The identification and characterisation of the target proteins of the anti-epileptic drug R-lacosamide

(2R)-N-Benzyl-2-acetamido-3-methoxypropionamide (lacosamide) is an anticonvulsant (Choi et al., 1996); under the brand name "Vimpat" this small molecule has recently been approved by the European Medicines Agency and the U.S. Food and Drug Administration for the treatment of epilepsy. The purpose of the research reported here is to develop and apply mass spectrometry approaches to the determination of protein targets of this novel therapeutic. The general strategy involves selecting potential target proteins using lacosamide analogues incorporating an 'affinity bait' to enable covalent modification binding to target proteins, and a 'chemical reporter' for the selective recovery of modified proteins. Lacosamide analogues are incubated with biological samples (primarily mouse brain extracts) and the modified proteins are recovered by introduction of a biotin tag (via the chemical reporter group). Streptavidin affinity chromatography is then used to enrich for bound molecules. The enriched proteins are subjected to tryptic digestion and the resultant peptides analysed by reversed phase liquid chromatography coupled with tandem MS, enabling recognition of proteins via database searching. Firstly, mass spectrometric characterisation of the biotinyl (R)-lacosamide analogue bound to model compounds was performed. Adducts with protected lysine, neurotensin and enolase were analysed. The data showed that ESI was more suitable for ionisation of modified peptides and proteins than MALDI. The biotin enrichment strategy was applied to mouse brain lysate to identify putative candidate target proteins. Twenty-eight candidate target proteins were identified. Moreover, the 14-3-3 protein family, CRMP2 and the sodium/potassium-transporting ATPase family showed preference for the biologically active(R)- isomer over the (S)- lacosamide analogue using a fluorescence tag. Three more biotinyl lacosamide analogues containing different affinity baits were used to enrich candidate target proteins of lacosamide. Most of the identified target proteins supported the findings of the analogue A. To indicate the binding sites, a method was developed for enriching peptides modified by the biotinyl (R)-lacosamide analogue, using streptavidin beads and subsequently analysed these biotinylated peptides using CID and ETD fragmentation methods. Neither fragmentation technique was optimal for elucidation of the sequence or site of modification of unknown target peptides. Purified recombinant proteins were therefore adducted with an AB-(R)-lacosamide analogue lacking the biotinprobe. This smaller (R)-lacosamide analogue underwent less fragmentation than the biotin analogue during CID and could be used for sequence and site identification of the modified peptides. In summary, the studies illustrated the power of MS to study drug mechanisms via the discovery of candidate protein targets.EThOS - Electronic Theses Online ServiceThe Royal Thai GovernmentGBUnited Kingdo

Proteomics profiling of cholangiocarcinoma exosomes: A potential role of oncogenic protein transferring in cancer progression

AbstractCholangiocarcinoma (CCA), a common primary malignant tumor of bile duct epithelia, is highly prevalent in Asian countries and unresponsive to chemotherapeutic drugs. Thus, a newly recognized biological entity for early diagnosis and treatment is highly needed. Exosomes are small membrane bound vesicles found in body fluids and released by most cell types including cancer cells. The vesicles contain specific subset of proteins and nucleic acids corresponding to cell types and play essential roles in pathophysiological processes. The present study aimed to assess the protein profiles of CCA-derived exosomes and their potential roles. We have isolated exosomes from CCA cells namely KKU-M213 and KKU-100 derived from Thai patients and their roles were investigated by incubation with normal human cholangiocyte (H69) cells. Exosomes were internalized into H69 cells and had no effects on viability or proliferation of the host cells. Interestingly, the exosomes from KKU-M213 cells only induced migration and invasion of H69 cells. Proteomic analysis of the exosomes from KKU-M213 cells disclosed multiple cancer related proteins that are not present in H69 exosomes. Consistent with the protein profile, treatment with KKU-M213 exosomes induced β-catenin and reduced E-cadherin expressions in H69 cells. Collectively, our results suggest that a direct cell-to-cell transfer of oncogenic proteins via exosomal pathway may be a novel mechanism for CCA progression and metastasis

Competition between Burkholderia pseudomallei and B. thailandensis.

