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Advertising agencies are hired to develop creative advertising for their clients. This paper explores the advertising creative process used by agencies when developing new creative work. Using in-depth interviews with 21 agency practitioners in the United Kingdom (UK) this study examines the stages that take place within the advertising creative process. Findings suggest the process is made up of a series of sequentially linked stages and illustrate how agencies validate advertising creative during development. The study provides insight into how agencies customise the process and identifies that agencies have different approaches to the level of client involvement. Implications for practitioners are discussed and areas for future research identified.

Canine rapid ventricular pacing produces a low output cardiomyopathic state which is similar to dilated cardiomyopathy. In this study dogs were paced at 245 beats per minute (bpm) for 3-4 weeks until signs of heart failure were apparent. Unpaced dogs were used as controls. A previous study identified myocardial protein changes in the pH region 4-7 following ventricular pacing by using two-dimensional electrophoresis (2-DE) (Heinke et al., Electrophoresis 1998 19, 2021-2030). Many of these proteins were associated with mitochondria, energy metabolism within the cardiomyocyte, the cytoskeleton and calcium cycling. The present study aimed to examine the proteins migrating in the more basic region of the 2-DE pattern using immobilised pH gradient 3-10 strips to separate myocardial proteins. The expression of 31 proteins was altered in the paced myocardium: 21 were decreased and 10 increased. Following the identification of 23 of these spots by either amino acid compositional analysis or peptide mass fingerprinting or a combination of both, we confirm that many of the proteins whose expression is altered following ventricular pacing are associated with the mitochondria and energy production within the cardiomyocyte, including creatine kinase M, triosephosphate isomerase, phosphoglycerate mutase, cytochrome c oxidase, cytochrome b5, hydroxymethyl glutaryl CoA synthase, myoglobin, and 3,2-trans-enoyl-CoA transferase. Additionally, the cytoskeletal protein actin was increased in the paced hearts. These results strongly support the notion that energy production is impaired and mitochondrial dysfunction is involved in the development of heart failure in the paced dog.

Background In the medical literature immunoglobulin (Ig)E-mediated sensitization to avocado is rarely reported. On the other hand, more than 50% of subjects having IgE-mediated natural rubber latex allergy are sensitized to avocado fruit as demonstrated by skin-prick testing and/or specific IgE measurements and about 10–20% report hypersensitivity reactions after ingesting avocado. Objective The underlying pathomechanism of latex-associated avocado allergy is still unknown. The conserved hevein domain of the major latex allergen prohevein (Hev b 6.01) is a ubiquitous chitin-binding protein structure that can be found in several plant proteins and may be responsible for the observed cross-reactivity between latex and avocado fruit. Methods Chitin-binding avocado proteins (CBAPs) were isolated by affinity-chromatography and their IgE-binding characteristics were studied by immunoblotting using the sera from 15 avocado-sensitized latex patients. Inhibition experiments using isolated hevein and CBAPs as inhibitor solutions were performed to study the immunological cross-reactivity between both protein species and to assess the role of the CBAPs as mediators in latex-associated avocado allergy. Results In 80% of avocado-sensitized subjects (n = 15), IgE antibodies directed against a 31-kDa allergen were detected by immunoblotting. This IgE-binding protein was identified by protein sequencing to be a class I endochitinase containing a hevein domain at the N-terminus. Purified native and digested (using simulated gastric fluid) endochitinase were able to completely block all avocado-specific IgE antibodies in six out of seven avocado patients. Conclusions Sensitization to endochitinase class I containing a hevein domain is the main underlying pathomechanism in latex-mediated avocado allergy.

Bovine hereditary dilated cardiomyopathy (bCMP) is endemic in Switzerland and hearts from diseased animals display important clinical and biochemical similarities to human DCM. Recent research has identified at least one protein (myoglobin) to be significantly reduced in bovine DCM. Using a proteomic approach, we have separated over 1125 protein species from bovine ventricular tissue. Gel analysis and protein characterisation have identified a number of proteins whose abundance is significantly altered in bovine DCM. Twenty-four proteins are of decreased abundance in diseased tissue, whilst 11 proteins are of increased abundance in the diseased state. A combination of amino acid compositional analysis, peptide mass profiling, N-terminal microsequencing and MultiIdent (http://www.expasy.ch/sprot/multiident. html) has been employed in order to elucidate the identities of the differentially expressed proteins. Using these techniques we have currently determined the identity of 12 of the 35 altered proteins. We have also detected three proteins that are differentially expressed in genotypically diseased but phenotypically normal animals, identifying a possible mechanism for the onset of the disease. The possibility that inappropriate ubiquination of proteins plays an important role in the disease is discussed. A database of bovine proteins is currently being established. The identity of the proteins affected, together with a comparison of the human and bovine expression patterns, is displayed.

