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Low vitamin D and insulin resistance are common in polycystic ovary syndrome (PCOS) and associated with higher inter- and intra-muscular adipose tissue (IMAT). We investigated associations between vitamin D, IMAT and insulin resistance in a cross-sectional study of 40 women with PCOS and 30 women without PCOS, and pre- and post-exercise in a 12-week intervention in 16 overweight participants (10 with PCOS and six without PCOS). A non-classical body mass index (BMI) threshold was used to differentiate lean and overweight women (BMI ≥ 27 kg/m²). Measurements included plasma 25-hydroxyvitamin D (25OHD), insulin resistance (glucose infusion rate (GIR; mg/m²/min), fasting glucose and insulin, and glycated haemoglobin), visceral fat, mid-thigh IMAT (computed tomography) and total body fat (dual-energy X-ray absorptiometry). Women with both PCOS and low 25OHD levels had the lowest GIR (all p < 0.05). Higher IMAT was associated with lower 25OHD (B = -3.95; 95% CI -6.86, -1.05) and GIR (B = -21.3; 95% CI -37.16, -5.44) in women with PCOS. Overweight women with pre-exercise 25OHD ≥30 nmol/L had significant increases in GIR, and decreases in total and visceral fat (all p < 0.044), but no associations were observed when stratified by PCOS status. Women with PCOS and low 25OHD levels have increased insulin resistance which may be partly explained by higher IMAT. Higher pre-training 25OHD levels may enhance exercise-induced changes in body composition and insulin resistance in overweight women., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Study Question: What is the degree of intrinsic insulin resistance (IR) in women with polycystic ovary syndrome (PCOS) and the relative contribution of BMI to overall IR based on meta-analysis of gold standard insulin clamp studies?, Summary Answer: We report an inherent reduction (-27%) of insulin sensitivity (IS) in PCOS patients, which was independent of BMI., What Is Already Known: PCOS is prevalent, complex and underpinned by IR but controversies surround the degree of intrinsic IR in PCOS, the effect of BMI and the impact of the different diagnostic criteria (NIH versus Rotterdam) in PCOS., Study Design, Size, Duration: A systematic review and meta-analysis of Medline and All EBM databases was undertaken of studies published up to 30 May 2015. Studies were included if premenopausal women diagnosed with PCOS were compared with a control group for IS, measured by the gold standard euglycaemic-hyperinsulinaemic clamp. The systematic review adheres to the principles of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Meta-analyses were performed using mixed modelling and magnitude-based inferences expressed as mean effect ±99% CI. We inferred the effect was small, moderate or large relative to a smallest important change of -3.7% or 3.8% derived by standardisation. Effects were deemed unclear when the CI overlapped smallest important positive and negative values. Effects were qualified with probabilities reflecting uncertainty in the magnitude of the true value (likely, 75-95%; very likely, 95-99.5%; most likely, >99.5%)., Participants/materials, Setting, Method: A total of 4881 articles were returned from the search. Of these, 28 articles were included in the meta-analysis., Main Results and the Role of Chance: Overall IS was lower in women with PCOS compared with controls (mean effect -27%, 99% CI ±6%; large, most likely lower). A higher BMI exacerbated the reduction in IS by -15% (±8%; moderate, most likely lower) in PCOS compared with control women. There was no clear difference in IS between women diagnosed by the original National Institutes of Health  (NIH) criteria alone compared with those diagnosed by the Rotterdam criteria. Low levels of sex hormone-binding globulin (SHBG) were associated with reduced levels of IS (-10%, ±10%; small, very likely negative), which was not confounded by BMI., Limitations, Reasons for Caution: This systematic review and meta-analysis inherited the confounding problems of small sample sizes, missing data (e.g. some hormones, waist and hip girths) and the lack of Rotterdam criteria phenotype reporting, limiting the evidence synthesis and meta-analysis., Wider Implications of the Findings: BMI has a greater impact on IS in PCOS than in controls. SHBG appears a potentially valuable marker of IR in PCOS, whereas testosterone after adjustment for BMI demonstrated an unexpected interplay with IS which warrants further investigation., Study Funding/competing Interests: This work was supported by grants from the National Health & Medical Research Council (NHMRC), grant number 606553 (H.J.T., N.K.S.), as well as Monash University. H.J.T. is an NHMRC Research Fellow. N.K.S. is supported through the Australian Government's Collaborative Research Networks (CRN) programme. The funding bodies played no role in the design, methods, data management or analysis or in the decision to publish. All authors declare no conflict of interests., Registration Number: N/A., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aging is associated with a reduction in osteoblast life span and the volume of bone formed by each basic multicellular unit. Each time bone is resorbed, less is deposited producing microstructural deterioration. Aging is also associated with insulin resistance and hyperglycemia, either of which may cause, or be the result of, a decline in undercarboxylated osteocalcin (ucOC), a protein produced by osteoblasts that increases insulin sensitivity. We examined whether glucose-loading reduces bone remodeling and ucOC in vivo and osteoblast function in vitro, and so compromises bone formation. We administered an oral glucose tolerance test (OGTT) to 18 pre and postmenopausal, nondiabetic women at rest and following exercise and measured serum levels of bone remodeling markers (BRMs) and ucOC. We also assessed whether increasing glucose concentrations with or without insulin reduced survival and activity