proteins, and residues in helices and beta-structures are subject to less positive selection than predicted. In addition, it appears that sites undergoing positive selection are more likely than expected to occur close to one another in the protein sequence. Finally, on a genome-wide scale, we have determined that positively selected sites are found more frequently toward the gene ends. Our results demonstrate that protein structures with a greater degree of organization and strong hydrophobicity, represented here as helices and beta-structures, are less tolerant to molecular adaptation than disordered, hydrophilic regions, across a diverse set of proteins.

The presence of heterozygous indels in a DNA sequence usually results in the sequence being discarded. If the sequence trace is of high enough quality, however, it will contain enough information to reconstruct the two constituent sequences with very little ambiguity. Solutions already exist using comparisons with a known reference sequence, but this is often unavailable for nonmodel organisms or novel DNA regions. I present a program which determines the sizes and positions of heterozygous indels in a DNA sequence and reconstructs the two constituent haploid sequences. No external data such as a reference sequence or other prior knowledge are required. Simulation suggests an accuracy of >99% from a single read, with errors being eliminable by the inclusion of a second sequencing read, such as one using a reverse primer. Diploid sequences can be fully reconstructed across any number of heterozygous indels, with two overlapping sequencing reads almost always sufficient to infer the entire DNA sequence. This eliminates the need for costly and laborious cloning, and allows data to be used which would otherwise be discarded. With no more laboratory work than is needed to produce two normal sequencing reads, two aligned haploid sequences can be produced quickly and accurately and with extensive phasing information., (© 2009 Blackwell Publishing Ltd.)

Colonization of the south-western European mountain ranges is suggested to have predominantly progressed from the Iberian Peninsula eastwards, but this hypothesis has never been tested in a statistical framework. Here, we test this hypothesis using Androsace vitaliana, a high elevation species with eight mostly allopatric subspecies, which is widely but disjunctly distributed across all major south-western European mountain ranges. To this end, we use plastid and nuclear sequence data as well as fingerprint (amplified fragment length polymorphisms) data and employ Bayesian methods, which allow co-estimation of genealogy and divergence times using explicit demographic models, as well as hypothesis testing via Bayes factors. Irrespective of the ambiguity concerning where A. vitaliana started to diversify -- both the Alps and the mountain ranges of the Iberian Peninsula outside the Pyrenees were possible -- colonization routes were not simply unidirectional, but involved Pleistocene connections between the Alps and mountain ranges of the Iberian Peninsula bypassing the interjacent Pyrenees via long-distance dispersal. In contrast, the species' post-glacial history is shaped by regional gene pool homogenization resulting in the genetic pattern showing good congruence with geographical proximity in agreement with a vicariance model, but only partly supporting current taxonomy.

The biogeographic history of polyploids and their lower-ploid ancestors is an important feature to achieve a better understanding of polyploid evolution. This is exemplified here using the ecologically congruent members of the Androsace adfinis group (Primulaceae) endemic to the southwestern European Alps. Employing relative genome size, AFLP fingerprint and chloroplast sequence haplotype data, we show that Androsace brigantiaca is a recent (probably no more than 0.2 million years) allopolyploid derivative of the geographically close A. adfinis and A. puberula, which formed reciprocally in a comparatively restricted area in the southern Southwestern Alps. Bayesian admixture analysis--also of artificial additive AFLP profiles--shows that the nuclear genome of A. brigantiaca is significantly biased towards the puberula-genome irrespective of maternal parentage. Nevertheless, there is no evidence for genetic interaction (hybridization, introgression) of A. brigantiaca with either of its ancestors, including the widely sympatric A. puberula. Sympatry might be facilitated by ecological displacement on a local scale or might be a transitory phase on the way to competitive replacement via, for instance, polyploid superiority.

Background: The topology of signaling cascades has been studied in quite some detail. However, how information is processed exactly is still relatively unknown. Since quite diverse information has to be transported by one and the same signaling cascade (e.g. in case of different agonists), it is clear that the underlying mechanism is more complex than a simple binary switch which relies on the mere presence or absence of a particular species. Therefore, finding means to analyze the information transferred will help in deciphering how information is processed exactly in the cell. Using the information-theoretic measure transfer entropy, we studied the properties of information transfer in an example case, namely calcium signaling under different cellular conditions. Transfer entropy is an asymmetric and dynamic measure of the dependence of two (nonlinear) stochastic processes. We used calcium signaling since it is a well-studied example of complex cellular signaling. It has been suggested that specific information is encoded in the amplitude, frequency and waveform of the oscillatory Ca(2+)-signal., Results: We set up a computational framework to study information transfer, e.g. for calcium signaling at different levels of activation and different particle numbers in the system. We stochastically coupled simulated and experimentally measured calcium signals to simulated target proteins and used kernel density methods to estimate the transfer entropy from these bivariate time series. We found that, most of the time, the transfer entropy increases with increasing particle numbers. In systems with only few particles, faithful information transfer is hampered by random fluctuations. The transfer entropy also seems to be slightly correlated to the complexity (spiking, bursting or irregular oscillations) of the signal. Finally, we discuss a number of peculiarities of our approach in detail., Conclusion: This study presents the first application of transfer entropy to biochemical signaling pathways. We could quantify the information transferred from simulated/experimentally measured calcium signals to a target enzyme under different cellular conditions. Our approach, comprising stochastic coupling and using the information-theoretic measure transfer entropy, could also be a valuable tool for the analysis of other signaling pathways.

Calcium has been established as a key messenger in both intra- and intercellular signaling. Experimentally observed intracellular calcium responses to different agonists show a variety of behaviors from simple spiking to complex oscillatory regimes. Here we study typical experimental traces of calcium oscillations in hepatocytes obtained in response to phenylephrine and ATP. The traces were analyzed with methods of nonlinear time series analysis in order to determine the stochastic/deterministic nature of the intracellular calcium oscillations. Despite the fact that the oscillations appear, visually, to be deterministic yet perturbed by noise, our analyses provide strong evidence that the measured calcium traces in hepatocytes are prevalently of stochastic nature. In particular, bursting calcium oscillations are temporally correlated Gaussian series distorted by a monotonic, instantaneous, time-independent function, whilst the spiking behavior appears to have a dynamical nonlinear component whereby the overall determinism level is still low. The biological importance of this finding is discussed in relation to the mechanisms incorporated in mathematical models as well as the role of stochasticity and determinism at cellular and tissue levels which resemble typical statistical and thermodynamic effects in physics.

Phylogeographical studies frequently detect range shifts, both expansions (including long-distance dispersal) and contractions (including vicariance), in the studied taxa. These processes are usually inferred from the patterns and distribution of genetic variation, with the potential pitfall that different historical processes may result in similar genetic patterns. Using a combination of DNA sequence data from the plastid genome, AFLP fingerprinting, and rigorous phylogenetic and coalescence-based hypothesis testing, we show that Androsace halleri (currently distributed disjunctly in the northwestern Iberian Cordillera Cantábrica, the eastern Pyrenees, and the French Massif Central and Vosges), or its ancestor, was once more widely distributed in the Pyrenees. While there, it hybridized with Androsace laggeri and Androsace pyrenaica, both of which are currently allopatric with A. halleri. The common ancestor of A. halleri and the north Iberian local endemic Androsace rioxana probably existed in the north Iberian mountain ranges with subsequent range expansion (to the French mountain ranges of the Massif Central and the Vosges) and allopatric speciation (A. rioxana, A. halleri in the eastern Pyrenees, A. halleri elsewhere). We have thus been able to use the reticulate evolution in this species group to help elucidate its phylogeographical history, including evidence of range contraction.