The aim of the investigation was to determine whether there are specific global quantitative and qualitative changes in protein expression in heart tissue from patients with dilated cardiomyopathy (DCM) compared with ischaemic heart disease and undiseased tissue. Two-dimensional (2-D) polyacrylamide gel electrophoresis and computer analysis was used to study protein alteration in DCM biopsy material (n=28) compared with donor heart biopsy samples (n=9) and explanted hearts from individuals suffering from ischaemic heart disease (IHD; n = 21). A total of 88 proteins displayed decreased abundance in DCM versus IHD material while five proteins had elevated levels in the DCM group (p

To investigate the receptor tolerances to N-terminal variation, novel analogues to Locusta AKH-I (adipokinetic hormone) have been synthesized with modifications at the N-terminus. Analogues were made where the N-terminal pyroglutamyl residue was spaced further from the remainder of the molecule by the insertion of glycine residues between either pGlu 1 and Leu 2 (Gly 1a –AKH-I) or Leu 2 and Asn 3 (Gly 2a –AKH-I and Gly 2ab –AKH-I). Other modified hormones with N-terminal extensions were: (Ahx) n –AKH-I (Ahx, aminohexanoic acid); HPP(Ahx) n –AKH-I (HPP, hydroxyphenyl propionate) and Ac(Ahx) n –AKH-I (where n =0–3). Finally, acetylated and non-acetylated amino acids were substituted for pGlu 1 : Glu, Pro, Ala and Tyr. The effects of these modifications on biological potency were tested in the lipid mobilization assay in vivo and acetate uptake assay in vitro . The potency of AKH-I was reduced much more by insertion of glycine between pGlu 1 and Leu 2 , than between Leu 2 and Asn 3 , perhaps suggesting that a hydrophobic residue is required adjacent to the pGlu for biological activity. In addition, a residue N-terminal to Leu 2 is necessary for activity (i.e., [despGlu]–AKH-I is inactive) unless the free N-terminus is acetylated: Ac[despGlu]–AKH-I is active, but has low potency. The potencies of HPP(Ahx) 0–3 –AKH-I, Ac(Ahx) 1–3 –AKH-I and glycine-inserted analogues decreased consistently with increasing extension of the N-terminus away from the remainder of the molecule. However, potencies of the unblocked (Ahx) n –AKH-I analogues did not, and potency in either assay did not appear related to the number of aminohexanoic residues. Similarly, while hormonal activity was retained by substitution of pGlu 1 by Tyr, Pro, Ala or Glu in both assays, acetylation of the resulting analogues did not provide a consistent increase in potency, but actually decreased for AcGlu 1 –AKH-I compared with its unblocked analogue. HPP 1 –AKH-I was the most potent of the modified peptides tested, with almost the same potency in the assay in vitro as the natural peptide.

A two-dimensional gel electrophoresis database of dog (Canis familiaris) proteins is presented. The database contains 1212 protein spots which have been characterised in terms of their pI and Mr. This database has been integrated into the HSC-2DPAGE database which is accessible on the Internet via the World Wide Web with the uniform resource location (URL): (http://www.harefield.nthames.nhs.uk/nhli/ protein/index.html). Identifications for 80 of the protein spots have been obtained by visual cross-matching with the human heart protein database in HSC-2DPAGE (42 spots), N-terminal microsequence analysis (25 spots) and peptide mass fingerprinting (20 spots). This database is being used in studies of alterations in protein expression in models of heart failure and heart disease.

The dissemination of information relating to the characterisation of proteins from two-dimensional electrophoresis (2-DE) gel databases is essential for their effective utilisation in the study of protein expression in cell biology. Since the inception of the World Wide Web and the pioneering development of SWISS-2DPAGE as a tool for retrieving information on proteins separated by 2-DE, the Internet has become the method of choice for disseminating and accessing information on 2-DE protein databases. At Harefield we have established HSC-2DPAGE which is an advanced interface for accessing protein databases relating to heart disease. The Web site currently includes databases of proteins from human, dog and rat ventricular tissue and a human endothelial cell line. The databases are searchable individually or as a whole by remote keyword searches. Each database is represented by both synthetic (computer generated) and real (scanned gel) clickable images upon which characterised protein spots are highlighted by hyperlinked symbols. The database conforms to all the rules proposed for federated 2-DE protein databases and individual protein entries are linked to other protein databases such as SWISS-PROT by active cross-references. This paper describes the construction of HSC-2DPAGE, its maintenance, and access via the Internet.

The use of infrared (IR) and ultraviolet (UV) matrix-assisted laser desorption (MALDI) mass spectrometry to analyse myocardial proteins separated by two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) is discussed. Proteins were electroblotted onto a FluoroTrans polyvinylidene difluoride (PVDF) membrane in order to facilitate analysis by MALDI, which represented the most efficient means of extracting large numbers of proteins simultaneously. Once on a FluoroTrans membrane, IR-MALDI was used to obtain spectra from selected protein spots, but no useful signals were obtained with UV MALDI. Spectra were generated from 46 of 50 spots analysed with protein masses from 13 to 82 kDa and isoelectric points (pI) 4.7-7.8. For those protein spots that had previously been characterised, and for which both sequence and post-translational modification data were known, IR-MALDI data was within plus or minus 0.5% of the expected mass. Some spots contained more than one protein signal, illustrating the increased information obtainable from MALDI, but also suggesting the limit of resolution of 2-D gels for separating large numbers of proteins. Attempts to digest proteins with specific proteases and generate peptide mass fingerprints by MALDI analysis on the membrane were unsuccessful with either IR or UV lasers. The peptides were extracted from the membrane and readily analysed by UV MALDI for peptide spectra. Poor data was obtained for peptide digests with IR-MALDI, probably because of matrix suppression by digest buffer. In order to obtain the maximum amount of information from blotted proteins, both IR and UV MALDI were required.

Amino acid compositional analysis and peptide mass fingerprinting by matrix assisted laser desorption mass spectrometry have been used to characterise proteins obtained from two-dimensional electrophoresis (2-DE) separations of human cardiac proteins. A group of twelve protein spots was selected for analysis. The identities of eight of the proteins had been determined by conventional protein characterisation methods, two were unknown proteins and two had putative identities from protein database spot comparison. Amino acid analysis and peptide mass fingerprinting gave corresponding identities for seven of the twelve proteins, which also agreed with our initial identifications. Three proteins which had been identified previously were not confirmed in this study and putative identities were obtained for the two unknown proteins. The advantages, problems and use of amino acid analysis and peptide mass fingerprinting for the analysis of proteins from 2-DE are discussed. The data highlight the need to use orthogonal techniques for the unequivocal identification of proteins from 2-DE gels.

We have investigated the feasibility of identifying homologous proteins in whole tissue protein extracts of dog, mouse and rat hearts by comparison with our human heart two-dimensional (2-D) database. Samples of ventricular myocardial tissue from each of these species were coelectrophoresed with a human tissue sample. Gels were silver stained and patterns were analysed using PDQUEST. The number of proteins comigrating with human proteins was 301, 201 and 356 for the dog, mouse and rat, respectively. In the dog pattern, 33 of these comigrating proteins were tentatively identified from the similarity between their migration properties and those of known human proteins. Twenty-nine such proteins were identified in the mouse pattern while 30 comigrating rat proteins were identified. While these tentative identifications require confirmation, we feel that this technique offers a useful shortcut in the characterisation of proteins present in similar tissue samples from different species and avoids the necessity for duplicating laborious procedures, such as protein microsequencing, otherwise used in the identification of these proteins in each species.

Two-dimensional gels offer a powerful method for separating complex protein mixtures, but subsequent methods for analysing individual components, such as protein sequencing and Western immunoblotting, are laborious and slow. The identification of proteins can be accelerated by using a combination of protease digestion and matrix assisted laser desorption-mass spectrometry (MALDI-MS). The peptide mass spectrum of a protein represents a unique fingerprint determined by the amino acid sequence and the cleavage properties of the protease. Software has been developed so that peptide masses can be used to search a mass-based peptide database generated from established protein sequence databases. A list of the closest matching proteins is produced to allow identification of the sample. The strategy was applied to 52 protein spots from human myocardial tissue separated by two-dimensional electrophoresis (2-DE) gels and analysed blind. Conditions for optimal trypsin digestion of proteins electroblotted onto polyvinylidene difluoride (PVDF) membranes are described. Mass data were generated from both Coomassie Brilliant Blue and sulforhodamine B-stained proteins, though the former required destaining prior to digestion. Alkylation of cysteine and oxidation of methionine were significant modifications that influenced the successful identification of a protein spot. Examples are presented to illustrate the advantages and disadvantages of this approach.

An updated human heart protein two-dimensional electrophoresis (2-DE) database is presented. The database, which contains some 1388 protein spots characterised in terms of M(r) and pI, has been analysed further by Western immunoblotting and protein sequencing. From a total of 103 protein spots analysed, 49 have been identified by immunoblotting and 32 have been identified by protein sequencing. A further six proteins have tentatively been assigned by comparison with the human heart 2-DE protein database of Jungblut et al. (Electrophoresis) 1994, 15, 685-607). This database is being used in studies of alterations in protein expression in the diseased and transplanted human heart.

An octapeptide which mobilizes lipid and inhibits the incorporation of [ 3 H]leucine into haemolymph proteins in both Acheta domesticus and Locusta migratoria has been purified from the corpora cardiaca of Acheta . The primary sequence of this Acheta adipokinetic hormone, determined by pulsed liquid-phase Edman sequencing after enzymatic removal of the N -terminal pyroglutamate is: (pGlu)-Val-Asn-Phe-Ser-Thr-Gly-Trp-NH 2 . This sequence is identical to that of an octapeptide isolated previously from Gryllus bimaculatus and Romalea microptera . Synthetic material with the assigned structure is biologically active and is chromatographically identical to the natural peptide. In both crickets and locusts, the sensitivity of [ 3 H]leucine incorporation to inhibition by Acheta adipokinetic hormone (EC 50 = 5 × 10 −11 M) is two orders of magnitude greater than that of the adipokinetic response (EC 50 = 3 × 10 −9 M). Acheta adipokinetic hormone elicits a full adipokinetic response in both Locusta and in Acheta .