of cultured human osteoblasts. Glucose-loading at rest and following exercise reduced BRMs in pre and postmenopausal women and reduced ucOC in postmenopausal women. Higher glucose correlated negatively, whereas insulin correlated positively, with baseline BRMs and ucOC. The increase in serum glucose following resting OGTT was associated with the reduction in bone formation markers. D-glucose (>10 mmol L(-1)) increased osteoblast apoptosis, reduced cell activity and osteocalcin expression compared with 5 mmol L(-1). Insulin had a protective effect on these parameters. Collagen expression in vitro was not affected in this time course. In conclusion, glucose exposure reduces BRMs in women and exercise failed to attenuate this suppression effect. The suppressive effect of glucose on BRMs may be due to impaired osteoblast work and longevity. Whether glucose influences material composition and microstructure remains to be determined., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Scope: Polycystic ovary syndrome (PCOS) is underpinned by insulin resistance (IR). In PCOS, the relationships between vitamin D, adiposity, and IR are unclear. We aim to explore these relationships in lean and overweight women with PCOS., Methods and Results: This is a cross-sectional study conducted in a tertiary medical center. Participants included 42 women with PCOS and 34 controls without PCOS. Vitamin D and metabolic markers were measured. Detailed body composition and gold standard hyperinsulinemic euglycemic clamps were performed. The main outcome measures were plasma levels of vitamin D, adiposity measures, and glucose infusion rate. Vitamin D levels were lower in overweight women with PCOS compared with overweight controls (31.6 and 46.1 nmol/L, respectively, p = 0.01). Vitamin D was not associated with IR after adjustment for confounders; however, there was a significant interaction between PCOS and percentage body fat. Further analysis by PCOS status revealed that vitamin D was associated with IR in the PCOS group (β coefficient 2.1, 95% CI 0.2-4.0, p = 0.03), but not in the non-PCOS group., Conclusion: Vitamin D is associated with IR in women with PCOS, but not in controls. Large intervention studies are needed to determine if vitamin D supplementation can improve IR in PCOS., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Zebrafish are an attractive vertebrate model due to their small size, transparency through organogenesis, and high fecundity. The zebrafish gastrointestinal (GI) tract is similar to the mammalian GI tract in gene expression, nervous system control, and response to chemical challenges. GI intolerance is a common preclinical finding and can be a serious clinical safety concern. Mammalian GI liability tests are conducted at the expense of time, test article, and labor. We developed a high-throughput method to predict mammalian GI safety issues using larval zebrafish., Methods: Fluorescent food is fed to larval zebrafish (7 days post fertilization). After feeding, larvae are placed individually into wells of a 96-well plate and dosed with test compounds. Fluorescence is measured from the bottom of the wells repeatedly over the course of 24h and thus fecal accumulation is tracked over time. The area under the curve is compared between treated and vehicle-treated groups., Results: Drugs with established clinical GI effects significantly impacted zebrafish GI transit time as measured by this method; tegaserod and metoclopramide accelerated transit time, while atropine and amitriptyline slowed transit time. This method is sensitive enough to reflect dose-level associated effects as demonstrated using atropine. Using a suite of 24 compounds with known (positive or negative) mammalian GI effects, we characterized this method as having a high positive predictive value., Discussion: Here we present an efficient assay for predicting mammalian GI transit liabilities using larval zebrafish. With this assay, an investigator can evaluate dozens of compounds in a single day using very little amount of each test article. As such, safe drug candidates can be prioritized for mammalian testing, which expedites the discovery process and provides 3 Rs impact., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Polycystic ovary syndrome (PCOS) is a common endocrine disorder associated with metabolic complications. Metabolic biomarkers with roles in obesity, glycaemic control and lipid metabolism are potentially relevant in PCOS. The aim was to investigate metabolic biomarkers in lean and overweight women with and without PCOS and to determine whether any biomarker was able to predict insulin resistance in PCOS., Design: Cross-sectional study., Patients: Eighty-four women (22 overweight and 22 lean women with PCOS, 18 overweight and 22 lean women without PCOS) were recruited from the community and categorized based on PCOS and BMI status., Measurements: Primary outcomes were metabolic biomarkers [ghrelin, resistin, visfatin, glucagon-like peptide-1 (GLP-1), leptin, plasminogen activator inhibitor -1 (PAI-1), glucose-dependent insulinotropic polypeptide (GIP) and C-Peptide] measured using the Bio-Plex Pro Diabetes assay and insulin sensitivity as assessed by glucose infusion rate on euglycaemic-hyperinsulinaemic clamp., Results: The biomarkers C-peptide, leptin, ghrelin and visfatin were different between overweight and lean women, irrespective of PCOS status. The concentration of circulating biomarkers did not differ between women with PCOS diagnosed by the Rotterdam criteria or National Institute of Health criteria. PAI-1 was the only biomarker that significantly predicted insulin resistance in both control women (P = 0.04) and women with PCOS (P = 0.01)., Conclusions: Biomarkers associated with metabolic diseases appear more strongly associated with obesity rather than PCOS status. PAI-1 may also be a novel independent biomarker and predictor of insulin resistance in women with and without PCOS., (© 2014 John Wiley & Sons Ltd.)

