Non-clathrin-coat protein alpha is a conserved subunit of coatomer and in Saccharomyces cerevisiae is essential for growth.

To complete the molecular characterization of coatomer, the preformed cytosolic complex that is involved in the formation of biosynthetic transport vesicles, we have cloned and characterized the gene for non-clathrin-coat protein alpha (alpha-COP) from Saccharomyces cerevisiae. The derived protein, molecular weight of 135,500, contains four WD-40 repeated motifs (Trp/Asp-containing motifs of approximately 40 amino acids). Disruption of the yeast alpha-COP gene is lethal. Comparison of the DNA-derived primary structure with peptides from bovine alpha-COP shows a striking homology. alpha-COP is localized to coated transport vesicles and coated buds of Golgi membranes derived from CHO cells.

Growth, survival, gene expression, microbiota and tryptic activity during feeding of Scophthalmus maximus first feeding larvae with beta-glucan (MacroGard)

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, beta-glucan (MacroGard®) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast beta-1,3/1,6-glucan in form of MacroGard® at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, trypsin activity and size measurements. Along with the feeding of beta-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard® fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by beta-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-alpha and il-1beta was observed. We conclude that the administration of beta-glucan induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.

Human growth hormone presented by K14hGH-transgenic skin grafts induces a strong immune response but no graft rejection

Although immune responses leading to rejection of transplantable tumours have been well studied, requirements for epithelial tumour rejection are unclear. Here, we use human growth hormone (hGH) expressed in epithelial cells (skin keratinocytes) as a model neo-self antigen to investigate the consequences of antigen presentation from epithelial cells. Mice transgenic for hGH driven from the keratin 14 promoter express hGH in skin keratinocytes. This hGH-transgenic skin is not rejected by syngeneic non-transgenic recipients, although an antibody response to hGH develops in grafted animals. Systemic immunization of graft recipients with hGH peptides, or local administration of stimulatory anti-CD40 antibody, induces temporary macroscopic graft inflammation, and an obvious dermal infiltrate of inflammatory cells, but not graft rejection. These results suggest that a neo-self antigen expressed in somatic cells in skin can induce an immune response that can be enhanced further by induction of specific immunity systemically or non-specific immunity locally. However, immune responses do not always lead to rejection, despite induction of local inflammatory changes. Therefore, in vitro immune responses and in vivo delayed type hypersensitivity are not surrogate markers for immune responses effective against epithelial cells expressing neoantigens.

The role of disulfide bonds in the structure and function of murine epidermal growth factor (mEGF)

A systematic study using solid phase peptide synthesis has been undertaken to examine the role of the disulfide bonds in the structure and function of mEGF. A combination of one, two and three native disulfide pair analogues of an active truncated (4-48) form of mEGF have been synthesised by replacing specific cysteine residues with isosteric alpha-amino-n-butyric acid (Abu). Oxidation of the peptides was performed using either conventional aerobic oxidation at basic pH, in DMSO under acidic conditions or via selective disulfide formation using orthogonal protection of the cysteine pairs. The contribution of individual, or pairs of, disulfide bonds to EGF structure was evaluated by CD and H-1-NMR spectroscopy. The mitogenic activity of each analogue was determined using Balb/c 3T3 mouse fibroblasts. As we have reported previously (Barnham et al. 1998), the disulfide bond between residues 6 and 20 can be removed with significant retention of biological activity (EC50 20-50 nM). The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. We now show that removal of any other disulfide bond, either singly or in pairs, results in a major disruption of the tertiary structure, and a large loss of activity (EC50>900 nM). Remarkably, the linear analogue appears to have greater activity (EC50 580 nM) than most one and two disulfide bond analogues although it does not have a definable tertiary structure.

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66-68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MITI, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pia ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1 mu M, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed beta-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors. (c) 2005 Elsevier Inc. All rights reserved.

Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells

Growth hormone (GH) regulates many of the factors responsible for controlling the development of bone marrow progenitor cells (BMPCs). The aim of this study was to elucidate the role of GH in osteogenic differentiation of BMPCs using GH receptor null mice (GHRKO). BMPCs from GHRKO and their wild-type (WT) littermates were quantified by flow cytometry and their osteogenic differentiation in vitro was determined by cell morphology, real-time RT-PCR, and biochemical analyses. We found that freshly harvested GHRKO marrow contains 3% CD34 (hernatopoietic lineage), 43.5% CD45 (monocyte/macrophage lineage), and 2.5% CD106 positive (CFU-F/BMPC) cells compared to 11.2%, 45%, and 3.4% positive cells for (WT) marrow cells, respectively. When cultured for 14 days under conditions suitable for CFU-F expansion, GHRKO marrow cells lost CD34 positivity, and were markedly reduced for CD45, but 3- to 4-fold higher for CD106. While WT marrow cells also lost CD34 expression, they maintained CD45 and increased CD106 levels by 16-fold. When BMPCs from GHRKO mice were cultured under osteogenic conditions, they failed to elongate, in contrast to WT cells. Furthermore, GHRKO cultures expressed less alkaline phosphatase, contained less mineralized calcium, and displayed lower osteocalcin expression than WT cells. However, GHRKO cells displayed similar or higher expression of cbfa-1, collagen 1, and osteopontin mRNA compared to WT. In conclusion, we show that GH has an effect on the proportions of hematopoietic and mesenchymal progenitor cells in the bone marrow, and that GH is essential for both the induction and later progression of osteogenesis. (c) 2005 Elsevier Inc. All rights reserved.

In vitro heterogeneity of osteogenic cell populations at various equine skeletal sites

Bone cell cultures were evaluated to determine if osteogenic cell populations at different skeletal sites in the horse are heterogeneous. Osteogenic cells were isolated from cortical and cancellous bone in vitro by an explant culture method. Subcultured cells were induced to differentiate into bone-forming osteoblasts. The osteoblast phenotype was confirmed by immunohistochemical testing for osteocalcin and substantiated by positive staining of cells for alkaline phosphatase and the matrix materials collagen and glycosaminoglycans. Bone nodules were stained by the von Kossa method and counted. The numbers of nodules produced from osteogenic cells harvested from different skeletal sites were compared with the use of a mixed linear model. On average, cortical bone sites yielded significantly greater numbers of nodules than did cancellous bone sites. Between cortical bone sites, there was no significant difference in nodule numbers. Among cancellous sites, the radial cancellous bone yielded significantly more nodules than did the tibial cancellous bone. Among appendicular skeletal sites, tibial metaphyseal bone yielded significantly fewer nodules than did all other long bone sites. This study detected evidence of heterogeneity of equine osteogenic cell populations at various skeletal sites. Further characterization of the dissimilarities is warranted to determine the potential role heterogeneity plays in differential rates of fracture healing between skeletal sites.

Immunogenicity of liposomes containing lipid core peptides and the adjuvant Quil A

Purpose. The purpose of this study was to investigate the immunogenicity of liposomes containing mannosylated lipid core peptide (manLCP) constructs, both in vitro and in vivo, with or without the addition of the immune stimulating adjuvant Quil A. Methods. Mouse bone marrow dendritic cells (BMDC) were cultured with liposome formulations for 48 h, and the resulting level of BMDC activation was determined by flow cytometry. BMDC pulsed with liposome formulations were incubated with 5,6-carboxyfluoroscein diacetate succinimidyl ester-labeled T cells for 72 h and the resulting T cell proliferation was determined by flow cytometry. To investigate the immunogenicity of formulations in vivo, groups of C57Bl/6J mice were immunized by subcutaneous injection, and the resulting antigen-specific cytotoxic and protective immune responses toward tumor challenge evaluated. Results. Despite being unable to demonstrate the activation of BMDC, BMDC pulsed with liposomes containing manLCP constructs were able to stimulate the proliferation of naive T cells in vitro. However, in vivo only liposomes containing both manLCP and Quil A were able to stimulate a strong antigen-specific cytotoxic immune response. Liposomes containing manLCP and Quil A within the same particle were able to protect against the growth of tumor cells to a similar level as if the antigen was administered in alum with CD4 help. Conclusion. ManLCPs administered in liposomes are able to stimulate strong cytotoxic and protective immune responses if Quil A is also incorporated as an adjuvant.