Direct and indirect assays of diuresis in insects are reviewed and their advantages and disadvantages discussed in relation to their use during the isolation and characterisation of diuretic factors. The nature of known and putative diuretic peptides is contrasted and points of controversy over the identity of these peptides highlighted. The isolation and characterisation of a putative diuretic peptide from Acheta domesticus is discussed as a case study in the purification and characterisation of such peptides.

Endoplasmic reticulum (ER) has been prepared and analysed from germinating and developing castor bean endosperm. A combination of one- and two-dimensional (1-D and 2-D) gel electrophoresis was used to study the complexity of sample and protein differences between the two stages. The ER of the developing oilseed is central to the synthesis, sorting and storage of protein and lipid reserves while the germinating seed is concerned with their degradation. Sample complexity has been reduced by separation of ER proteins into lumenal, peripheral membrane and integral membrane subfractions. Membrane proteins pose specific problems in aggregation and binding to passive surfaces. We have overcome this by collection of membranes at density gradient interfaces and by silanization of plastic ware. Several major components have been identified from 1-D gels by N-terminal sequencing and matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprints. These include protein disulphide isomerase (PDI), calreticulin and developing-ER-specific oleate-12-hydroxylase involved in the biosynthesis of ricinoleic acid. In excess of 300 spots are detectable in each developmental fraction by high sensitivity 2-D gels. This is the first 2-D electrophoretic analysis of plant ER. These gels reveal significant differences between germinating and developing ER. Preparative loading 2-D gels of germinating ER have been run and 14 selected spots characterized by quadrupole time of flight tandem mass spectrometry (Q-TOF MS/MS). Ten of these proteins were assigned function on the basis of identity with existing castor database entries, or by homology with other species. Two proteins, aspartate proteinase precursor and N-carbamyl-L-aminohydrolase-like protein, appear to be absent from developing profiles. Most of the proteins identified are concerned with roles in protein processing and storage, and lipid metabolism which occur in the ER. Data from three of the assigned spots included unidentified peptides indicating the presence of more than one protein in these spots following 2-D electrophoresis. More extensive analysis will have to await developments in genomics but the basic separation technologies to simplify sample identity for a plant ER preparation have been established.

Recent increases in the number of genome sequencing projects means that the amount of protein sequence in databases is increasing at an astonishing pace. In proteome studies, this is facilitating the identification of proteins from molecularly well-defined organisms. However, in studies of proteins from the majority of organisms, proteins must be identified by comparing analytical data to sequences in databases from other species. This process is known as cross-species protein identification. Here we present a new program, MultiIdent, which uses multiple protein parameters such as amino acid composition, peptide masses, sequence tags, estimated protein pI and mass, to achieve cross-species protein identification. The program is structured so that protein amino acid composition, which is highly conserved across species boundaries, first generates a set of candidate proteins. These proteins are then queried with other protein parameters such as sequence tags and peptide masses. A final list of database entries which considers all analytical parameters is presented, ranked by an integrated score. We illustrate the power of the approach with the identification of a set of standard proteins, and the identification of proteins from dog heart separated by two-dimensional gel electrophoresis. The MultiIdent program is available on the world-wide web at: http://www.expasy.ch/sprot/multiident.h tml.

Rapid ventricular pacing in dogs results in a low output cardiomyopathic state which is similar to idiopathic dilated cardiomyopathy in man. However, the pathophysiological mechanisms which cause this failure following pacing are unknown. Five dogs underwent rapid ventricular pacing. Hearts were stimulated at 245 beats per min (bpm) for four weeks and then reduced to 190 bpm to stabilize the failure. Six unoperated dogs were used as controls. This paper compares the two-dimensional gel electrophoresis (2-DE) protein patterns of left ventricular samples from the paced myocardium with the control dogs. Changes in protein expression were analyzed qualitatively and semi-quantitatively. In the paced dog samples 69 protein spots were significantly altered of which 42 were decreased and 27 were elevated. One qualitative change was observed: elongation factor Tu was present only the control hearts. Of these proteins, 20 have been identified by a combination of N-terminal protein microsequencing, peptide mass profiling by mass spectrometry, amino acid compositional analysis, and by comparison with databases of canine and human ventricular proteins. Ten of these are associated with mitochondria and energy production, including: pyruvate dehydrogenase E1 component, isocitrate dehydrogenase subunit alpha, HSP60 and HSP70, creatine kinase M and fatty acid binding protein. The cytoskeletal protein desmin was detected in reduced quantities and a spot corresponding to a fragment of desmin was increased. These results indicate that the development of heart failure in the paced dog involves alterations in mitochondrial energy production, the cytoskeleton and calcium activation.

Two-dimensional (2-D) electrophoresis followed by immunoblotting and N-terminal protein microsequencing were used to characterize and identify the IgE-reactive proteins of Hevea latex that are the main cause of the latex type I allergy affecting especially health care workers and spina bifida children. This approach generated a comprehensive latex allergen database, which facilitated the integration of most of the latex allergen data presented in the literature. The major latex allergens Hev b 1, Hev b 3, Hev b 6 and Hev b 7 have been localized on our 2-D maps. Moreover, we were able to identify six previously undescribed IgE-binding latex proteins, namely enolase, superoxide dismutase, proteasome subunit C5, malate dehydrogenase, triosephosphate isomerase and endochitinase. The generated latex 2-D maps will provide valuable information to develop strategies for the isolation of the novel IgE binding proteins in order to study the frequency of sensitization among both risk groups. Detailed knowledge of all proteins involved in latex allergy will allow better diagnosis of latex allergy and to monitor the success of prevention strategies that are needed to reduce the high prevalence of latex allergy among both risk groups.

Background: Proteins of natural rubber latex cause IgE-mediated sensitization in 3% to 18% of health care workers and in up to 50% of patients with spina bifida. Objective: This study was aimed at the generation of a comprehensive latex protein database by two-dimensional electrophoresis (2-DE). Methods: Proteins extracted from fresh Hevea brasiliensis latex were separated by 2-DE. IgE-reactive proteins were analyzed by immunoblotting with sera of health care workers with latex allergy. Protein microsequencing and monoclonal antibodies were used to identify the latex allergens. Results: The latex C-serum 2-DE map was very complex and exhibited about 200 distinct polypeptides. The proteins eluted from the latex particles consisted primarily of two groups of acidic proteins located in the 8 to 14 kd and 22 to 24 kd areas of the 2-DE map. Major IgE-reactivity was detected with C-serum proteins in the 56, 45, 30, 20, 14, and

Heart transplantation is the clinically acceptable treatment for end-stage heart failure. Since the development of better immunosuppressive regimes and particularly the introduction of Cyclosporin A in 1983, which combats cellular rejection, short-term survival for heart transplant recipients has increased steadily with survival at 1 year being in the order of 85% (Kriett and Kaye 1990). The major medium to long-term complication is development of a proliferative occlusive disease of the vasculature of the allograft. This disease, variously described as transplant associated coronary artery disease (TxCAD; Rose and Dunn 1993), cardiac allograft vasculopathy (CAV; Hosenpud et al. 1992), accelerated coronary artery sclerosis (ACS; Yacoub and Rose, 1994) and graft coronary artery sclerosis (GCA; Schoen and Libby 1991), is a rapidly progressing disease which causes blockage of the coronary arteries. The incidence of TxCAD varies between heart transplant centres. Harefield Hospital, U.K. has an incidence of 6% at 1 year increasing to 17% at 3 years (see Dunn and Rose, 1993), however, other centres have reported incidences as high as 10%–20% at 1 year, 25%–40% at 3 years and at least 40%–50% at 5 years (Gao et al. 1989; Uretsky et al. 1987).

Micropreparative two-dimensional (2-D) gel electrophoresis with immobilized pH gradients (4-8) in the first dimension (IPG-DALT) was optimized for the separation of salt-soluble wheat grain proteins, associated with bakers' asthma disease. The resolved polypeptides were electroblotted onto a polyvinylidene difluoride (PVDF) membrane and incubated with the pooled sera from four asthmatic bakers. Bound IgE was demonstrated by alkaline phosphatase conjugated anti-human IgE. Major IgE binding was detected in the 27 kDa, 37 kDa and, to a lesser extent, in the 14-18 kDa area of the 2-D immunoblots, respectively. Since the main purpose of our study was to determine the N-terminal amino acid sequences of the major wheat grain allergens, N-terminal sequencing was performed for six out of a total of eleven major allergens located in the 27 kDa area, for one out of two 37 kDa allergens, and for two out of four 14-18 kDa allergens. Our results revealed that two of the 27 kDa polypeptides are clearly related to several Acyl-CoA oxidase variants of barley and rice, whereas no significant homologies were found for the remaining four 27 kDa allergens analyzed. The N-terminus of the 37 kDa allergen appeared to be blocked so that no sequence information was obtained, while the two 14-18 kDa allergens analyzed were identified as members of the wheat alpha-amylase-inhibitor family.

A biologically active 125 I-labeled analogue of AK-II (3′-hydroxyphenyl propionic-Gly-Gly-Gly-Phe-Ser-Pro-Trp-Gly-NH 2 ) was used to investigate the properties of achetakinin binding sites on plasma membranes from Malpighian tubules of Acheta domesticus . With optimized conditions, binding was rapid, reversible, and specific, and saturation studies revealed a single class of binding sites with K d 0.55 n M and B max 39.9 fmol/mg membrane protein. The affinities of achetakinins for binding sites on tubule membranes ranked AK-V > AK III > AK-II > AK-I ≥ AK-IV, in general agreement with their potencies in functional assays. However, IC 50 values were several orders of magnitude higher than corresponding values for EC 50 , which suggests a considerable receptor reserve.

