Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves

Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis ( 2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a > 1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel alpha 2 delta-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S(35)methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.

New scientific paradigms for probiotics and prebiotics

The inaugural meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP) was held May 3 to May 5 2002 in London, Ontario, Canada. A group of 63 academic and industrial scientists from around the world convened to discuss current issues in the science of probiotics and prebiotics. ISAPP is a non-profit organization comprised of international scientists whose intent is to strongly support and improve the levels of scientific integrity and due diligence associated with the study, use, and application of probiotics and prebiotics. In addition, ISAPP values its role in facilitating communication with the public and healthcare providers and among scientists in related fields on all topics pertinent to probiotics and prebiotics. It is anticipated that such efforts will lead to development of approaches and products that are optimally designed for the improvement of human and animal health and well being. This article is a summary of the discussions, conclusions, and recommendations made by 8 working groups convened during the first ISAPP workshop focusing on the topics of: definitions, intestinal flora, extra-intestinal sites, immune function, intestinal disease, cancer, genetics and genomics, and second generation prebiotics. Humans have evolved in symbiosis with an estimated 1014 resident microorganisms. However, as medicine has widely defined and explored the perpetrators of disease, including those of microbial origin, it has paid relatively little attention to the microbial cells that constitute the most abundant life forms associated with our body. Microbial metabolism in humans and animals constitutes an intense biochemical activity in the body, with profound repercussions for health and disease. As understanding of the human genome constantly expands, an important opportunity will arise to better determine the relationship between microbial populations within the body and host factors (including gender, genetic background, and nutrition) and the concomitant implications for health and improved quality of life. Combined human and microbial genetic studies will determine how such interactions can affect human health and longevity, which communication systems are used, and how they can be influenced to benefit the host. Probiotics are defined as live microorganisms which, when administered in adequate amounts confer a health benefit on the host.1 The probiotic concept dates back over 100 years, but only in recent times have the scientific knowledge and tools become available to properly evaluate their effects on normal health and well being, and their potential in preventing and treating disease. A similar situation exists for prebiotics, defined by this group as non-digestible substances that provide a beneficial physiological effect on the host by selectively stimulating the favorable growth or activity of a limited number of indigenous bacteria. Prebiotics function complementary to, and possibly synergistically with, probiotics. Numerous studies are providing insights into the growth and metabolic influence of these microbial nutrients on health. Today, the science behind the function of probiotics and prebiotics still requires more stringent deciphering both scientifically and mechanistically. The explosion of publications and interest in probiotics and prebiotics has resulted in a body of collective research that points toward great promise. However, this research is spread among such a diversity of organisms, delivery vehicles (foods, pills, and supplements), and potential health targets such that general conclusions cannot easily be made. Nevertheless, this situation is rapidly changing on a number of important fronts. With progress over the past decade on the genetics of lactic acid bacteria and the recent, 2,3 and pending, 4 release of complete genome sequences for major probiotic species, the field is now armed with detailed information and sophisticated microbiological and bioinformatic tools. Similarly, advances in biotechnology could yield new probiotics and prebiotics designed for enhanced or expanded functionality. The incorporation of genetic tools within a multidisciplinary scientific platform is expected to reveal the contributions of commensals, probiotics, and prebiotics to general health and well being and explicitly identify the mechanisms and corresponding host responses that provide the basis for their positive roles and associated claims. In terms of human suffering, the need for effective new approaches to prevent and treat disease is paramount. The need exists not only to alleviate the significant mortality and morbidity caused by intestinal diseases worldwide (especially diarrheal diseases in children), but also for infections at non-intestinal sites. This is especially worthy of pursuit in developing nations where mortality is too often the outcome of food and water borne infection. Inasmuch as probiotics and prebiotics are able to influence the populations or activities of commensal microflora, there is evidence that they can also play a role in mitigating some diseases. 5,6 Preliminary support that probiotics and prebiotics may be useful as intervention in conditions including inflammatory bowel disease, irritable bowel syndrome, allergy, cancer (especially colorectal cancer of which 75% are associated with diet), vaginal and urinary tract infections in women, kidney stone disease, mineral absorption, and infections caused by Helicobacter pylori is emerging. Some metabolites of microbes in the gut may also impact systemic conditions ranging from coronary heart disease to cognitive function, suggesting the possibility that exogenously applied microbes in the form of probiotics, or alteration of gut microecology with prebiotics, may be useful interventions even in these apparently disparate conditions. Beyond these direct intervention targets, probiotic cultures can also serve in expanded roles as live vehicles to deliver biologic agents (vaccines, enzymes, and proteins) to targeted locations within the body. The economic impact of these disease conditions in terms of diagnosis, treatment, doctor and hospital visits, and time off work exceeds several hundred billion dollars. The quality of life impact is also of major concern. Probiotics and prebiotics offer plausible opportunities to reduce the morbidity associated with these conditions. The following addresses issues that emerged from 8 workshops (Definitions, Intestinal Flora, Extra-Intestinal Sites, Immune Function, Intestinal Disease, Cancer, Genomics, and Second Generation Prebiotics), reflecting the current scientific state of probiotics and prebiotics. This is not a comprehensive review, however the study emphasizes pivotal knowledge gaps, and recommendations are made as to the underlying scientific and multidisciplinary studies that will be required to advance our understanding of the roles and impact of prebiotics, probiotics, and the commensal microflora upon health and disease management.

Cetobacterium somerae sp nov from human feces and emended description of the genus Cetobacterium

Phenotypic and phylogenetic studies were performed on four isolates of an unidentified gram-negative, microaerotolerant, non-spore-forming, rod-shaped bacterium isolated from the feces of children. The unknown organism was bile resistant and produced acetic acid as the major end product of metabolism of peptides and carbohydrates. It possessed a low DNA G + C content of 31 mol %. Comparative 16S rRNA gene sequencing demonstrated that the four isolates were phylogenetically identical (100% 16S rRNA sequence similarity) and represent a hitherto unknown sub-line within the genus Cetobacterium. The novel bacterium displayed approximately 5% sequence divergence with Cetobacterium ceti, and can be readily distinguished from the latter by physiological and biochemical criteria. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown fecal bacterium be classified in the genus Cetobacterium, as Cetobacterium somerae sp. nov. The proposed type strain of Cetobacterium somerae is WAL 14325(T) (ATCC BAA-474(T) = CCUG 46254T).

Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell

Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.

Comparison of bottom-up protein identification methods

With the rapid development of proteomics, a number of different methods appeared for the basic task of protein identification. We made a simple comparison between a common liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow using an ion trap mass spectrometer and a combined LC-MS and LC-MS/MS method using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and accurate peptide masses. To compare the two methods for protein identification, we grew and extracted proteins from E. coli using established protocols. Cystines were reduced and alkylated, and proteins digested by trypsin. The resulting peptide mixtures were separated by reversed-phase liquid chromatography using a 4 h gradient from 0 to 50% acetonitrile over a C18 reversed-phase column. The LC separation was coupled on-line to either a Bruker Esquire HCT ion trap or a Bruker 7 tesla APEX-Qe Qh-FTICR hybrid mass spectrometer. Data-dependent Qh-FTICR-MS/MS spectra were acquired using the quadrupole mass filter and collisionally induced dissociation into the external hexapole trap. Proteins were in both schemes identified by Mascot MS/MS ion searches and the peptides identified from these proteins in the FTICR MS/MS data were used for automatic internal calibration of the FTICR-MS data, together with ambient polydimethylcyclosiloxane ions.

Fibrillisation of hydrophobically modified amyloid peptide fragments in an organic solvent

The self-assembly of a hydrophobically modified fragment of the amyloid beta(A beta) peptide has been studied in methanol. The peptide FFKLVFF is based on A beta(16-20) extended at the N terminus by two phenylalanine residues. The formation of amyloid-type fibrils is confirmed by Congo Red staining, thioflavin T fluorescence and circular dichroism experiments. FTIR points to the formation of beta-sheet structures in solution and in dried films and suggests that aggregation occurs at low concentration and is not strongly affected by further increase in concentration, i.e. the peptide is a strong fibril-former in methanol. UV fluorescence experiments on unstained peptide and CD point to the importance of aromatic interactions between phenylalanine groups in driving aggregation into beta-sheets. The CD spectrum differs from that usually observed for beta-sheet assemblies formed by larger peptides or proteins and this is discussed for solutions in methanol and also trifluoroethanol. The fibril structure is imaged by transmission electron microscopy and scanning electron microscopy on dried samples and is confirmed by small-angle X-ray scattering experiments in solution.

Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin- was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

Non-genomic effects of PPARgamma ligands: inhibition of GPVI-stimulated platelet activation

BACKGROUND: Peroxisome proliferator-activated receptor-(gamma) (PPAR(gamma)) is expressed in human platelets although in the absence of genomic regulation in these cells, its functions are unclear. OBJECTIVE: In the present study, we aimed to demonstrate the ability of PPAR(gamma) ligands to modulate collagen-stimulated platelet function and suppress activation of the glycoprotein VI (GPVI) signaling pathway. METHODS: Washed platelets were stimulated with PPAR(gamma) ligands in the presence and absence of PPAR(gamma) antagonist GW9662 and collagen-induced aggregation was measured using optical aggregometry. Calcium levels were measured by spectrofluorimetry in Fura-2AM-loaded platelets and tyrosine phosphorylation levels of receptor-proximal components of the GPVI signaling pathway were measured using immunoblot analysis. The role of PPAR(gamma) agonists in thrombus formation was assessed using an in vitro model of thrombus formation under arterial flow conditions. RESULTS: PPAR(gamma) ligands inhibited collagen-stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P-selectin exposure. PPAR(gamma) ligands inhibited thrombus formation under arterial flow conditions. The incorporation of GW9662 reversed the inhibitory actions of PPAR(gamma) agonists, implicating PPAR(gamma) in the effects observed. Furthermore, PPAR(gamma) ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway. PPAR(gamma) was found to associate with Syk and LAT after platelet activation. This association was prevented by PPAR(gamma) agonists, indicating a potential mechanism for PPAR(gamma) function in collagen-stimulated platelet activation. CONCLUSIONS: PPAR(gamma) agonists inhibit the activation of collagen-stimulation of platelet function through modulation of early GPVI signalling.

Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells

Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule-1 (PECAM-1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcR-chain signaling via recruitment of SHP-2 to phosphorylated immunoreceptor tyrosine-based inhibitory motifs in PECAM-1. Objective: To investigate the possibility that PECAM-1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI-mediated platelet activation in platelets. Methods: The ability of PECAM-1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling. We therefore propose that PECAM-1-mediated inhibition of GPVI-dependent platelet responses result, at least in part, from recruitment of SHP-2–p85 complexes to tyrosine-phosphorylated PECAM-1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.

Differential effects of overexpression of ERalpha and ERbeta in MCF10A immortalised, non-transformed human breast epithelail cells

Background: Cellular effects of oestrogen are mediated by two intracellular receptors ERα and ERβ. However, to compare responses mediated through these two receptors, experimental models are needed where ERα and ERβ are individually stably overexpressed in the same cell type. Methods: We compared the effects of stable overexpression of ERα and ERβ in the MCF10A cell line, which is an immortalised but non-transformed breast epithelial cell line without high endogenous ER expression. Results: Clones of MCF10A cells were characterised which stably overexpressed ERα (10A-ERα2, 10A-ERα13) or which stably overexpressed ERβ (10A-ERβ12, 10A-ERβ15). Overexpression of either ERα or ERβ allowed induction of an oestrogen-regulated ERE-LUC reporter gene by oestradiol which was not found in the untransfected cells. Oestradiol also increased proliferation of 10A-ERα13 and 10A-ERβ12 cells, but not untransfected cells, by 1.3-fold over 7 days. The phytoestrogen, genistein, which is reported to bind more strongly to ERβ than to ERα, could induce luciferase gene expression from an ERE-LUC reporter gene at concentrations of 10−6 M and 10−5 M but only in the clones overexpressing ERβ and not in those overexpressing ERα. Clone 10A-ERβ12 also yielded growth stimulation with 10-6 M genistein. Finally, the overexpression of ERα, but not ERβ, gave rise to increased growth in semi-solid methocel suspension culture in the presence of 70 nM oestradiol, suggesting that overexpression of ERα, but not ERβ, produces characteristics of a transformed phenotype. Conclusions: This provides a model system to compare effects of oestradiol with other oestrogenic ligands in cells stably overexpressing individually ERα or ERβ.

Intra-ovarian roles of activins and inhibins

Granulosa cells are the main ovarian source of inhibins, activins and activin-binding protein (follistatin) while germ (oogonia, oocytes) and somatic (theca, granulosa, luteal) cells express activin receptors, signaling components and inhibin co-receptor (betaglycan). Activins are implicated in various intra-ovarian roles including germ cell survival and primordial follicle assembly; follicle growth from preantral to mid-antral stages; suppression of thecal androgen production; promotion of granulosa cell proliferation, FSHR and CYP19A1 expression; enhancement of oocyte developmental competence; retardation of follicle luteinization and/or atresia and involvement in luteolysis. Inhibins (primarily inhibin A) are produced in greatest amounts by preovulatory follicles (and corpus luteum in primates) and suppress FSH secretion through endocrine negative feedback. Together with follistatin, inhibins act locally to oppose auto-/paracrine activin (and BMP) signaling thus modulating many of the above processes. The balance between activin-inhibin shifts during follicle development with activin signalling prevailing at earlier stages but declining as inhibin and betaglycan expression rise.

Surface activity of surfactin recovered and purified from fermentation broth using a two-step ultrafiltration (UF) process

B. subtilis under certain types of media and fermentation conditions can produce surfactin, a biosurfactant which belongs to the lipopeptide class. Surfactin has exceptional surfactant activity, and exhibits some interesting biological characteristics such as antibacterial activity, antitumoral activity against ascites carcinoma cells, and a hypocholesterolemic activity that inhibits cAMP phosphodiesterase, as well as having anti-HIV properties. A cost effective recovery and purification of surfactin from fermentation broth using a two-step ultrafiltration (UF) process has been developed in order to reduce the cost of surfactin production. In this study, competitive adsorption of surfactin and proteins at the air-water interface was studied using surface pressure measurements. Small volumes of bovine serum albumin (BSA) and β-casein solutions were added to the air-water interface on a Langmuir trough and allowed to stabilise before the addition of surfactin to the subphase. Contrasting interfacial behaviour of proteins was observed with β-casein showing faster initial adsorption compared to BSA. On introduction of surfactin both proteins were displaced but a longer time were taken to displace β-casein. Overall the results showed surfactin were highly surface-active by forming a β-sheet structure at the air-water interface after reaching its critical micelle concentration (CMC) and were effective in removing both protein films, which can be explained following the orogenic mechanism. Results showed that the two-step UF process was effective to achieve high purity and fully functional surfactin.

Combined transcriptomic-(1)H NMR metabonomic study reveals yhat monoethylhexyl phthalate stimulates adipogenesis and glyceroneogenesis in human adipocytes

Adipose tissue is a major storage site for lipophilic environmental contaminants. The environmental metabolic disruptor hypothesis postulates that some pollutants can promote obesity or metabolic disorders by activating nuclear receptors involved in the control of energetic homeostasis. In this context, monoethylhexyl phthalate (MEHP) is of particular concern since it was shown to activate the peroxisome proliferator-activated receptor γ (PPARγ) in 3T3-L1 murine preadipocytes. In the present work, we used an untargeted, combined transcriptomic-(1)H NMR-based metabonomic approach to describe the overall effect of MEHP on primary cultures of human subcutaneous adipocytes differentiated in vitro. MEHP stimulated rapidly and selectively the expression of genes involved in glyceroneogenesis, enhanced the expression of the cytosolic phosphoenolpyruvate carboxykinase, and reduced fatty acid release. These results demonstrate that MEHP increased glyceroneogenesis and fatty acid reesterification in human adipocytes. A longer treatment with MEHP induced the expression of genes involved in triglycerides uptake, synthesis, and storage; decreased intracellular lactate, glutamine, and other amino acids; increased aspartate and NAD, and resulted in a global increase in triglycerides. Altogether, these results indicate that MEHP promoted the differentiation of human preadipocytes to adipocytes. These mechanisms might contribute to the suspected obesogenic effect of MEHP.

Natural food and beverage flavour enhancers

Flavour enhancers are of great importance to the food industry and the consumer, in terms of achieving strong, balanced and preferred product flavour. The understanding and use of flavour enhancers can help to avoid excessive use of individual ingredients such as salt or specific character impact flavours. This chapter first discusses savoury flavour enhancement through the use of ingredients rich in amino acids and 5’nucleotides. It later includes the potential role of peptides and Maillard derived compounds. The role of volatile flavour compounds in the enhancement of salt and sweet taste is also discussed.

Endosomes: a legitimate platform for the signaling train

Although long regarded as a conduit for the degradation or recycling of cell surface receptors, the endosomal system is also an essential site of signal transduction. Activated receptors accumulate in endosomes, and certain signaling components are exclusively localized to endosomes. Receptors can continue to transmit signals from endosomes that are different from those that arise from the plasma membrane, resulting in distinct physiological responses. Endosomal signaling is widespread in metazoans and plants, where it transmits signals for diverse receptor families that regulate essential processes including growth, differentiation and survival. Receptor signaling at endosomal membranes is tightly regulated by mechanisms that control agonist availability, receptor coupling to signaling machinery, and the subcellular localization of signaling components. Drugs that target mechanisms that initiate and terminate receptor signaling at the plasma membrane are widespread and effective treatments for disease. Selective disruption of receptor signaling in endosomes, which can be accomplished by targeting endosomal-specific signaling pathways or by selective delivery of drugs to the endosomal network, may provide novel therapies for disease.