Inhibition of transforming growth factor-beta 1 signaling attenuates ataxia telanglectasia mutated activity in response to genotoxic stress

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGF beta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF beta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf beta 1 nail murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF beta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gamma H2AX radiation-induced foci; and increased radiosensitivity compared with TGF beta competent cells. We determined that loss of TGF beta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF beta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf beta 1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF beta may be used to advantage in cancer therapy.

A polymorphism in the growth hormone-releasing hormone receptor gene: clinical significance?

Two forms of the growth hormone-releasing hormone (GHRH) receptor (GHRH-R) exist in terms of a polymorphism at codon 57. The most common allele possesses GCG, coding for Ala. This codon can also be ACG, replacing the Ala with Thr. The present study demonstrates that the latter occurs in about 20% of pituitary somatotrophinomas, removed from patients with acromegaly. Somatotrophinomas possessing the alternative allele respond, on average, more strongly to GHRH in terms of GH secretion in vitro than tumors which are homozygous for the more common allele. The distribution of the two allelic forms of the GHRH-R did not significantly differ between acromegalic and non-acromegalic subjects. Thus, while the alternative allelic forms may, at least partially, contribute to the variable response of serum GH levels to i.v. GHRH observed in acromegalic and normal subjects, it is unlikely that subjects possessing the rarer form containing Thr in place of Ala at residue 57 are at increased risk of developing acromegaly.

Do changes in transglutaminase activity alter latent transforming growth factor beta activation in experimental diabetic nephropathy?

Background. Diabetic nephropathy is the leading cause of end-stage kidney failure worldwide. It is characterized by excessive extracellular matrix accumulation. Transforming growth factor beta 1 (TGF-ß1) is a fibrogenic cytokine playing a major role in the healing process and scarring by regulating extracellular matrix turnover, cell proliferation and epithelial mesanchymal transdifferentiation. Newly synthesized TGF-ß is released as a latent, biologically inactive complex. The cross-linking of the large latent TGF-ß to the extracellular matrix by transglutaminase 2 (TG2) is one of the key mechanisms of recruitment and activation of this cytokine. TG2 is an enzyme catalyzing an acyl transfer reaction leading to the formation of a stable e(?-glutamyl)-lysine cross-link between peptides.Methods. To investigate if changes in TG activity can modulate TGF-ß1 activation, we used the mink lung cell bioassay to assess TGF-ß activity in the streptozotocin model of diabetic nephropathy treated with TG inhibitor NTU281 and in TG2 overexpressing opossum kidney (OK) proximal tubular epithelial cells.Results. Application of the site-directed TG inhibitor NTU281 caused a 25% reduction in kidney levels of active TGF-ß1. Specific upregulation of TG2 in OK proximal tubular epithelial cells increased latent TGF-ß recruitment and activation by 20.7% and 19.7%, respectively, in co-cultures with latent TGF-ß binding protein producing fibroblasts.Conclusions. Regulation of TG2 directly influences the level of active TGF-ß1, and thus, TG inhibition may exert a renoprotective effect by targeting not only a direct extracellular matrix deposition but also TGF-ß1 activation and recruitment.