The issues surrounding the use of prostate-specific antigen (PSA) in the diagnosis of prostate cancer (PCa) are well documented and the need for a molecular diagnostic test with greater discriminatory power is clear. The development of autoantibodies associated with prostate cancer has also been described. In general, the appearance of such antibodies can precede disease symptoms by many years, making them attractive as potential biomarkers for early diagnosis. We have developed a unique “functional protein” array platform which utilises correctly folded proteins and has the ability to display native, discontinuous epitopes. The reproducibility of the platform is exceptionally good, making it possible to screen statistically meaningful numbers of samples. Following on from a successful pilot study, where panels of autoantibody biomarkers exhibiting a specificity and sensitivity for PCa superior to PSA were identified using the platform1, we have validated this approach in a large-scale analytical study. The current analytical study involving approximately 1800 samples was primarily designed to identify panels of biomarkers with the ability to distinguish between PCa (n=400) and control samples from patients with benign prostatic hypertrophy (BPH, n=406). BPH can present with similar symptoms to PCa and can also result in elevated PSA levels. Additional sample cohorts included prostatitis, other cancers of various origin and non-disease/healthy controls (n=400). All samples were age, ethnicity and gender matched. The products of 1296 unique genes (1330 proteins), chosen for their association with disease, signal transduction and cancer autoimmunity, are immobilized on each array. Serum samples (n=1781) were analysed using the arrays as described previously1. Data were split into test and training sets and analyzed with several classification algorithms to identify classifiers which would successfully distinguish case from control samples. Data were repeatedly split into test and training sets and analysis cycles repeated until a stable set of classifiers was identified. At the time of writing, data is still under analysis; however, early indications are encouraging and suggest that panels of biomarkers with a performance that significantly exceeds that of PSA may be identified. Full results of the study will be presented. 1 McAndrew et al Development of a panel of biomarkers for the diagnosis of prostate cancer (2010) Molecular Diagnostics in Cancer Therapeutic Development conference Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5071. doi:10.1158/1538-7445.AM2011-5071

Insect diuretic peptides differ chemically and, even within a single species, several forms of diuretic peptide may be present (Wheeler and Coast, 1989). Thus, in Locusta migratoria, diuretic peptides have been described as AVP-like, ACTH-like, and as novel peptides. Only two insect diuretic peptides have been fully characterised, one from Locusta (Proux et al., 1987), and the other from Manduca sexta (Kataoka et al., 1989). They have no structural analogy to each other, but each has similarities with some vertebrate peptides. Fractionation of aqueous extracts of the corpora cardiaca of Acheta domesticus yields a number of peptides which stimulate fluid secretion by isolated Malpighian tubules (Coast and Wheeler, 1989). One peptide, Acheta peptide-I (AP-I), is associated with a major UV absorbing peak, and was selected for sequence analysis.

In locusts, adipokinetic hormones have well defined actions: they mobilise diacylglycerols from the fat body and stimulate oxidation of lipids by the flight muscles (Goldsworthy and Wheeler, 1989). Their actions presumably depend on interactions with specific receptors in the tissues, but there have been no direct studies of AKH receptors. Some information about receptors can, however, be inferred from biological assay data. For most actions, AKH-I and to a lesser extent AKH-II are both effective agonists, but AKH-II stimulates cAMP accumulation in the fat body more than AKH-I (Goldsworthy et al., 1986), and AKH-II releases carbohydrate from the fat body, whereas AKH-I does not (Loughton and Orchard, 1981). These differences in actions suggest that AKH-I and -II may act via different receptors at the fat body, although their high degree of cross reactivity in other assays indicates that they may also stimulate the same receptors. The availability of synthetic naturally occurring analogues of adipokinetic peptides has prompted an examination of their structure/activity relationships.

Natural adipokinetic peptides I and II have been isolated from corpora cardiaca of Locusta, and their effects on hyperlipaemia, fat body phosphorylase activation, and lipoprotein-A+ formation investigated. At saturation doses for each of these assays, adipokinetic hormone I is more effective than hormone II. In these systems, however, adipokinetic hormone II is unable to induce, even at high doses, the magnitude of response elicited by hormone I. When assayed for their hyperlipaemic effects, two adipokinetic peptides from the corpora cardiaca of Schistocerca also have similar differential potency to those in Locusta. Interestingly, although adipokinetic hormone I from Locusta is more potent in eliciting the above responses, adipokinetic hormone II is more effective than hormone I in bringing about an accumulation of cAMP in the fat body.

Changes in haemolymph proteins and lipoproteins during adipokinetic hormone action have been studied using polyacrylamide gel electrophoresis (PAGE) and a heparin/EDTA precipitation technique. During hormone action, the formation of A + takes place at the expense of Ayellow and C L -proteins, which decrease in free concentration in the haemolymph. Ayellow is heparin precipitable, whereas A + precipitates with EDTA after prior treatment with heparin. After injection of adipokinetic hormone, heparin-precipitable protein (HPP) decreases after a delay of 10–15 min, but heparin/EDTA precipitable protein (HEPP) increases immediately. These changes occur in response to extracts of corpora cardiaca and to synthetic adipokinetic hormone, and are dose-dependent. Both the lipid and the C L -protein content of the HEPP rise as its protein content increases. A + formation does not occur in fifth-instar nymphs or newly emerged adults, but this response to adipokinetic hormone develops slowly as the adults mature.

Lipoprotein lipases in the flight muscles of Locusta migratoria show a marked substrate specificity: diacylglycerols associated with the adipokinetic hormone (AKH)-induced lipoprotein, A+, are hydrolysed at 4 to 5 times the rate of those associated with the lipoprotein in resting (non-hormone-stimulated) locusts, Ayellow. To determine the basis for this discrimination, the effect on the activity of flight muscle lipoprotein lipase of CL-proteins, a major constituent of lipoprotein A+, but not of Ayellow, has been investigated; they inhibit the flight muscle enzyme in a competitive manner whether activity is measured with a natural lipoprotein substrate, a lipid emulsion or a water soluble substrate. Experiments in vivo suggest that the flight muscle enzyme is normally inhibited in resting (non-AKH-stimulated) locusts but, interestingly, injection of synthetic AKH-I relieves the inhibition and increases the activity by 30 to 40%. This is not a direct effect of the hormone on the enzyme, but appears to be related to the hormone-induced formation of lipoprotein A+, so that the majority of CL-proteins in the haemolymph become bound to this lipoprotein and the concentration of free CL-proteins is markedly reduced. We suggest that CL-proteins play a major role in the regulation of lipoprotein lipase in locust flight muscle.

Although the existence of hormones in insects has been known for many years, it is only comparatively recently — with the benefit of sensitive new techniques of assay, isolation, and sequencing — that an overall picture of their nature has begun to emerge. From this it is clear that, like vertebrate hormones —with which they have some structural affinity — they fall into related families. Apart from their great intrinsic scientific interest, these studies suggest new lines of approach to the development of more potent or discriminating drugs and insecticides.

Detailed studies of lipoprotein A+ formation during AKH action have been made in Locusta migratoria L. using gel filtration combined with the use of radiolabeled haemolymph protein probes. In addition, we have assessed the quantitative contribution of the CL-proteins to A+ formation by direct measurement of the changes in concentration of free CL-proteins and report some properties of the C-I and C-II proteins: they appear to be glycoproteins of 20,000 and 16,000 MW respectively, but do not bind to concanavalin A. We have confirmed earlier observations (using different techniques) which showed that liproprotein Ayellow is not involved per se in A+ formation during the first 15 min of AKH action. In contrast, the (two) CL-proteins take part in A+ formation without any apparent delay after hormone injection. Our observations show that A+ formation is essentially complete within 30 min of AKH injection, although further CL-protein binding and lipid-loading do occur subsequently. After 30 min there is no further decrease in the Ayellow titre. It is argued that much, if not all, CL-protein is located at the surface of the A+ particle. From the changes in titres which occur in Ayellow and CL-proteins during AKH action we estimate that A+ is formed from 1 mole of Ayellow and approximately 28 moles of CL-proteins. Using these figures we calculate an apparent molecular weight for A+ within the range of 1.65–2.12×106, which is in reasonable agreement with estimates derived from gel exclusion chromatography data. These studies emphasize the dynamic and fully reversible nature of lipoprotein A+ formation and highlight the complex nature of the lipoprotein transformations occurring during hormone-stimulated lipid transport in locusts.

The involvement of C L -proteins in the formation of lipoprotein A + during adipokinetic hormone action has been investigated using radiolabelling experiments. Injected [ 3 H]-C L -proteins associate rapidly with lipoprotein A + during its formation. Both [ 3 H]-C L -proteins and [ 3 H]-A yellow are liberated from [ 3 H]-A + during its natural degradation in the haemolymph (when adipokinetic hormone action is declining). It appears that [ 3 H]-C L -proteins bind reversibly to A + , since they are easily displaced in vivo and in vitro by competing concentrations of non-labelled C L -proteins. It is suggested that A yellow is an integral component of the A + lipoprotein complex, whereas C L -proteins may play only a relatively minor part in its structural organisation. Possible functions of the binding of C L -proteins to A + are discussed.

Naturally occurring peptides from the corpora cardiaca of various insect species are compared on the basis of their ability to cause hyperlipaemia in adult male migratory locusts. The peptides studied are the decapeptide adipokinetic hormone I and the octapeptide adipokinetic hormone II from the migratory locust, hypertrehalosaemic hormones I and II from the cockroach (octapeptides which are identical to the myoactive factors I and II), and the nonapeptide hypertrehalosaemic factor II from the stick insect Carausius morosus. For comparison, a crustacean octapeptide, red pigment concentrating hormone, was also tested. The results show clearly that over the range of its dose-response curve, the stick insect nonapeptide, whose structure is thought to be almost identical to that of adipokinetic hormone I, has the biological activity most closely comparable with this hormone, but Locusta adipokinetic hormone II has comparable potency at low doses (although high doses fail to achieve maximum lipid mobilisation). The octapeptides red pigment concentrating hormone, myoactive factors I and II display almost identical dose-response curves and elicit near maximal adipokinetic responses (comparable to those with adipokinetic hormone I), but only when a 5-fold greater dose is injected compared with that required of this hormone. However adipokinetic hormone II, which lacks proline, gives only about a 65% response compared with adipokinetic hormone I, even when injected at doses more than 20-fold higher than those of adipokinetic hormone I required to cause maximal rates of lipid mobilisation. The possible relationships between their amino acid sequencies, the presence or absence of proline at position 6, and the different biological potencies of the peptides are discussed.

