Characterization and effects on cAMP accumulation of adrenomedullin and calcitonin gene-related peptide (CGRP) receptors in dissociated rat spinal cord cell culture

1 Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) have structural similarities, interact with each others receptors (calcitonin receptor-like receptor (CLR)/receptor-activity-modifying proteins (RAMPs)) and show overlapping biological activities. AM and CGRP receptors are chiefly coupled to cAMP production. In this study, a method of primary dissociated cell culture was used to investigate the presence of AM and CGRP receptors and their effects on cAMP production in embryonic spinal cord cells. 2 Both neuronal and non-neuronal CLR immunopositive cells were present in our model. 3 High affinity, specific [ 125I]-AM binding sites (K(d) 79±9 pM and B(max) 571±34 fmol mg -1 protein) were more abundant than specific [ 125I]-CGRP binding sites (K(d) 12±0.7 pM and B(max) 32±2 fmol mg -1 protein) in embryonic spinal cord cells. 4 Specific [ 125I]-AM binding was competed by related molecules with a ligand selectivity profile of rAM>hAM(22-52)>rCGRPα>CGRP(8-37) ≫[r-(r*,s*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl] carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1, 4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-piperidinecarboxamide (BIBN4096BS). 5 Specific [ 125I]-CGRP binding was competed by rCGRPα>rAM≥ CGRP(8-37)≥BIBN4096BS>hAM(22-52). 6 Cellular levels of cAMP were increased by AM (pEC"5"0 10.2±0.2) and less potently by rCGRPα (pEC"5"0 8.9±0.4). rCGRPα-induced cAMP accumulation was effectively inhibited by CGRP(8-37) (pA"2 7.63±0.44) and hAM(22-52) (pA"2 6.18±0.21) while AM-stimulation of cAMP levels was inhibited by CGRP(8-37) (pA"2 7.41±0.15) and AM(22-52) (pA"2 7.26±0.18). BIBN4096BS only antagonized the effects of CGRP (pA"2 8.40±0.30) on cAMP accumulation. 7 These pharmacological profiles suggest that effects of CGRP are mediated by the CGRP"1 (CLR/RAMP1) receptor in our model while those of AM are related to the activation of the AM"1 (CLR/RAMP2) receptor subtype. © 2006 Nature Publishing Group All rights reserved.

Characterization of tumour necrosis factor alpha release by human granulocytes in response to procainamide challenge

The role of human granulocytes in the promotion of procainamide (PA) toxicity in vitro has been studied and one of the agents responsible for DNA strand scission and cell death in human target cells has been characterized. Crude peripheral blood mononuclear cells (cPBMNs) isolated by density centrifugation, and the lymphocyte cell lines--CCRF-HSB2 and WIL-2NS--were exposed to PA, and DNA strand breaks were quantified by fluorescent analysis of DNA unwinding. Therapeutic plasma concentrations of PA (0-50 microM) caused dose-dependent cytotoxicity, determined by dye exclusion, and strand breaks in cPBMNs incubated for 3 and 1.5 hr at 37 degrees, respectively. Using 50 microM PA a five-fold increase in DNA strand breaks was observed after 1.5 hr, with significant induction of strand breaks also being observed for 10 and 25 microM concentrations. Toxicity was much reduced in lymphocyte cell lines (maximal killing = 3.0% at 50 microM PA compared with 13.2% in cPBMNs). A similar decrease in toxicity was observed where N-acetyl procainamide (NAPA) was substituted for PA (less than 50% of strand breaks at all concentrations). Further investigations showed that the presence of a contaminating granulocyte population in the cPBMN fraction was responsible for the induction of PA toxicity. Incubation of a highly enriched granulocyte population with PA for 1 hr prior to exposure to purified peripheral blood mononuclear cells (pPBMNs) led to the complete restoration of the toxic effects. The resulting cyto- and genotoxicity were not significantly different to levels observed in cPBMNs. Significantly, incubation of granulocytes with NAPA did not induce toxicity in target pPBMNs. Ultrafiltration of granulocyte supernatants led to the identification of two toxic fractions of < 3000 and > 30,000 Da. Temporal studies showed that the toxicity associated with the < 3000 Da fraction appeared during the first 10-15 min incubation with PA whereas the > 30,000 Da fraction did not display significant toxicity until the 40-60 min period. Further assessment of the nature of these agents indicated that the 30,000 Da fraction was a protein. SDS-PAGE analysis showed an inducible 17,800 Da species appearing in granulocyte supernatants after 40 min incubation with PA. Dot blot analysis indicated that tumour necrosis factor alpha (TNF alpha) was present in the > 30,000 Da fraction. Evidence that TNF alpha was the high-molecular weight species responsible for PA-induced toxicity was obtained from neutralization assays employing an anti-TNF alpha antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Casebook on contract law

The law of contract can be a complex and technical subject, rvt the new edition of Jill Poole's Casebook on Contract provides a clear and well-structured exposition of the principles and rules through a comprehensive selection of case law, addressing all aspects encountered on undergraduate courses. Opening with a chapter of valuable advice and guidance on how to successfully develop and improve the essential skills of case-reading, featuring two worked examples, the coverage in this sixth edition expands to incorporate all recent significant decisions and judgments made by the House of Lords and Court of Appeal such as, Director General of Fair Trading v First National Bank plc, Farley v Skinner, Royal Bank of Scotland v Etridge and UCB Corporate Services v Williams. Interesting recent decisions in relation to battle of forms, terms, exemption clauses and misrepresentation are also included. Extracts have been chosen from a wide range of historical and contemporary cases to illustrate the reasoning processes of the court, why decisions are made and how legal principles are developed - enabling cases to be analysed and discussed independently while, taken as a whole, the chapters provide a sound understanding of the modern law of contract. The section on damages for breach of contract has been expanded to reassess Ruxley Electronics and Construction Ltd v Forsyth in the light of Farley v Skinner, the future of Addis v Gramophone Co Ltd is considered in the light of Johnson v Unisys Ltd and Attorney General v Blake is examined in the light of the decision in Esso Petroleum Co Ltd v Niad Ltd. Succinct author comment focuses the reader on the key elements within the extracts, while thought-provoking questions are posed throughout to develop more in-depth analysis. The logical and clear organization of topics has been further improved to more accurately echo the order adopted within the author's popular textbook and closer crossreferencing to this text has been incorporated to highlight where more detailed discussion of issues arising from the caselaw can be explored. As a result, this new edition can be used both as a traditional casebook and as a companion volume to Poole's Textbook on Contract. This edition is also supported by a new companion web site that offers the benefits of essential updating of key materials, sample questions, lists for key further reading sources and relevant web links, additional relevant cases and materials and guidance on successful exam technique. As with previous editions, Casebook on Contract is an invaluable primary source and an essential study aid for all those following elements of contract law as part of the LLB and CPE, as well as for students from related disciplines such as Accounting and Business.

Factors affecting corneoscleral topography

PURPOSE: To evaluate factors affecting corneoscleral profile (CSP) using Anterior Segment Optical Coherence Tomography (AS-OCT) in combination with conventional videokeratoscopy. METHODS: OCT data were collected from 204 subjects of mean age 34.9 years (SD: ±15.2 yrs, range 18 to 65) using the Zeiss Visante AS-OCT and Medmont M300 corneal topographer. Measurements of corneal diameter (CD), corneal sagittal height (CS), iris diameter (ID), corneoscleral junction angle (CSJ) and scleral radius (SR) were extracted from multiple OCT images. Horizontal visible iris diameter (HVID) and vertical palpebral aperture (PA) were measured using a slit lamp graticule. Subject body height was also measured. Associations were then sought between CSP variables and age, height, ethnicity, sex and refractive error data collected. Results: Significant correlations were found between age and ocular topography variables of HVID, PA, CSJ, SR and ID (P<0.0001), while height correlated with HVID, CD and ID, and power vector terms only with vertical plane keratometry, CD and CS. Significant differences were noted between ethnicities with respect to CD (P=0.0046), horizontal and vertical CS (P=0.0068 and P=0.0095), and also horizontal ID (P=0.0010), while the same variables, with the exception of vertical CS, also varied with sex; horizontal CD (P=0.0018), horizontal CS (P=0.0018) and ID (P=0.0012). Age accounted for up to 36% of the variance in CSP variables. Conclusion: Age is the main factor influencing corneoscleral topography; consequently, this should be taken into consideration in contact lens design, in the optimization of surgical procedures involving the cornea and sclera and in IOL selection.