Control of the endocrine pancreas by gastrointestinal peptides

This thesis concerns the mechanism through which enteral delivery of glucose results in a larger insulin response than an equivalent parenteral glucose load. Preliminary studies in which mice received a glucose solution either intragastrically or intraperitoneally confirmed this phenomenon. An important regulatory system in this respect is the entero-insular axis, through which insulin secretion is influenced by neural and endocrine communication between the gastrointestinal tract and the pancreatic islets of Langerhans. Using an in vitro system involving static incubation of isolated (by collagenase digestion) islets of Langerhans, the effect of a variety of gastrointestinal peptides on the secretion of the four main islet hormones, namely insulin, glucagon, somatostatin and pancreatic polypeptide, was studied. The gastrointestinal peptides investigated in this study were the secretin family, comprising secretin, glucagon, gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI) and growth hormone releasing factor (GRF). Gastrin releasing peptide (GRP) was also studied. The results showed that insulin release was stimulated by all peptides studied except PHI, glucagon release was stimulated by all peptides tested, except GRF which suppressed glucagon release, somatostatin release was stimulated by GIP and GRF but suppressed by VIP, PHI, glucagon and secretin, and PP release was stimulated by GIP and GRF, but suppressed by PHI. The insulinotropic effect of GRP was investigated further. A perifusion system was used to examine the time-course of insulin release from isolated islets after stimulation with GRP. GRP was shown to be insulinotropic only in the presence of physiologically elevated glucose concentrations and both first and second phases of insulin release were augmented. There was no effect at substimulatory or very high glucose concentrations. Studies using a cultured insulin-secreting islet cell line, the RINm5F cell line, were undertaken to elucidate the intracellular mechanism of action of GRP. This peptide did not enhance insulin release via an augmentation of glucose metabolism, or via the adenylate cyclase/cyclic AMP secondary messenger system. The pattern of changes of cytosolic free calcium in response to GRP, which involved both mobilization of intracellular stores and an influx of extracellular calcium, suggested the involvement of phosphatidylinositol bisphosphate breakdown as a mediator of the effect of GRP on insulin secretion.

The integration of ProxiMAX randomisation with CIS display for the high-throughput production of novel peptides

Saturation mutagenesis is a powerful tool in modern protein engineering. This can allow the analysis of potential new properties thus allowing key residues within a protein to be targeted and randomised. However, the creation of large libraries using conventional saturation mutagenesis with degenerate codons (NNN or NNK) has inherent redundancy and disparities in residue representation. In this we describe the combination of ProxiMAX randomisation and CIS display for the use of generating novel peptides. Unlike other methods ProxiMAX randomisation does not require any intricate chemistry but simply utilises synthetic DNA and molecular biology techniques. Designed ‘MAX’ oligonucleotides were ligated, amplified and digested in an iterative cycle. Results show that randomised ‘MAX’ codons can be added sequentially to the base sequence creating a series of randomised non-degenerate codons that can subsequently be inserted into a gene. CIS display (Isogencia, UK) is an in vitro DNA based screening method that creates a genotype to phenotype link between a peptide and the nucleic acid that encodes it. The use of straight forward in vitro transcription/translation and other molecular biology techniques permits ease of use along with flexibility making it a potent screening technique. Using ProxiMAX randomisation in combination with CIS display, the aim is to produce randomised anti-nerve growth factor (NGF) and calcitonin gene-related (CGRP) peptides to demonstrate the high-throughput nature of this combination.

Cholecystokinin (CCK) receptor and CCK gene expression in human pituitary adenomas and in vitro effects of CCK peptides on GH and gonadotrophin secretion

There is growing evidence that cholecystokinin (CCK) affects growth and differentiation of anterior pituitary cells, via the CCK-B receptor. The possibility of an autocrine / paracrine role for CCK to modulate hormone secretion in human pituitary tumour cells is demonstrated here by RT-PCR and direct sequencing. In support of this conclusion, a neutralising antibody against the CCK peptide exhibited a dose dependent inhibition of hormone secretion by functionless pituitary adenomas. Total RNA was extracted from human pituitary adenomas, reverse transcribed into cDNA and subjected to PCR using primers specific for the gene for CCK, CCK-A and CCK-B receptors. PCR bands of the predicted length were observed in all tumours using human CCK gene and CCK-B receptor primers. Restriction digestion and direct sequence analysis provided further evidence that they represented both the human CCK peptide along with the CCK-A and/B receptor mRNA. CCK-33 and CCK octapeptide sulphate (CCK-8s) both powerfully stimulated phosphatidylinositol hydrolysis, providing evidence for functional activity of the CCK-A and/B receptors. A direct stimulatory effect of CCK peptides on both LH and FSH secretion is reported for the first time, whereas stimulatory effects on GH were blocked by antagonists to CCK. These results may indicate an autocrine role for CCK in the functioning and perhaps development of human pituitary tumours. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart.