The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure) mediates activation of the immune response in bacterial infection of the central nervous system (CNS). The chemotactic G-protein-coupled receptor (GPCR) formyl-peptide-receptor like-1 (FPRL1) plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear.

A peptide derived from an extracellular domain selectively inhibits receptor internalization: Target sequences on insulin and insulin-like growth factor 1 receptors

Certain peptides derived from the α1 domain of the major histocompatibility class I antigen complex (MHC-I) inhibit receptor internalization, increasing the steady-state number of active receptors on the cell surface and thereby enhancing the sensitivity to hormones and other agonists. These peptides self-assemble, and they also bind to MHC-I at the same site from which they are derived, suggesting that they could bind to receptor sites with significant sequence similarity. Receptors affected by MHC-I peptides do, indeed, have such sequence similarity, as illustrated here by insulin receptor (IR) and insulin-like growth factor-1 receptor. A synthetic peptide with sequence identical to a certain extracellular receptor domain binds to that receptor in a ligand-dependent manner and inhibits receptor internalization. Moreover, each such peptide is selective for its cognate receptor. An antibody to the IR peptide not only binds to IR and competes with the peptide but also inhibits insulin-dependent internalization of IR. These observations, and binding studies with deletion mutants of IR, indicate that the sequence QILKELEESSF encoded by exon 10 plays a key role in IR internalization. Our results illustrate a principle for identifying receptor-specific sites of importance for receptor internalization, and for enhancing sensitivity to hormones and other agonists.

New insights into appetite, inflammation and the use of fish oil in hemodialysis patients