BACKGROUND: Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, an often fatal disease in tropical countries. Burkholderia thailandensis is a non-virulent but closely related species. Both species are soil saprophytes but are almost never isolated together. RESULTS: We identified two mechanisms by which B. pseudomallei affects the growth of B. thailandensis. First, we found that six different isolates of B. pseudomallei inhibited the growth of B. thailandensis on LB agar plates. Second, our results indicated that 55% of isolated strains of B. pseudomallei produced a secreted compound that inhibited the motility but not the viability of B. thailandensis. Analysis showed that the active compound was a pH-sensitive and heat-labile compound, likely a protein, which may affect flagella processing or facilitate their degradation. Analysis of bacterial sequence types (STs) demonstrated an association between this and motility inhibition. The active compound was produced from B. pseudomallei during the stationary growth phase. CONCLUSION: Taken together, our results indicate that B. pseudomallei inhibits both the growth and motility of its close relative B. thailandensis. The latter phenomenon appears to occur via a previously unreported mechanism involving flagellar processing or degradation

Competition between and

Syftet med denna litteraturstudie var att beskriva behov av information och stöd hos kvinnor med bröstcancer. Metoden som användes var en litteraturstudie och inkluderade både kvalitativa och kvantitativa artiklar. Tio studier inkluderades och resultatet presenterades i tre huvudkategorier: Information, stöd och sjuksköterskans uppfattning om behov av information och stöd, detta behov måste identifieras individuellt efter varje kvinna med bröstcancer. Dessa kvinnor uppskattade information om möjligheten att bli botad, behandlingsalternativ samt recidiv. Yngre kvinnor hade större behov av information om sexualitet än äldre. Detta informationsbehov beräknades ändå som mindre viktigt av alla kvinnor med bröstcancer oberoende av ålder. Det var många studier som visade samma resultat att kvinnor med bröstcancer har stort behov av information och stöd. Resultatet av litteraturstudien kan ge sjuksköterskorna ökad förståelse om vilka behov av information och stöd kvinnor med bröstcancer har. Ökad medvetenhet om dessa behov kan ligga till grund för en god omvårdnad.The aim of this study was to describe the need for information and support in women with breast cancer. The method used is a literature study with both of qualitative and quantitative articles. Ten studies were included and the results were presented in three main categories: information, support, and the nurse's perception of the need for information and support, this need must be identified individually by each woman with breast cancer. These women appreciated the information about chances of cure, treatment and recurrence. Young women had a greater need for information about sexuality than older. This information was calculated, however, as less important for all women with breast cancer regardless of age. There were many studies that showed the same results that women with breast cancer have great need for information and support. The results of the literature study can give nurses greater understanding of the needs for information and support women with breast cancer have. Increased awareness of these needs can be the basis for good care

Nitric Oxide Synthesis Inhibition and Anti-Inflammatory Effect of Polypeptide Isolated from Chicken Feather Meal in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Dušikov oksid ima ključnu ulogu u mehanizmu nastanka upalnog procesa te uzrokuje ozljedu tkiva. Brašno od perja kokoši bogato je aminokiselinama te može poslužiti za inhibiciju sinteze dušikovog oksida kao hidrolizat peptida. Pripremljeni su hidrolizati brašna od perja s protuupalnim svojstvima, koji su razdvojeni u pet skupina prema molekulskoj masi. Najmanja je frakcija (<0,65 kDa) najbolje inhibirala sintezu dušikovog oksida, a da nije djelovala citotoksično na stanice mikrofaga RAW 264.7. Daljnjim razdvajanjem dobivene su subfrakcije u količinama dovoljnim za provedbu analize aminokiselinskog slijeda metodom Q-TOF LC-MS/MS ESI. Peptid SNPSVAGVR (885,97 Da) i odgovarajući čisti sintetizirani peptid inhibirali sintezu dušikovog oksida (IC50=(55,2±0,2)) u makrofagu RAW 264.7, a da nisu imale citotoksični učinak. Rezultati dobiveni pomoću metoda RT-PCR i kvantitativne RT-PCR pokazuju da dobiveni peptid SNPSVAGVR smanjuje ekspresiju upalnih citokina iNOS, TNF-α, COX-2 i IL-6 u stanicama RAW 264.7 stimuliranim pomoću lipopolisaharida. Time je potvrđeno da se peptidi iz proteina brašna od perja mogu primijeniti kao dodatak funkcionalnoj hrani ili nutraceuticima, a za smanjenje upalnih procesa.Nitric oxide (NO) plays a key role in the pathogenesis of inflammation and has been implicated in endotoxin-induced tissue injury. Chicken feather meal is a rich source of amino acids that may serve as a peptide hydrolysate to inhibit NO activity. Anti-inflammatory hydrolysates of chicken feather meal were prepared and fractionated into five samples based on molecular mass. The smallest fraction (<0.65 kDa) exhibited the highest NO inhibitory activity without cytotoxicity towards macrophage RAW 264.7 cells. Further subfractions were sufficient to obtain amino acid sequences by Q-TOF LC-MS/MS ESI analysis. Of these, the SNPSVAGVR (885.97 Da) peptide and its corresponding pure synthetic peptide have inhibitory activity against NO production by RAW 264.7 cells (IC50=(55.2±0.2) mM) without cytotoxicity. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR results revealed that the peptide of the obtained fraction reduced transcript expression levels of the pro-inflammatory cytokines iNOS, TNF-α, COX-2 and IL-6 in lipopolysaccharide-stimulated RAW 264.7 cells. These results suggest that the peptides derived from the chicken feather meal protein could potentially be used as a promising ingredient in functional foods or nutraceuticals against inflammatory diseases