Epidermal growth factor (EGF) stimulation of cell cycle progression in cultured primary hepatocytes has previously been reported to be dependent on the mammalian target of rapamycin (mTOR) elements of the phosphoinositide 3-kinase (PI3K) signaling cascade and not the Akt pathway. Here we have established conditions of combined treatment of rat hepatocytes with insulin and EGF that favor cell cycle progression. The resulting cell population expresses albumin and retains receptor regulation of the signaling pathways leading to glycogen phosphorylase activation. We then investigated the hypothesis that the Akt limb of the PI3K pathway plays a central role in this insulin/EGF enhancement of cell cycle progression. The phosphorylation of Akt, central to the PI3K pathway, was increased by both insulin (sustained) and EGF (transient). The stimulation of Akt phosphorylation was inhibited in a concentration-dependent manner by the PI3K inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). Cell cycle progression in these cultures was reduced, but not abolished, by this inhibitor. The mTOR inhibitor, rapamycin, also inhibited entry into S phase. The novel Akt inhibitor A-443654 [(S)-1-(1H-indol-3-ylmethyl)-2-[5-(3-methyl-1H-indazol-5-yl)-pyridin-3-yloxy]-ethylamine] blocked both EGF-stimulated cell cycle progression and phosphorylation of the Akt substrate glycogen synthase kinase-3. Infection of cells with an adenoviral vector expressing a constitutively active form of Akt but not a kinase-dead form increased hepatocyte proliferation probably through enhanced cell cycle progression and reduced apoptosis. These results show that the Akt element of the PI3K cascade is necessary for EGF-stimulated cell cycle progression and provide evidence that the sustained elevation of Akt alone generates a hyperproliferative window in hepatocyte cultures.

Simulation and modeling is becoming more and more important when studying complex biochemical systems. Most often, ordinary differential equations are employed for this purpose. However, these are only applicable when the numbers of participating molecules in the biochemical systems are large enough to be treated as concentrations. For smaller systems, stochastic simulations on discrete particle basis are more accurate. Unfortunately, there are no general rules for determining which method should be employed for exactly which problem to get the most realistic result. Therefore, we study the transition from stochastic to deterministic behavior in a widely studied system, namely the signal transduction via calcium, especially calcium oscillations. We observe that the transition occurs within a range of particle numbers, which roughly corresponds to the number of receptors and channels in the cell, and depends heavily on the attractive properties of the phase space of the respective systems dynamics. We conclude that the attractive properties of a system, expressed, e.g., by the divergence of the system, are a good measure for determining which simulation algorithm is appropriate in terms of speed and realism.

In the rat both short-term liver function, such as glycogen metabolism, and long-term events such as proliferation after partial hepatectomy, are in part controlled by release of nucleotides such as ATP acting on hepatocyte P2Y(1) and P2Y(2) receptors (members of a family of P2Y receptors for extracellular nucleotides such as ATP and UTP). Here, we have studied P2Y receptor regulation of signaling pathways involved in glycogen phosphorylase activation and proliferation of primary human hepatocytes. Stimulation of cultured hepatocytes with either ATP and UTP, but not UDP or 2-methylthio ADP, led to concentration-dependent increases in cytosolic free Ca(2+) concentration ([Ca(2+)](c); EC(50) for ATP = 3.3 microM, for UTP = 2.3 microM) and [(3)H]inositol (poly)phosphates (EC(50) for ATP = 9.4 microM, for UTP = 15.4 microM). ATP and UTP also stimulated glycogen phosphorylase in human hepatocytes, each with a threshold for activation of less than 1 microM. Application of 2-methylthio ADP up to 100 microM was ineffective. Phosphorylation of both extracellular signal-related kinase and c-Jun N-terminal kinase was stimulated by ATP and UTP, but not by 2-methylthio ADP or UDP, either alone or when costimulated with epidermal growth factor. In conclusion, in human hepatocytes P2Y receptors control both glycogen metabolism and proliferation-associated responses such as increased [Ca(2+)](c) and mitogen-activated protein kinase cascades. Regulation seems to be primarily through P2Y(2) receptors. In contrast with previous studies on rat hepatocytes, there is an absence of responses mediated by P2Y(1) receptors.

Hepatocyte function is regulated by several P2Y receptor subtypes. Here we report that 2-methylthioadenosine 5'-diphosphate (2-MeSADP), an agonist at P2Y(1), P2Y(12), and P2Y(13) receptors, potently (threshold 30 nM) stimulates glycogen phosphorylase in freshly isolated rat hepatocytes. Antagonism by N(6)-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS 2179) confirms that this response is mediated by P2Y(1) receptors. In addition, in these cells, both 2-MeSADP and UTP inhibited glucagon-stimulated cyclic AMP accumulation. This inhibitory effect of 2-MeSADP was not reversed by the P2Y(1) antagonists, adenosine-3'-phosphate-5'-phosphate (A3P5P) or MRS 2179, both in the range 1 to 300 microM, indicating that it was not mediated by P2Y(1) receptors. This contrasts with the increase in cytosolic free Ca(2+) concentration ([Ca(2+)](c)) induced by 2-MeSADP, which has shown to be inhibited by A3P5P. Pertussis toxin abolished the inhibitory effect of both UTP and 2-MeSADP. After culture of cells for 48 h, the ability of 2-MeSADP to inhibit cyclic AMP accumulation was greatly diminished. Reverse transcriptase-polymerase chain reaction analysis revealed that during this culture period, there was a decline in the ability to detect transcripts for P2Y(12) and P2Y(13) receptors, both of which are activated by 2-MeSADP and negatively coupled to adenylyl cyclase. However, in freshly isolated cells, the P2Y(12) and P2Y(13) receptor antagonist, 2-propylthio-beta,gamma-dichloromethylene-d-ATP (AR-C67085) (10 nM to 300 microM) did not alter the ability of 2-MeSADP to inhibit glucagon-stimulated cyclic AMP accumulation. We conclude that 2-MeSADP regulates rat hepatocyte glycogen phosphorylase by acting on P2Y(1) receptors coupled to raised [Ca(2+)](c), and by inhibiting cyclic AMP levels by an unknown G(i)-coupled receptor subtype, distinct from P2Y(1), P2Y(12), or P2Y(13) receptors.

1 Accumulation of inositol (poly)phosphates (InsP(x)) has been studied in rat hepatocytes labelled with [(3)H]inositol. Stimulation with ADP resulted in a significant increase in total [(3)H]InsP(x), whereas 2-MeSADP had only a small effect and ADPbetaS was ineffective. UTP and ITP also stimulated substantial increases in [(3)H]InsP(x). 2 The dose-response curve to ADP was largely unaltered by the presence of the P2Y(1) antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P). Similarly, inclusion of MRS 2179, a more selective P2Y(1) antagonist, had no effect on the dose-response curve to ADP. 3 The inclusion of hexokinase in the assay reduced, but did not abolish, the response to ADP. 4 HPLC analysis revealed that ADP in the medium was rapidly converted to AMP and ATP. The inclusion of hexokinase removed ATP, but exacerbated the decline in ADP concentration, leading to increased levels of AMP. 2-MeSADP was stable in the medium and ATP was largely unaffected. 5 The addition of the adenylate kinase inhibitor, diadenosine pentaphosphate (Ap(5)A) significantly reduced the ADP response. HPLC analysis conducted in parallel demonstrated that this treatment inhibited conversion of ADP to ATP and AMP. 6 Inclusion of the P1 antagonist CGS 15943 had no effect on the dose-response curve to ADP. 7 These observations indicate that hepatocytes respond to ADP with an increase in inositol (poly)phosphates following conversion to ATP. P2Y(1) activation in hepatocytes does not appear to be coupled to inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) production.

We present a new model for calcium oscillations based on experiments in hepatocytes. The model considers feedback inhibition on the initial agonist receptor complex by calcium and activated phospholipase C, as well as receptor type-dependent self-enhanced behavior of the activated G(alpha) subunit. It is able to show simple periodic oscillations and periodic bursting, and it is the first model to display chaotic bursting in response to agonist stimulations. Moreover, our model offers a possible explanation for the differences in dynamic behavior observed in response to different agonists in hepatocytes.

In the absence of selective antagonists, pharmacological characterization of P2Y receptor subtypes has relied heavily upon their distinct agonist profiles. 2-methylthioADP (2-MeSADP) is a selective agonist for the P2Y(1) receptor. The agonist action of 2-MeSATP at the P2Y(1) receptor has recently been questioned. The effects of both 2-MeSADP and 2-MeSATP have been studied on rat hepatocytes injected with the bioluminescent Ca(2+) indicator, aequorin. Single hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca(2+)](i)) when stimulated with agonists acting through the phosphoinositide signalling pathway. The transients induced by 2-MeSADP and 2-MeSATP in the same cell were indistinguishable, indicating that they act at a common receptor. In contrast the transients evoked by ATP and UTP had very different profiles. Treatment of 2-MeSATP with an ATP-regenerating system to remove contaminating 2-MeSADP did not abolish its agonist activity. Application of the P2Y(1) antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P) inhibited the transients induced by both 2-MeSADP and 2-MeSATP. In contrast the transients induced by ATP and UTP were enhanced by the addition of A3P5P. These results indicate that both 2-MeSADP and 2-MeSATP are agonists at the rat hepatocyte P2Y(1) receptor.

Previous studies have indicated the expression of multiple P2Y receptors by rat hepatocytes although they have not been identified. Here we show by reverse transcriptase-polymerase chain reaction (RT - PCR) that rat hepatocytes express mRNA encoding all of the four cloned rat P2Y receptors (P2Y(1), P2Y(2), P2Y(4) and P2Y(6)). The effects of UTP have been examined on single aequorin-injected rat hepatocytes. The [Ca(2+)](i) transients induced by UTP were indistinguishable from those induced by ATP in the same cell. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both UTP- and ATP-induced [Ca(2+)](i) transients. UDP, an agonist at the P2Y(6) receptor, failed to induce transients in hepatocytes, indicating that functional P2Y(6) receptors coupled to increased [Ca(2+)](i) are not expressed. The transients evoked by ADP were more sensitive to inhibition by suramin than those induced by either ATP or UTP. Within an individual cell, the transients induced by ATP and UTP were inhibited by the same concentration of suramin. This sensitivity of ATP and UTP responses to suramin suggests action through P2Y(2) rather than P2Y(4) receptors. Co-application of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) caused a decrease in frequency and amplitude of transients induced by ADP. ATP- and UTP-induced transients also displayed a decrease in amplitude in response to addition of PPADS, but this was accompanied by an increase in frequency of transients. In conclusion the data presented here are consistent with the co-expression of P2Y(1) and P2Y(2) receptors by rat hepatocytes.

We report the results using bioluminescent and fluorescent indicators to investigate maitotoxin-induced free Ca changes in single rat hepatocytes. Maitotoxin generated a steadily rising free Ca increase after a long lag period. The free Ca increase was dependent on extracellular calcium and could be antagonised by chelation of extracellular calcium or the inclusion of nickel in the superfusate. Manganese-induced quench of cytoplasmic Fura2 dextran revealed an accelerated rate of calcium entry during the final period of the lag phase, immediately prior to the free Ca increase. Imaging experiments demonstrated a markedly different part of free Ca mobilisation compared with glycogenolytic stimuli. Moreover, the use of a combination of hormonal stimuli and maitotoxin revealed that some cells could exhibit free Ca oscillations despite steadily rising intracellular free Ca level. The significance of these observations in terms of the mechanism of action of maitotoxin and the mechanism of free Ca transient generation is discussed.

1. Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y2 receptors by human keratinocytes, the cells which comprise the epidermis. 2. Reverse transcriptase-polymerase chain reaction (RT - PCR) revealed expression of mRNA for the G-protein-coupled, P2Y2 receptor in primary cultured human keratinocytes. 3. In situ hybridization studies of skin sections revealed that P2Y2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. 4. Increases in intracellular free Ca2+ concentration ([Ca2+]i) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. 5. In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPgammaS were found to stimulate the proliferation of keratinocytes. 6. Using a real-time firefly luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. 7. These findings indicate that P2Y2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis.

Extracellular nucleotides acting through specific P2 receptors activate intracellular signaling cascades. Consistent with the expression of G protein-coupled P2Y receptors in skeletal tissue, the human osteosarcoma cell line SaOS-2 and primary osteoblasts express P2Y1 and P2Y2 receptors, respectively. Their activation by nucleotide agonists (ADP and ATP for P2Y1; ATP and UTP for P2Y2) elevates [Ca2+]i and moderately induces expression of the c-fos proto-oncogene. A synergistic effect on c-fos induction is observed by combining ATP and parathyroid hormone, a key bone cell regulator. Parathyroid hormone elevates intracellular cAMP levels and correspondingly activates a stably integrated reporter gene driven by the Ca2+/cAMP-responsive element of the human c-fos promoter. Nucleotides have little effect on either cAMP levels or this reporter, instead activating luciferase controlled by the full c-fos promoter. This induction is reproduced by a stably integrated serum response element reporter independently of mitogen-activated protein kinase activation and ternary complex factor phosphorylation. This novel example of synergy between the cAMP-dependent protein kinase/CaCRE signaling module and a non-mitogen-activated protein kinase/ternary complex factor pathway that targets the serum response element shows that extracellular ATP, via P2Y receptors, can potentiate strong responses to ubiquitous growth and differentiative factors.

Single rat hepatocytes, microinjected with the Ca(2+)-sensitive photoprotein aequorin, respond to agonists acting through the phosphoinositide signalling pathway by the generation of oscillations in cytosolic free Ca2+ concentration ([Ca2+]i). The duration of [Ca2+]i transients generated is characteristic of the stimulating agonist; the differences lie in the rate of fall of [Ca2+]i from its peak. We considered that differential sensitivity of the InsP3 receptor may underlie agonist specificity. The thiol reagent, thimerosal, is known to increase the sensitivity of the Ca2+ stores to InsP3 by increasing the affinity of the InsP3 receptor for InsP3 in rat hepatocytes. We show here that a low dose of thimerosal (1 microM), insufficient alone to elevate [Ca2+]i, potentiates [Ca2+]i oscillations induced by phenylephrine or ATP in single, aequorin-injected, rat hepatocytes. Moreover, thimerosal enhances both the frequency and amplitude of phenylephrine-induced oscillations, whereas, in contrast, ATP-induced oscillations undergo an increase in the duration of the falling phase of individual [Ca2+]i transients. Thimerosal, therefore, enhances, rather than eliminates, agonist-specific differences in the hepatocyte [Ca2+]i oscillator.

Extracellular nucleotides acting through P2 receptors elicit a range of responses in many cell types. Previously, we have cloned the G-protein coupled P2Y2 receptor from a human osteoclastoma complementary deoxyribonucleic acid (cDNA) library and demonstrated its expression by reverse transcription linked (RT)-PCR and Southern analysis in a number of skeletal tissues, including a purified population of giant cells. In this study we have localized the expression of P2Y2 receptor transcripts to osteoclasts of giant cell tumor of bone by in situ hybridization. In osteoblasts and other cell types, the P2Y2 receptor is coupled to Ins(1,4,5)P3-mediated Ca2+ release from intracellular stores. In this study, the P2Y2 receptor agonists adenosine triphosphate (ATP) and uridine triphosphate (UTP) did not increase cytosolic free calcium concentration ([Ca2+]i) in giant cells isolated from osteoclastoma, while the G-protein coupled calcium sensing receptor agonist, Ni2+, elevated [Ca2+]i in the same cells. These data indicate that P2Y2 receptor transcripts expressed by giant cells are not presented at the surface of cells as functional receptors, or alternatively, functional receptors are coupled to an effector other than [Ca2+]i. ATPgammaS (10 micromol/L), but not UTP (10 micromol/L), significantly stimulated resorption by an enriched giant cell population. These results indicate that ATP-induced effects on resorption, following direct osteoclastic activation, are mediated by a P2 receptor other than the P2Y2 subtype. Nucleotides, released locally in the bone microenvironment in response to acute trauma or transient physical stress, will interact with a complement of P2 receptors expressed by both osteoclasts and osteoblasts to influence the remodeling process.