Protein-energy wasting (PEW) is commonly seen in patients with chronic kidney disease (CKD). The condition is characterised by chronic, systemic low-grade inflammation which affects nutritional status by a variety of mechanisms including reducing appetite and food intake and increasing muscle catabolism. PEW is linked with co-morbidities such as cardiovascular disease, and is associated with lower quality of life, increased hospitalisations and a 6-fold increase in risk of death1. Significant gender differences have been found in the severity and effects of several markers of PEW. There have been limited studies testing the ability of anti-inflammatory agents or nutritional interventions to reduce the effects of PEW in dialysis patients. This thesis makes a significant contribution to the understanding of PEW in dialysis patients. It advances understanding of measurement techniques for two of the key components, appetite and inflammation, and explores the effect of fish oil, an anti-inflammatory agent, on markers of PEW in dialysis patients. The first part of the thesis consists of two methodological studies conducted using baseline data. The first study aims to validate retrospective ratings of hunger, desire to eat and fullness on visual analog scales (VAS) (paper and pen and electronic) as a new method of measuring appetite in dialysis patients. The second methodological study aims to assess the ability of a variety of methods available in routine practice to detect the presence of inflammation. The second part of the thesis aims to explore the effect of 12 weeks supplementation with 2g per day of Eicosapentaenoic Acid (EPA), a longchain fatty acid found in fish oil, on markers of PEW. A combination of biomarkers and psychomarkers of appetite and inflammation are the main outcomes being explored, with nutritional status, dietary intake and quality of life included as secondary outcomes. A lead in phase of 3 months prior to baseline was used so that each person acts as their own historical control. The study also examines whether there are gender differences in response to the treatment. Being an exploratory study, an important part of the work is to test the feasibility of the intervention, thus the level of adherence and factors associated with adherence are also presented. The studies were conducted at the hemodialysis unit of the Wesley Hospital. Participants met the following criteria: adult, stage 5 CKD on hemodialysis for at least 3 months, not expected to receive a transplant or switch to another dialysis modality during the study, absence of intellectual impairment or mental illness impairing ability to follow instructions or complete the intervention. A range of intermediate, clinical and patient-centred outcome measures were collected at baseline and 12 weeks. Inflammation was measured using five biomarkers: c-reactive protein (CRP), interleukin-6 (IL6), intercellular adhesion molecule (sICAM-1), vascular cell adhesion molecule (sVCAM-1) and white cell count (WCC). Subjective appetite was measured using the first question from the Appetite and Dietary Assessment (ADAT) tool and VAS for measurements of hunger, desire to eat and fullness. A novel feature of the study was the assessment of the appetite peptides leptin, ghrelin and peptide YY as biomarkers of appetite. Nutritional status/inflammation was assessed using the Malnutrition-Inflammation Score (MIS) and the Patient-Generated Subjective Global Assessment (PG-SGA). Dietary intake was measured using 3-day records. Quality of life was measured using the Kidney Disease Quality of Life Short Form version 1.3 (KDQOL-SF™ v1.3 © RAND University), which combines the Short-Form 36 (SF36) with a kidney-disease specific module2. A smaller range of these variables was available for analysis during the control phase (CRP, ADAT, dietary intake and nutritional status). Statistical analysis was carried out using SPSS version 14 (SPSS Inc, Chicago IL, USA). Analysis of the first part of the thesis involved descriptive and bivariate statistics, as well as Bland-Altman plots to assess agreement between methods, and sensitivity analysis/ROC curves to test the ability of methods to predict the presence of inflammation. The unadjusted (paired ttests) and adjusted (linear mixed model) change over time is presented for the main outcome variables of inflammation and appetite. Results are shown for the whole group followed by analyses according to gender and adherence to treatment. Due to the exploratory nature of the study, trends and clinical significance were considered as important as statistical significance. Twenty-eight patients (mean age 61±17y, 50% male, dialysis vintage 19.5 (4- 101) months) underwent baseline assessment. Seven out of 28 patients (25%) reported sub-optimal appetite (self-reported as fair, poor or very poor) despite all being well nourished (100% SGA A). Using the VAS, ratings of hunger, but not desire to eat or fullness, were significantly (p<0.05) associated with a range of relevant clinical variables including age (r=-0.376), comorbidities (r=-0.380) nutritional status (PG-SGA score, r=-0.451), inflammatory markers (CRP r=-0.383; sICAM-1 r=-0.387) and seven domains of quality of life. Patients expressed a preference for the paper and pen method of administering VAS. None of the tools (appetite, MIS, PG-SGA, albumin or iron) showed an acceptable ability to detect patients who are inflamed. It is recommended that CRP should be tested more frequently as a matter of course rather than seeking alternative methods of measuring inflammation. 27 patients completed the 12 week intervention. 20 patients were considered adherent based on changes in % plasma EPA, which rose from 1.3 (0.94)% to 5.2 (1.1)%, p<0.001, in this group. The major barriers to adherence were forgetting to take the tablets as well as their size. At 12 weeks, inflammatory markers remained steady apart from the white cell count which decreased (7.6(2.5) vs 7.0(2.2) x109/L, p=0.058) and sVCAM-1 which increased (1685(654) vs 2249(925) ng/mL, p=0.001). Subjective appetite using VAS increased (51mm to 57mm, +12%) and there was a trend towards reduction in peptide YY (660(31) vs 600(30) pg/mL, p=0.078). There were some gender differences apparent, with the following adjusted change between baseline and week 12: CRP (males -3% vs females +17%, p=0.19), IL6 (males +17% vs females +48%, p=0.77), sICAM-1 (males -5% vs females +11%, p=0.07), sVCAM-1 (males +54% vs females +19%, p=0.08) and hunger ratings (males 20% vs females -5%, p=0.18). On balance, males experienced a maintainence or reduction in three inflammatory markers and an improvement in hunger ratings, and therefore appeared to have responded better to the intervention. Compared to those who didn’t adhere, adherent patients maintained weight (mean(SE) change: +0.5(1.6) vs - 0.8(1.2) kg, p=0.052) and fat-free mass (-0.1 (1.6) vs -1.8 (1.8) kg, p=0.045). There was no difference in change between the intervention and control phase for CRP, appetite, nutritional status or dietary intake. The thesis makes a significant contribution to the evidence base for understanding of PEW in dialysis patients. It has advanced knowledge of methods of assessing inflammation and appetite. Retrospective ratings of hunger on a VAS appear to be a valid method of assessing appetite although samples which include patients with very poor appetite are required to confirm this. Supplementation with fish oil appeared to improve subjective appetite and dampen the inflammatory response. The effectiveness of the intervention is influenced by gender and adherence. Males appear to be more responsive to the primary outcome variables than females, and the quality of response is improved with better adherence. These results provide evidence to support future interventions aimed at reducing the effects of PEW in dialysis patients.

The mechanism of maturation of insulin-like growth factor-1

This study, to elucidate the role of des(1-3)IGF-I in the maturation of IGF-I,used two strategies. The first was to detect the presence of enzymes in tissues, which would act on IGF-I to produce des(1-3)IGF-I, and the second was to detect the potential products of such enzymic activity, namely Gly-Pro-Glu(GPE), Gly-Pro(GP) and des(l- 3)IGF-I. No neutral tripeptidyl peptidase (TPP II), which would release the tripeptide GPE from IGF-I, was detected in brain, urine nor in red or white blood cells. The TPPlike activity which was detected, was attributed to a combined action of a dipeptidyl peptidase (DPP N) and an aminopeptidase (AP A). A true TPP II was, however, detected in platelets. Two purified TPP II enzymes were investigated but they did not release GPE from IGF-I under a variety of conditions. Consequently, TPP II seemed unlikely to participate in the formation of des(1-3)IGF-I. In contrast, an acidic tripeptidyl peptidase activity (TPP I) was detected in brain and colostrum, the former with a pH optimum of 4.5 and the latter 3.8. It seems likely that such an enzyme would participate in the formation of des( 1-3 )IGF-I in these tissues in vitro, ie. that des(1-3)IGF-I may have been produced as an artifact in the isolation of IGF-I from brain and colostrum in acidic conditions. This contrasts with suggestions of an in vivo role for des(1-3)IGF-I, as reported by others. The activity of a dipeptidyl peptidase N (DPP N) from urine, which should release the dipeptide GP from IGF-I, was assessed under a variety of conditions and with a variety of additives and potential enzyme stimulants, but there was no release of GP. The DPP N also exhibited a transferase activity with synthetic substrates in the presence of dipeptides, at lower concentrations than previously reported for other acceptors or other proteolytic enzymes. In addition, a low concentration of a product,possibly the tetrapeptide Gly-Pro-Gly-Leu, was detected with the action of the enzyme on IGF-I in the presence of the dipeptide Gly-Leu. As part of attempts to detect tissue production of des(1-3)IGF-I, a monoclonal antibody (MAb ), directed towards the GPE- end ofiGF-I was produced by immunisation with a 10-mer covalently attached to a carrier protein. By the use of indirect ELISA and inhibitor studies, the MAb was shown to selectively recognise peptides with anNterminal GPE- sequence, and applied to the indirect detection of des(1-3)IGF-I. The concentration of GPE in brain, measured by mass spectrometry ( MS), was low, and the concentration of total IGF-I (measured by ELISA with a commercial polyclonal antibody [P Ab]) was 40 times higher at 50 nmol/kg. This also, was not consistent with the action of a tripeptidyl peptidase in brain that converted all IGF-I to des(1-3)IGF-I plus GPE. Contrasting ELISA results, using the MAb prepared in this study, suggest an even higher concentration of intact IGF-I of 150 nmollkg. This would argue against the presence of any des( 1-3 )IGF-I in brain, but in turn, this indicates either the presence of other substances containing a GPE amino-terminus or other cross reacting epitope. Although the results of the specificity studies reported in Chapter 5 would make this latter possibility seem unlikely, it cannot be completely excluded. No GP was detected in brain by MS. No GPE was detected in colostrum by capillary electrophoresis (CE) but the interference from extraneous substances reduced the detectability of GPE by CE and this approach would require further, prior, purification and concentration steps. A molecule, with a migration time equal to that of the peptide GP, was detected in colostrum by CE, but the concentration (~ 10 11mo/L) was much higher than the IGF-I concentration measured by radio-immunoassay using a PAb (80 nmol/L) or using a Mab (300-400 nmolL). A DPP IV enzyme was detected in colostrum and this could account for the GP, derived from substrates other than IGF-1. Based on the differential results of the two antibody assays, there was no indication of the presence of des(1-3)IGF-I in brain or colostrum. In the absence of any enzyme activity directed towards the amino terminus of IGF-I and the absence any potential products, IGF-I, therefore, does not appear to "mature" via des(1-3)IGF-I in the brain, nor in the neutral colostrum. In spite of these results which indicate the absence of an enzymic attack on IGF-I and the absence of the expected products in tissues, the possibility that the conversion of IGF-I may occur in neutral conditions in limited amounts, cannot be ruled out. It remains possible that in the extracellular environment of the membrane, a complex interaction of IGF-I, binding protein, aminopeptidase(s) and receptor, produces des(1- 3)IGF-I as a transient product which is bound to the receptor and internalised.

The ghrelin receptor isoforms (GHS-R1a and GHS-R1b) and GPR39 : an investigation into receptor dimerisation

Prostate cancer is the second most common cause of cancer-related deaths in Western males. Current diagnostic, prognostic and treatment approaches are not ideal and advanced metastatic prostate cancer is incurable. There is an urgent need for improved adjunctive therapies and markers for this disease. GPCRs are likely to play a significant role in the initiation and progression of prostate cancer. Over the last decade, it has emerged that G protein coupled receptors (GPCRs) are likely to function as homodimers and heterodimers. Heterodimerisation between GPCRs can result in the formation of novel pharmacological receptors with altered functional outcomes, and a number of GPCR heterodimers have been implicated in the pathogenesis of human disease. Importantly, novel GPCR heterodimers represent potential new targets for the development of more specific therapeutic drugs. Ghrelin is a 28 amino acid peptide hormone which has a unique n-octanoic acid post-translational modification. Ghrelin has a number of important physiological roles, including roles in appetite regulation and the stimulation of growth hormone release. The ghrelin receptor is the growth hormone secretagogue receptor type 1a, GHS-R1a, a seven transmembrane domain GPCR, and GHS-R1b is a C-terminally truncated isoform of the ghrelin receptor, consisting of five transmembrane domains. Growing evidence suggests that ghrelin and the ghrelin receptor isoforms, GHS-R1a and GHS-R1b, may have a role in the progression of a number of cancers, including prostate cancer. Previous studies by our research group have shown that the truncated ghrelin receptor isoform, GHS-R1b, is not expressed in normal prostate, however, it is expressed in prostate cancer. The altered expression of this truncated isoform may reflect a difference between a normal and cancerous state. A number of mutant GPCRs have been shown to regulate the function of their corresponding wild-type receptors. Therefore, we investigated the potential role of interactions between GHS-R1a and GHS-R1b, which are co-expressed in prostate cancer and aimed to investigate the function of this potentially new pharmacological receptor. In 2005, obestatin, a 23 amino acid C-terminally amidated peptide derived from preproghrelin was identified and was described as opposing the stimulating effects of ghrelin on appetite and food intake. GPR39, an orphan GPCR which is closely related to the ghrelin receptor, was identified as the endogenous receptor for obestatin. Recently, however, the ability of obestatin to oppose the effects of ghrelin on appetite and food intake has been questioned, and furthermore, it appears that GPR39 may in fact not be the obestatin receptor. The role of GPR39 in the prostate is of interest, however, as it is a zinc receptor. Zinc has a unique role in the biology of the prostate, where it is normally accumulated at high levels, and zinc accumulation is altered in the development of prostate malignancy. Ghrelin and zinc have important roles in prostate cancer and dimerisation of their receptors may have novel roles in malignant prostate cells. The aim of the current study, therefore, was to demonstrate the formation of GHS-R1a/GHS-R1b and GHS-R1a/GPR39 heterodimers and to investigate potential functions of these heterodimers in prostate cancer cell lines. To demonstrate dimerisation we first employed a classical co-immunoprecipitation technique. Using cells co-overexpressing FLAG- and Myc- tagged GHS-R1a, GHS-R1b and GPR39, we were able to co-immunoprecipitate these receptors. Significantly, however, the receptors formed high molecular weight aggregates. A number of questions have been raised over the propensity of GPCRs to aggregate during co-immunoprecipitation as a result of their hydrophobic nature and this may be misinterpreted as receptor dimerisation. As we observed significant receptor aggregation in this study, we used additional methods to confirm the specificity of these putative GPCR interactions. We used two different resonance energy transfer (RET) methods; bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), to investigate interactions between the ghrelin receptor isoforms and GPR39. RET is the transfer of energy from a donor fluorophore to an acceptor fluorophore when they are in close proximity, and RET methods are, therefore, applicable to the observation of specific protein-protein interactions. Extensive studies using the second generation bioluminescence resonance energy transfer (BRET2) technology were performed, however, a number of technical limitations were observed. The substrate used during BRET2 studies, coelenterazine 400a, has a low quantum yield and rapid signal decay. This study highlighted the requirement for the expression of donor and acceptor tagged receptors at high levels so that a BRET ratio can be determined. After performing a number of BRET2 experimental controls, our BRET2 data did not fit the predicted results for a specific interaction between these receptors. The interactions that we observed may in fact represent ‘bystander BRET’ resulting from high levels of expression, forcing the donor and acceptor into close proximity. Our FRET studies employed two different FRET techniques, acceptor photobleaching FRET and sensitised emission FRET measured by flow cytometry. We were unable to observe any significant FRET, or FRET values that were likely to result from specific receptor dimerisation between GHS-R1a, GHS-R1b and GPR39. While we were unable to conclusively demonstrate direct dimerisation between GHS-R1a, GHS-R1b and GPR39 using several methods, our findings do not exclude the possibility that these receptors interact. We aimed to investigate if co-expression of combinations of these receptors had functional effects in prostate cancers cells. It has previously been demonstrated that ghrelin stimulates cell proliferation in prostate cancer cell lines, through ERK1/2 activation, and GPR39 can stimulate ERK1/2 signalling in response to zinc treatments. Additionally, both GHS-R1a and GPR39 display a high level of constitutive signalling and these constitutively active receptors can attenuate apoptosis when overexpressed individually in some cell types. We, therefore, investigated ERK1/2 and AKT signalling and cell survival in prostate cancer the potential modulation of these functions by dimerisation between GHS-R1a, GHS-R1b and GPR39. Expression of these receptors in the PC-3 prostate cancer cell line, either alone or in combination, did not alter constitutive ERK1/2 or AKT signalling, basal apoptosis or tunicamycin-stimulated apoptosis, compared to controls. In summary, the potential interactions between the ghrelin receptor isoforms, GHS-R1a and GHS-R1b, and the related zinc receptor, GPR39, and the potential for functional outcomes in prostate cancer were investigated using a number of independent methods. We did not definitively demonstrate the formation of these dimers using a number of state of the art methods to directly demonstrate receptor-receptor interactions. We investigated a number of potential functions of GPR39 and GHS-R1a in the prostate and did not observe altered function in response to co-expression of these receptors. The technical questions raised by this study highlight the requirement for the application of extensive controls when using current methods for the demonstration of GPCR dimerisation. Similar findings in this field reflect the current controversy surrounding the investigation of GPCR dimerisation. Although GHS-R1a/GHS-R1b or GHS-R1a/GPR39 heterodimerisation was not clearly demonstrated, this study provides a basis for future investigations of these receptors in prostate cancer. Additionally, the results presented in this study and growing evidence in the literature highlight the requirement for an extensive understanding of the experimental method and the performance of a range of controls to avoid the spurious interpretation of data gained from artificial expression systems. The future development of more robust techniques for investigating GPCR dimerisation is clearly required and will enable us to elucidate whether GHS-R1a, GHS-R1b and GPR39 form physiologically relevant dimers.

The expanding roles of the ghrelin-gene derived peptide obestatin in health and disease

Obestatin is a 23 amino acid, ghrelin gene-derived peptide hormone produced in the stomach and a range of other tissues throughout the body. While it was initially reported that obestatin opposed the actions of ghrelin with regards to appetite and food intake, it is now clear that obestatin is not an endogenous ghrelin antagonist of ghrelin, but it is a multi-functional peptide hormone in its own right. In this review we will discuss the controversies associated with the discovery of obestatin and explore emerging central and peripheral roles of obestatin, roles in adipogenesis, pancreatic homeostasis and cancer.

Association study of the calcitonin gene-related polypeptide-alpha (CALCA) and the receptor activity modifying 1 (RAMP1) genes with migraine

Migraine is a common neurovascular brain disorder characterised by recurrent attacks of severe headache that may be accompanied by various neurological symptoms. Migraine is thought to result from activation of the trigeminovascular system followed by vasodilation of pain-producing intracranial blood vessels and activation of second-order sensory neurons in the trigeminal nucleus caudalis. Calcitonin gene-related peptide (CGRP) is a mediator of neurogenic inflammation and the most powerful vasodilating neuropeptide, and has been implicated in migraine pathophysiology. Consequently, genes involved in CGRP synthesis or CGRP receptor genes may play a role in migraine and/or increase susceptibility. This study investigates whether variants in the gene that encodes CGRP, calcitonin-related polypeptide alpha (CALCA) or in the gene that encodes a component of its receptor, receptor activity modifying protein 1 (RAMP1), are associated with migraine pathogenesis and susceptibility. The single nucleotide polymorphisms (SNPs) rs3781719 and rs145837941 in the CALCA gene, and rs3754701 and rs7590387 at the RAMP1 locus, were analysed in an Australian Caucasian population of migraineurs and matched controls. Although we find no significant association of any of the SNPs tested with migraine overall, we detected a nominally significant association (p = 0.031) of the RAMP1 rs3754701 variant in male migraine subjects, although this is non-significant after Bonferroni correction for multiple testing.

Comparative analysis reveals loss of the appetite-regulating peptide hormone ghrelin in falcons

Ghrelin and leptin are key peripherally secreted appetite-regulating hormones in vertebrates. Here we consider the ghrelin gene (GHRL) of birds (class Aves), where it has been reported that ghrelin inhibits rather than augments feeding. Thirty-one bird species were compared, revealing that most species harbour a functional copy of GHRL and the coding region for its derived peptides ghrelin and obestatin. We provide evidence for loss of GHRL in saker and peregrine falcons, and this is likely to result from the insertion of an ERVK retrotransposon in intron 0. We hypothesise that the loss of anorexigenic ghrelin is a predatory adaptation that results in increased food-seeking behaviour and feeding in falcons.

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

Assessment and control of Bacillus cereus emetic toxin in food

Despite of improving levels of hygiene, the incidence of registered food borne disease has been at the same level for many years: there were 40 to 90 epidemics in which 1000-9000 persons contracted food poisoning through food or drinking water in Finland. Until the year 2004 salmonella and campylobacter were the most common bacterial causes of food borne diseases, but in years 2005-2006 Bacillus cereus was the most common. Similar developement has been published i.e. in Germany already in the 1990´s. One reason for this can be Bacillus cereus and its emetic toxin, cereulide. Bacillus cereus is a common environmental bacterium that contaminates raw materials of food. Otherwise than salmonella and campylobacter, Bacillus cereus is a heat resistant bacterium, capable of surviving most cooking procedures due to the production of highly thermo resistant spores. The food involved has usually been heat treated and surviving spores are the source of the food poisoning. The heat treatment induces germination of the spore and the vegetative cells then produce toxins. This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing Bacillus cereus. The biochemistry and physiology of cereulide production was investigated and the results were targeted to offer tools for minimizing toxin risk in food during the production. I developed methods for the extraction and quantitative analysis of cereulide directly from food. A prerequisite for that is knowledge of the chemical and physical properties of the toxin. Because cereulide is practically insoluble in water, I used organic solvents; methanol, ethanol and pentane for the extraction. For extraction of bakery products I used high temperature (100C) and pressure (103.4 bars). Alternaties for effective extraction is to flood the plain food with ethanol, followed by stationary equilibration at room temperature. I used this protocol for extracting cereulide from potato puree and penne. Using this extraction method it is also possible also extract cereulide from liquid food, like milk. These extraction methods are important improvement steps for studying of Bacillus cereus emetic food poisonings. Prior my work, cereulide extraction was done using water. As the result, the yield was poor and variable. To investigate suspected food poisonings, it is important to show actual toxicity of the incriminated food. Many toxins, but not cereulide, inactivate during food processing like heating. The next step is to identify toxin by chemical methods. I developed with my colleague Maria Andesson a rapid assay for the detection of cereulide toxicity, within 5 to 15 minutes. By applying this test it is possible to rapidly detect which food was causing the food poisoning. The chemical identification of cereulide was achieved using mass spectrometry. I used cereulide specific molecular ions, m/z (+/-0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 microg/g wet wt) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, if stored at non refrigerated temperatures (21-23C). Rice and meat pastries are frequently consumed under conditions where no cooled storage is available e.g. picnics and outdoor events. Bacillus cereus is a ubiquitous spore former and is therefore difficult to eliminate from foods. It is therefore important to know which conditions will affect the formation of cereulide in foods. My research showed that the cereulide content was strongly (10 to 1000 fold differences in toxin content) affected by the growth environment of the bacterium. Storage of foods under nitrogen atmosphere (> 99.5 %) prevented the production of cereulide. But when also carbon dioxide was present, minimizing the oxygen contant (< 1%) did not protect the food from formation of cereulide in preliminary experiments. Also food supplements affected cereulide production at least in the laboratory. Adding free amino acids, leucine and valine, stimulated cereulide production 10 to 20 fold. In peptide bonded form these amino acids are natural constituents in all proteins. Interestingly, adding peptide bonded leucine and valine had no significant effect on cereulide production. Free amino acids leucine and valine are approved food supplements and widely used as flawour modifiers in food technology. My research showed that these food supplements may increase food poisoning risk even though they are not toxic themselves.

Spontaneous and reversible self-assembly of a polypeptide fragment of insulin-like growth factor binding protein-2 into fluorescent nanotubular structures

In this communication, we report the spontaneous and reversible in vitro self-assembly of a polypeptide fragment derived from the C-terminal domain of Insulin-like Growth Factor Binding Protein (IGFBP-2) into soluble nanotubular structures several micrometres long via a mechanism involving inter-molecular disulfide bonds and exhibiting enhanced fluorescence.