High-intensity interval exercise (HIIE) has gained popularity in recent years for patients with cardiovascular and metabolic diseases. Despite potential benefits, concerns remain about the safety of the acute response (during and/or within 24 hours postexercise) to a single session of HIIE for these cohorts. Therefore, the aim of this study was to perform a systematic review to evaluate the safety of acute HIIE for people with cardiometabolic diseases. Electronic databases were searched for studies published prior to January 2015, which reported the acute responses of patients with cardiometabolic diseases to HIIE (≥80% peak power output or ≥85% peak aerobic power, VO2peak). Eleven studies met the inclusion criteria (n = 156; clinically stable, aged 27-66 years), with 13 adverse responses reported (~8% of individuals). The rate of adverse responses is somewhat higher compared to the previously reported risk during moderate-intensity exercise. Caution must be taken when prescribing HIIE to patients with cardiometabolic disease. Patients who wish to perform HIIE should be clinically stable, have had recent exposure to at least regular moderate-intensity exercise, and have appropriate supervision and monitoring during and after the exercise session.

Objective: Polycystic ovary syndrome (PCOS) is a complex endocrine disorder associated with insulin resistance, hyperandrogenism, obesity, altered gonadotrophin release and anovulatory infertility. Anti-Müllerian hormone (AMH) has been proposed as a marker of ovarian function and fertility. Across a cohort of lean and overweight women with and without PCOS, we investigated the association of AMH with insulin resistance and body composition using gold standard measures. A secondary aim was to examine whether AMH was useful to determine PCOS status., Design: Cross-sectional study., Patients: A total of 22 lean and 21 overweight women with PCOS and 19 lean and 16 overweight non-PCOS healthy controls were recruited. PCOS was diagnosed based on the Rotterdam criteria., Measurements: Euglycaemic-hyperinsulinaemic clamp for assessing insulin resistance, dual energy X-ray absorptiometry and computed tomography for assessing adiposity, and blood sampling for the assessment of androgens, gonadotrophins and AMH., Results: Anti-Müllerian hormone levels were increased in women with PCOS (P <0·001) regardless of adiposity, with this increase associated with testosterone (P <0·001) rather than insulin resistance (P = 0·79), adiposity (P = 0·98) or gonadotrophins. In assessing the ability of AMH to predict PCOS, a value of 30 pmol/l or higher indicated 79% of women with PCOS were correctly identified as having the condition., Conclusion: Anti-Müllerian hormone appears primarily related to androgen status suggesting a direct and predominant role of androgens in the pathophysiology of reproductive dysfunction in PCOS. As AMH reflects PCOS status, it may also be useful in PCOS diagnosis., (© 2014 John Wiley & Sons Ltd.)