During trivial flight in locusts, carbohydrates are the main energy source for the flight muscles, but during long-term (migratory) flight, lipids mobilised from the fat body are utilised. The supply of fuels to the flight muscles, and the balance between fuels, is determined by peptidic adipokinetic hormones, AKH-I and AKH-II, released from the corpora cardiaca. These peptides also indirectly effect changes in lipid-transporting mechanisms in the haemolymph, and regulate a lipoprotein lipase necessary for lipid uptake by the flight muscles. The sequences of the AKHs of locusts are known and they are members of a family of invertebrate neuropeptides. Extensive structure/activity studies for the action of AKHs on lipid mobilisation have been carried out and, together with studies on secondary structure, the structural features necessary for biological activity are being elucidated. Such information will be of great importance in receptor studies for these hormones, and in the development of powerful agonists and antagonists.

The structures of 3 locust adipokinetic hormones are known: the decapeptide adipokinetic hormone I is common to all species of locust investigated so far, but the octapeptide AKH-II’s of Locusta (AKH-IIL) and Schistocerca (AKH-IIS) differ by a single amino acid (see Table 1). In addition to these locust peptides, structurally related but often functionally different peptides from other insect species have been sequenced. These include the adipokinetic peptide from Heliothis zea (Jaffe et al., 1986), which is identical to that in Manduca sexta (Ziegler et al., 1985); hypertrehalosaemic peptides I and II from Periplaneta americana (sequenced independently by various groups; see Goldsworthy et al., 1986a); and hypertrehalosaemic factor II from Carausius morosus (Gade and Rinehart, 1986). Together with a crustacean peptide, Red Pigment Concentrating Hormone, these peptides apparently represent a group of closely related molecules, the AKH/RPCH family: they possess striking sequence and structural similarities, including blocked amino and carboxy terminals. We have compared the activities of these peptides in the locust hyperlipaemic assay to give an insight into the structural features necessary for the fit of the AKH molecule to its receptor on the fat body.

Adipokinetic hormones (AKH) I and II are released into the haemolymph during long-term flight in locusts and have several well defined sites of action: they increase the rate of release of diacylglycerols from the fat body and stimulate the uptake and utilisation of these lipids in the flight muscles (see Goldsworthy, 1983)• Adipokinetic hormones also promote indirect effects such as haemolymph lipoprotein transformations (Goldsworthy et al., 1985). Lipids released from the fat body are transported as part of lipoproteins; in resting locusts the majority of haemolymph diacylglycerol is carried as part of lipoprotein Ayellow, but after hormone injection Ayellow becomes lipid-loaded and associates with non-lipid carrying apoproteins to become lipoprotein A+, which carries 10 to 15X as much lipid as Ayellow (Mwangi and Goldsworthy, 1977; Van Der Horst et al., 1981a,b; Wheeler and Goldsworthy, 1983a,b).

Using natural lipoproteins as substrates, lipase activity has been measured in leg muscle, fat body, midgut and flight muscles of Locusta migratoria. The enzymic activity in the flight muscles is higher than in those other tissues tested, confirming the potential of the flight muscles to utilise lipids at high rates. In addition, a membrane-bound lipoprotein lipase can be extracted from flight muscle. The flight muscle enzyme activity shows a marked substrate specificity; at lipoprotein concentrations equivalent to those found normally in flown or resting locusts respectively, the enzyme hydrolyses diacylglycerols associated with lipoprotein A+ (present in the haemolymph of flown or adipokinetic hormone-injected locusts) at about 4 times the rate of those associated with lipoprotein Ayellow (which is the major lipoprotein in resting locusts). In addition, the hydrolysis of lipids carried by lipoprotein Ayellow is dramatically reduced in the presence of lipoprotein A+. These observations indicate that the enzyme plays a specific role in the uptake of lipids at the flight muscles to ensure a smooth transition from carbohydrate to lipid based metabolism during flight.

A diuretic peptide Locusta-DP, identified by its ability to increase cyclic AMP production in locust Malpighian tubules in vitro, has been isolated and characterized from whole heads of Locusta migratoria. The purified peptide stimulates fluid secretion by Malpighian tubules maximally in vitro. The primary structure of Locusta-DP was established as a 46 residue amidated peptide: MGMGPSLSIVNPMDVLRQRLLLEIARRRLRDAEEQIKANKDFLQQI-NH2. Locusta-DP has 48% sequence identity with Acheta-DP and 49% identity with Manduca-DH, and provides further evidence for the presence of a family of diuretic peptides in insects.