An unusual C-H center dot center dot center dot O hydrogen bond mediated reversal of polypeptide chain direction in a synthetic peptide helix

An unusual C-terminal conformation has been detected in a synthetic decapeptide designed to analyze the stereochemistry of helix termination in polypeptides. The crystal structure of the decapeptide Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-(D)Ala-(D)Leu-Aib-OMe reveals a helical segment spanning residues 1-7 and helix termination by formation of a Schellman motif, generated by (D)Ala(8) adopting the left-handed helical (alpha(L)) conformation. The extended conformation at (D)Leu(9) results in a compact folded structure, stabilized by a potentially strong C-H ... O hydrogen bond between Ala(4) (CH)-H-alpha and (D)Leu(9)CO. The parameters for C-H ... O interaction are Ala(4) (CH)-H-alpha .. O=C (D)Leu(9) distance 3.27 Angstrom C-alpha-H .. O angle 176 degrees, and O .. H-alpha distance 2.29 Angstrom. This structure suggests that insertion of contiguous D-residues may provide a handle for the generation of designed structures containing more than one helical segment folded in a compact manner. (C) 2000 Academic Press.

A C-H…O hydrogen bond stabilized polypeptide chain reversal motif at the C-terminus helices in proteins.

The serendipitous observation of a C–Hcdots, three dots, centeredO hydrogen bond mediated polypeptide chain reversal in synthetic peptide helices has led to a search for the occurrence of a similar motif in protein structures. From a dataset of 634 proteins, 1304 helices terminating in a Schellman motif have been examined. The C–Hcdots, three dots, centeredO interaction between the T−4 CαH and T+1 C=O group (Ccdots, three dots, centeredO≤3.5 Å) becomes possible only when the T+1 residue adopts an extended β conformation (T is defined as the helix terminating residue adopting an αL conformation). In all, 111 examples of this chain reversal motif have been identified and the compositional and conformational preferences at positions T−4, T, and T+1 determined. A marked preference for residues like Ser, Glu and Gln is observed at T−4 position with the motif being further stabilized by the formation of a side-chain–backbone Ocdots, three dots, centeredH–N hydrogen bond involving the side-chain of residue T−4 and the N–H group of residue T+3. In as many as 57 examples, the segment following the helix was extended with three to four successive residues in β conformation. In a majority of these cases, the succeeding β strand lies approximately antiparallel with the helix, suggesting that the backbone C–Hcdots, three dots, centeredO interactions may provide a means of registering helices and strands in an antiparallel orientation. Two examples were identified in which extended registry was detected with two sets of C–Hcdots, three dots, centeredO hydrogen bonds between (T−4) CαHcdots, three dots, centeredC=O (T+1) and (T−8) CαHcdots, three dots, centeredC=O (T+3).

A C-H center dot center dot center dot O hydrogen bond stabilized polypeptide chain reversal motif at the C terminus of helices in proteins

The serendipitous observation of a C-H...O hydrogen bond mediated polypeptide chain reversal in synthetic peptide helices has led to a search for the occurrence of a similar motif in protein structures. From a dataset of 634 proteins, 1304 helices terminating in a Schellman motif have been examined. The C-H...O interaction between the T - 4 (CH)-H-alpha and T + 1 C=O group (C...O 3.5 Angstrom) becomes possible only when the T + 1 residue adopts an extended beta conformation (T is defined as the helix terminating residue adopting an alpha(L) conformation). In all, 111 examples of this chain reversal motif have been identified and the compositional and conformational. preferences at positions T - 4, T, and T + 1 determined. A marked preference for residues like Set, Glu and Gln is observed at T - 4 position with the motif being further stabilized by the formation of a side-chain-backbone O...H-N hydrogen bond involving the side-chain of residue T - 4 and the N-H group of residue T + 3. In as many as 57 examples, the segment following the helix was extended with three to four successive residues in beta conformation. In a majority of these cases, the succeeding beta strand lies approximately antiparallel with the helix, suggesting that the backbone C-H...O interactions may provide a means of registering helices and strands in an antiparallel orientation. Two examples were identified in which extended registry was detected with two sets of C-H...O hydrogen bonds between (T - 4) (CH)-H-alpha...C=O (T + 1) and (T - 8) (CH)-H-alpha...C=O (T + 3). 0 2002 Published by Elsevier Science Ltd.