Purpose: This paper assesses the patients' healthcare information needs and expectations when they attend a rheumatology outpatient clinic. It proposes that obtaining the patients' perspectives about the services they use is an essential service-development tool. The aim of this paper is to expand the current domains used to evaluate these perspectives., Design/methodology/approach: This paper is an exploratory study, looking at quality assessment and improvement based on Donabedian's quality model in a rheumatology outpatient setting. A structured interview schedule addressing care pathways was used and 70 consecutive patients were recruited., Findings: The article provides insights about how relevant change can be brought about when service development is contemplated. It suggests that patients are important stakeholders in the ongoing service development process., Research Limitations/implications: Because rheumatological conditions tend to be chronic and require long-term follow-up, the results may lack generalisability. Therefore, researchers are encouraged to test propositions in different clinical settings., Practical Implications: The article highlights healthcare delivery areas that are not meeting patient expectations. Some recommendations (such as informing waiting patients regularly about any delays) require minimal additional resources for successful implementation. Service providers need to obtain the patients' healthcare perspectives to ensure that services are built around their needs., Originality/value: This article fulfils an identified need to study how patients perceive service quality.

The earliest stages of osteoarthritis are characterized by peripheral pathology; however, during disease progression chronic pain emerges-a major symptom of osteoarthritis linked to neuroplasticity. Recent clinical imaging studies involving chronic pain patients, including osteoarthritis patients, have demonstrated that functional properties of the brain are altered, and these functional changes are correlated with subjective behavioral pain measures. Currently, preclinical osteoarthritis studies have not assessed if functional properties of supraspinal pain circuitry are altered, and if these functional properties can be modulated by pharmacological therapy either by direct or indirect action on brain systems. In the current study, functional connectivity was first assessed in order to characterize the functional neuroplasticity occurring in the rodent medial meniscus tear (MMT) model of osteoarthritis-a surgical model of osteoarthritis possessing peripheral joint trauma and a hypersensitive pain state. In addition to knee joint trauma at week 3 post-MMT surgery, we observed that supraspinal networks have increased functional connectivity relative to sham animals. Importantly, we observed that early and sustained treatment with a novel, peripherally acting broad-spectrum matrix metalloproteinase (MMP) inhibitor (MMPi) significantly attenuates knee joint trauma (cartilage degradation) as well as supraspinal functional connectivity increases in MMT animals. At week 5 post-MMT surgery, the acute pharmacodynamic effects of celecoxib (selective cyclooxygenase-2 inhibitor) on brain function were evaluated using pharmacological magnetic resonance imaging (phMRI) and functional connectivity analysis. Celecoxib was chosen as a comparator, given its clinical efficacy for alleviating pain in osteoarthritis patients and its peripheral and central pharmacological action. Relative to the vehicle condition, acute celecoxib treatment in MMT animals yielded decreased phMRI infusion responses and decreased functional connectivity, the latter observation being similar to what was detected following chronic MMPi treatment. These findings demonstrate that an assessment of brain function may provide an objective means by which to further evaluate the pathology of an osteoarthritis state as well as measure the pharmacodynamic effects of therapies with peripheral or peripheral and central pharmacological action., (Copyright © 2012 Elsevier Inc. All rights reserved.)

This is the second in a two-part series on quality of life (QoL) after percutaneous coronary intervention (PCI). Part I presented the research methodology of this retrospective cross-sectional descriptive study that investigated the QoL of a systematic sample of clients after PCI with a response rate of 64% (n=228; menn=169, women n=59; age 40-89 years). Quantitative data was collected using the Maltese version of the mailed WHOQoL-BREF questionnaire. The study aimed to identify significant differences in QoL between subgroups of clients according to demographic characteristics and their perceived cardiovascular risk factors.Significant differences were found in the overall QoL between subgroups of gender,education, occupation, friends' support and stress. The domain of social support,which is characteristic of Maltese culture, appeared to yield high mean scores of the overall QoL, irrespective of cardiovascular risk factors, such as smoking and alack of physical exercise. Hence, social relationships may enhance QoL as they mayyield security in life, feelings of love and belonging to a community. Perceived stress and social support seemed to generate significant differences in QoL. Although these findings were consistent with existing research, the use of different operational definitions of QoL means they cannot be compared accurately. While considering the limitations of this study, recommendations addressed the need for holistic assessment of patients' needs, education, follow-up monitoring and community services. Further comparative longitudinal research is recommended to identify confounding variables and fluctuation of results across time.