Immunoproteomics to identify species-specific antigens in Neospora caninum recognised by infected bovine sera

Bovine neosporosis is a disease of concern due to its global distribution and significant economic impact through massive losses in the dairy and meat industries. To date, there is no effective chemotherapeutic drug or vaccine to prevent neosporosis. Control of this disease is therefore dependent on efficient detection tests that may affect treatment management strategies. This study was conducted to identify the specific immunoreactive proteins of Neospora caninum tachyzoites recognised by sera from cattle infected with N. caninum, Toxoplasma gondii, Cryptosporidium parvum, Babesia bovis and B. bigemina, and by sera from uninfected cattle using two-DE dimensional gel electrophoresis (2-DE) combined with immunoblot and mass spectrometry (LC-MS/MS). Among 70 protein spots that reacted with all infected sera, 20 specific antigenic spots corresponding to 14 different antigenic proteins were recognised by N. caninum-positive sera. Of these immunoreactive antigens, proteins involved in cell proliferation and invasion process were highly immunogenic, including HSP90-like protein, putative microneme 4 (MIC4), actin, elongation factor 1-alpha and armadillo/beta-catenin-like repeat-containing protein. Interestingly, we discovered an unnamed protein product, rhoptry protein (ROP1), possessing strong immunoreactivity against N. caninum but with no data on function available. Moreover, we identified cross-reactive antigens among these apicomplexan parasites, especially N. caninum, T. gondii and C. parvum. Neospora caninum-specific immunodominant proteins were identified for immunodiagnosis and vaccine development. The cross-reactive antigens could be evaluated as potential common vaccine candidates or drug targets to control the diseases caused by these apicomplexan protozoan parasites

Identification of a Lacosamide Binding Protein Using an Affinity Bait and Chemical Reporter Strategy: 14-3-3 ζ

We have advanced a useful strategy to elucidate binding partners of ligands (drugs) with modest binding affinity. Key to this strategy is attaching to the ligand an affinity bait (AB) and a chemical reporter (CR) group, where the AB irreversibly attaches the ligand to the receptor upon binding and the CR group is employed for receptor detection and isolation. We have tested this AB&CR strategy using lacosamide ((R)-1), a low-molecular-weight antiepileptic drug. We demonstrate that using a (R)-lacosamide AB&CR agent ((R)-2) 14-3-3 ζ in rodent brain soluble lysates is preferentially adducted, adduction is stereospecific with respect to the AB&CR agent, and adduction depends upon the presence of endogenous levels of the small molecule metabolite xanthine. Substitution of lacosamide AB agent ((R)- 5) for (R)-2 led to the identification of the 14-3-3 ζ adduction site (K120) by mass spectrometry. Competition experiments using increasing amounts of (R)-1 in the presence of (R)-2 demonstrated that (R)-1 binds at or near the (R)-2 modification site on 14-3-3 ζ. Structure-activity studies of xanthine derivatives provided information concerning the likely binding interaction between this metabolite and recombinant 14-3-3 ζ. Documentation of the 14-3-3 ζ-xanthine interaction was obtained with isothermal calorimetry using xanthine and the xanthine analogue 1,7-dimethylxanthine

Potential antihypertensive activity of novel peptides from green basil leaves

Abstract Hypertension is among the risk factors of death globally. Novel antihypertensive peptides are alternative choices of antihypertensive assistance. This study aimed to discover novel antihypertensive peptides from green basil leaves. Two bioactive peptides with high angiotensin-converting enzyme inhibition (Asp-Leu-Ser-Ser-Ala-Pro; peptide 1) and antioxidant (Asp-Ser-Val-Ser-Ala-Ser-Pro; peptide 2) activities were gavaged to male Wistar rats induced with N G -nitro-l-arginine methyl-ester (L-NAME). L-NAME-treated rats (HT) had decreased body weights and levels of nitrite and nitrate, which are metabolites of nitric oxide. The levels of their glucose and liver function indicators increased as compared to normal rats. HT rats receiving antihypertensive drugs (HTD) showed higher low-density lipoprotein and low-density lipoprotein/high-density lipoprotein levels than HT rats. Peptide 1 seems to benefit the rat lipid profiles, liver functions, antioxidant, nitrite, nitrate, and angiotensin II peptide levels but not peptide 2. In conclusion, our findings indicate the antihypertensive potential related to vasodilation of peptides from green basil leaves. Graphical Abstrac

All systems are go

A report on 'Genomes to Systems', the Fourth Conference of the Consortium for Post-Genome Science, Manchester, UK, 17-19 March 2008