Single rat hepatocytes, microinjected with the Ca(2+)-sensitive photoprotein aequorin, respond to agonists acting through the phosphoinositide signalling pathway by the generation of oscillations in cytosolic free Ca2+ concentration ([Ca2+]i). The duration of [Ca2+]i transients generated is characteristic of the receptor species activated; the variability results in differences in the rate of fall of [Ca2+]i from its peak. It is conceivable that the plasma membrane Ca(2+)-ATPase (PM Ca2+ pump) may have an important role in the mechanism underlying agonist specificity. It has recently been shown that an esterified form of carboxyeosin, an inhibitor of the red cell PM Ca2+ pump, is suitable for use in whole cell studies. Glucagon-(19-29) (mini-glucagon) inhibits the Ca2+ pump in liver plasma membranes, mediated by Gs. We show here that carboxyeosin and mini-glucagon inhibit Ca2+ efflux from populations of intact rat hepatocytes. We show that carboxyeosin and mini-glucagon enhance the frequency of oscillations induced by Ca(2+)-mobilizing agonists in single hepatocytes, but do not affect the duration of individual transients. Furthermore, we demonstrate that inhibition of the hepatocyte PM Ca2+ pump enables the continued generation of [Ca2+]i oscillations for a prolonged period following the removal of extracellular Ca2+.

1. Human osteoblasts responded to the application of extracellular nucleotides, acting at P2-receptors, with increases in cytosolic free calcium concentration ([Ca2+]i). 2. In populations of human osteoblasts, adenosine 5'-diphosphate (ADP) evoked a rise in [Ca2+]i with less than 40% of the amplitude of that induced by adenosine 5'-triphosphate (ATP). 3. ATP and uridine 5'-triphosphate (UTP) were applied to single human osteoblasts and induced [Ca2+]i rises of comparable amplitude in every cell tested. 4. However, from the results of single cell studies with ADP (and 2-methylthioATP (2-meSATP)) two groups of cells were delineated; one group responded to ADP (or 2-meSATP) with a rise in [Ca2+]i indistinguishable from that evoked by ATP; whereas the second group failed completely to respond to ADP (or 2-meSATP). 5. Therefore heterogeneity of receptor expression exists within this population of human osteoblasts. The limited distribution of the ADP-responsive receptor underlies the small response to ADP, compared with ATP, recorded in populations of human osteoblasts. This heterogeneity may reflect differences in the differentiation status of individual cells.

Single rat hepatocytes microinjected with the photoprotein aequorin generate oscillations in the cytosolic free Ca2+ concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway. We show here that, in single rat hepatocytes, bovine growth hormone (bGH) is able to induce [Ca2+]i oscillations which display similarities with oscillations induced by phenylephrine. Thus the rate of rise of intracellular Ca2+ in each oscillation closely resembles that induced by Ins(1,4,5)P3-mediated agonists. However, the duration of bGH-induced oscillations increases with agonist concentration, in contrast to phenylephrine-induced oscillations, which undergo an increase in frequency as the agonist concentration is raised, without any increase in the duration of individual oscillations.

1. Aequorin-injected, single rat hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway, including ADP and ATP. We have previously described differences in the [Ca2+]i responses of aequorin-injected hepatocytes to ADP and ATP. 2. The effects of the phosphorothioate analogue of ATP, 2-methylthioATP (2-meSATP), have been examined on single rat hepatocytes. This analogue is belived to be the most potent agonist at the P2Y1 subclass of purinoceptor. 3. The [Ca2+]i transients induced by 2-meSATP were indistinguishable from those induced by ADP, and in contrast to those induced by ATP. 4. At hig concentrations, 2-meSATP and ADP both induced transients at high frequency. In contrast, hepatocytes responded to high concentrations of ATP with an initial rapid rise in [Ca2+]i, followed by a slowly decaying fall. 5. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both 2-meSATP- and ADP-induced [Ca2+]i transients; the peak height and frequency of transients were enhanced. ATP-induced transients, however, underwent either an increase in duration or conversion into a sustained rise in [Ca2+]i. 6. ATP-induced transients were specifically potentiated by the co-addition of alpha, beta-methyleneATP, whereas 2-meSATP- and ADP-induced transients were unaffected by this treatment. 7. We conclude that 2-meSATP acts at the same receptor as ADP on rat hepatocytes, and that this is distinct from teh receptor(s) mediating the effects of ATP.