Quality of life (QOL) is a complex concept comprised of biopsychosocial, spiritual and environmental dimensions. However, the majority of research addresses only its physical function perspectives. This two-part series examines the holistic perspective of QOL of patients after percutaneous coronary intervention (PCI). Part 1 explains the research process of a cross-sectional descriptive study and its limitations. Data were collected by a mailed WHOQOL-BREF questionnaire in Maltese from a systematic sample of patients who had undergone PCI; the response rate was 64% (n=228; males n=169, females n=59, age 40-89 years). Part 1 also considers limitations, such as its cross-sectional design and retrospective data collection. The hierarchy of human needs theory (Maslow, 1999) guided the study. Part 2 gives the findings on the holistic view of QOL. Having social and family support, as a characteristic of Maltese culture appeared to contribute towards a better QOL.

Genistein and 5-hydroxyindole (5-HI) potentiate the α7 nicotinic acetylcholine receptor current by primarily increasing peak amplitude, a property of type I α7 positive allosteric modulation. In this study, the effects of these two compounds were investigated at two different α7/5-HT(3) chimeras (chimera 1, comprising of extracellular α7 N-terminus fused to the remainder of 5-HT(3A), and chimera 2 containing an additional α7 encoded M2-M3 loop), and wild-type α7 and 5-HT(3A) receptors. Agonist-evoked responses, examined by expression of the chimeras in Xenopus laevis oocytes or HEK-293 cells, revealed that currents decayed slower and compounds {rank order: N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987)~2-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5-phenyl-1,3,4-oxadiazole (NS6784)>acetylcholine>choline} were more potent in chimera 2 than chimera 1 or α7 receptors. In chimera 2, genistein and 5-HI potentiated agonist-evoked responses (EC(50): 4-5 μM for genistein and 300-500 μM for 5-HI) and at higher concentrations evoked current directly consistent with ago-allosteric modulation. At chimera 1 and 5-HT(3A) receptors, neither compound directly evoked any current and 5-HI, only at chimera 1, was able to potentiate agonist-evoked responses. Genistein and 5-HI did not inhibit the binding of the α7 agonist [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1] heptane ([(3)H]A-585539) to rat brain or chimera 2. In summary, this study supports the role of the M2-M3 loop being critical for the positive allosteric effect of genistein, but not 5-HI, and in agonist-evoked response fine-tuning. The identification of distinct α7 receptor modulatory sites offers unique opportunities for developing CNS therapeutics and understanding its pharmacology., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The alpha7 nicotinic acetylcholine receptor (nAChR), a homopentameric, rapidly activating and desensitizing ligand-gated ion channel with relatively high degree of calcium permeability, is expressed in the mammalian central nervous system, including regions associated with cognitive processing. Selective agonists targeting the alpha7 nAChR have shown efficacy in animal models of cognitive dysfunction. Use of positive allosteric modulators selective for the alpha7 receptor is another strategy that is envisaged in the design of active compounds aiming at improving attention and cognitive dysfunction. The recent discovery of novel positive allosteric modulators such as 1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethylphenyl)urea (NS-1738) and 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea (PNU-120596) that are selective for the alpha7 nAChRs but display significant phenotypic differences in their profile of allosteric modulation, suggests that these molecules may act at different sites on the receptor. Taking advantage of the possibility to obtain functional receptors by the fusion of proteins domains from the alpha7 and the 5-HT(3) receptor, we examined the structural determinants required for positive allosteric modulation. This strategy revealed that the extracellular N-terminal domain of alpha7 plays a critical role in allosteric modulation by NS-1738. In addition, alpha7-5HT(3) chimeras harboring the M2-M3 segment showed that spontaneous activity in response to NS-1738, which confirmed the critical contribution of this small extracellular segment in the receptor gating. In contrast to NS-1738, positive allosteric modulation by PNU-120596 could not be restored in the alpha7-5HT(3) chimeras but was selectively observed in the reverse 5HT(3)-alpha7 chimera. All together, these data illustrate the existence of distinct allosteric binding sites with specificity of different profiles of allosteric modulators and open new possibilities to investigate the alpha7 receptor function.

Objective: The purpose of this series was to evaluate the role of spectral Doppler and color flow Doppler sonography in the evaluation of partial testicular torsion., Methods: Eight cases of partial testicular torsion, diagnosed on the basis of abnormal spectral Doppler waveforms or abnormal color flow Doppler findings, were retrospectively pooled from 2 teaching hospitals., Results: The age group ranged from 4 to 85 years. Testicles with partial testicular torsion showed variable spectral Doppler patterns, including increased, similar, or decreased amplitude of the arterial waveform relative to the contralateral testicle. Two cases showed reversal of arterial diastolic flow, and 1 case showed diastolic flow variability within the same testicle. Decreased blood flow was observed on color flow Doppler sonography in 7 of the 8 patients., Conclusions: Variability of the Doppler waveform when compared with the contralateral testicle and reversal of diastolic blood flow are indirect clues that aid in the diagnosis of partial testicular torsion.

Sperm-specific CatSper1 and CatSper2 proteins are critical to sperm-hyperactivated motility and male fertility. Although architecturally resembling voltage-gated ion channels, neither CatSper1 nor CatSper2 alone forms functional ion channels in heterologous expression systems, which may be related to the absence of yet unidentified accessory subunits. Here we isolated CatSper1- and CatSper2-associated protein(s) from human sperm and analyzed their identities by a multidimensional protein identification technology approach. We identified the T-type voltage-gated calcium channel Ca(v)3.3 as binding to both CatSper1 and CatSper2. The specificity of their interactions was verified by co-immunoprecipitation in transfected mammalian cells. Electrophysiological studies revealed that the co-expression of CatSper1 or CatSper2 specifically inhibited the amplitude of Ca(v)3.3-evoked T-type calcium current without altering other biophysical properties of Ca(v)3.3. Immunostaining studies revealed co-localization of CatSper1 and Ca(v)3.3 on the principal piece of human sperm tail. Furthermore, fluorescence resonance energy transfer analysis revealed close proximity and physical association of these two proteins on the sperm tail. These studies demonstrate that CatSper1 and CatSper2 can associate with and modulate the function of the Ca(v)3.3 channel, which might be important in the regulation of sperm function.

1. A-349821 is a selective histamine H3 receptor antagonist/inverse agonist. Herein, binding of the novel non-imidazole H3 receptor radioligand [3H]A-349821 to membranes expressing native or recombinant H3 receptors from rat or human sources was characterized and compared with the binding of the agonist [3H]N--methylhistamine ([3H]NMH). 2. [3H]A-349821 bound with high affinity and specificity to an apparent single class of saturable sites and recognized human H3 receptors with 10-fold higher affinity compared to rat H3 receptors. [3H]A-349821 detected larger populations of receptors compared to [3H]NMH. 3. Displacement of [3H]A-349821 binding by H3 receptor antagonists/inverse agonists was monophasic, suggesting recognition of a single binding site, while that of H3 receptor agonists was biphasic, suggesting recognition of both high- and low-affinity H3 receptor sites. 4. pKi values of high-affinity binding sites for H3 receptor competitors utilizing [3H]A-349821 were highly correlated with pKi values obtained with [3H]NalphaMH, consistent with labelling of H3 receptors by [3H]A-349821. 5. Unlike assays utilizing [3H]NMH, addition of GDP had no effect on saturation parameters measured with [3H]A-349821, while displacement of [3H]A-349821 binding by the H3 receptor agonist histamine was sensitive to GDP. 6. In conclusion, [3H]A-349821 labels interconvertible high- and low-affinity states of the H3 receptor, and displays improved selectivity over imidazole-containing H3 receptor antagonist radioligands. [3H]A-349821 competition studies showed significant differences in the proportions and potencies of high- and low-affinity sites across species, providing new information about the fundamental pharmacological nature of H3 receptors.

Pharmacological effects of cannabinoid ligands are thought to be mediated through cannabinoid CB1 and CB2 receptor subtypes. Sequence analysis revealed that rat and human cannabinoid CB2 receptors are divergent and share 81% amino acid homology. Pharmacological analysis of the possible species differences between rat and human cannabinoid CB2 receptors was performed using radioligand binding and functional assays. Pronounced species selectivity at the rat cannabinoid CB2 receptor (50- to 140-fold) was observed with AM-1710 (3-(1,1-Dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo[c]chromen-6-one) and AM-1714 (3-(1,1-Dimethyl-heptyl)-1-9-dihydroxy-benzo[c]chromen-6-one). In contrast, JWH-015 ((2-Methyl-1-propyl-1H-indol-3-yl)-napthalen-1-yl-methanone) was 3- to 10-fold selective at the human cannabinoid CB2 receptor. Endocannabinoid ligands were more human receptor selective. Cannabinoid CB2 receptor antagonist, AM-630 ((6-Iodo-2-methyl-1-(2-morpholin-4-yl-ethyl)-1H-indol-3-yl)-(4-methoxy-phenyl)-methanone) was more potent at the rat receptor in radioligand binding and functional assays than that of the human receptor. The findings of the pharmacological differences between the human and rat cannabinoid CB2 receptors in this study provide critical information for characterizing cannabinoid ligands in in vivo rodent models for drug discovery purpose.

Species differences have been described previously for histamine H(3) receptor pharmacology. Rat selective histamine H(3) receptor ligands such as ciproxifan and A-304121 (2-amino-1-[4-[3-(4-cyclopropanecarbonyl-phenoxy)-propyl]-piperazin-1-yl]-propan-1-one) show over 100-fold selectivity for the rat receptor compared to the human receptor. To date, however, the pharmacology of the cloned monkey histamine H(3) receptor has not been examined. In this study, we cloned the monkey histamine H(3) receptor gene (H(3)R) and evaluated the receptor pharmacology in binding and functional assays. The monkey histamine H(3) receptor is highly homologous to the human receptor with 438 identities in their 445 amino acid sequences, but less homologous to the rat receptor. However, unlike the human or rat, we found no evidence for additional splicing for the monkey H(3)R. Pharmacological analysis indicated that the monkey receptor exhibited similar pharmacological profiles to those of the human receptor, providing critical information for characterizing histamine H(3) receptor ligands in monkey behavioral models.

Axotomized motoneurons regenerate their axons regardless of whether axotomy occurs proximally or distally from their cell bodies. In contrast, regeneration of rubrospinal axons into peripheral nerve grafts has been detected after cervical but not after thoracic injury of the rubrospinal tract. By using in situ hybridization (ISH) combined with reliable retrograde tracing methods, we compared regeneration-associated gene expression after proximal and distal axotomy in spinal motoneurons versus rubrospinal neurons. Regardless of whether they were axotomized at the iliac crest (proximal) or popliteal fossa (distal), sciatic motoneurons underwent highly pronounced changes in ISH signals for Growth Associated Protein 43 (GAP-43) (10-20x increase) and neurofilament M (60-85% decrease). In contrast, tubulin ISH signals substantially increased only after proximal axotomy (3-5x increase). To compare these changes in gene expression with those of axotomized rubrospinal neurons, the rubrospinal tract was transected at the cervical (proximal) or thoracic (distal) levels of the spinal cord. Cervically axotomized rubrospinal neurons showed three- to fivefold increases in ISH signals for GAP-43 and tubulins (only transient) and a 75% decrease for neurofilament-M. In sharp contrast, thoracic axotomy had only marginal effects. After implantation of peripheral nerve transplants into the spinal cord injury sites, retrograde labeling with the sensitive retrograde tracer Fluoro-Gold identified regenerating rubrospinal neurons only after cervical axotomy. Furthermore, rubrospinal neurons specifically regenerating into the transplants were hypertrophied and expressed high levels of GAP-43 and tubulins. Taken together, these data support the concept that, even if central nervous system (CNS) axons are presented with a permissive/supportive environment, appropriate cell body responses to injury are a prerequisite for CNS axonal regeneration., (Copyright 1999 Wiley-Liss, Inc.)

A population-based study of the biology of the thin-level melanoma according to site, Breslow's thickness, and Clark's level was undertaken. Two hundred fifteen patients were studied with a mean follow-up of 41 months. Overall, 23 patients (10.7%) had recurrences, 8 locally, 9 regionally, and 6 systemically, despite an adequate local excision. A multivariate analysis was done. In the patients with thin lesions (less than 1 mm), increasing level (p < 0.002) and head and neck site (p < 0.04) increased the risk of recurrence. Increasing thickness of melanoma up to 1 mm did not influence the risk. This study identifies a group of high-risk melanoma patients for whom adjuvant therapy to decrease recurrences should be studied.