Proteomic analysis of plasma identifies the Toll-like receptor agonists S100A8/A9 as a novel possible marker for systemic sclerosis phenotype.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterised by fibrosis of the skin and the internal organs. Except for anticentromere, antitopoisomerase I and antipolymerase III antibodies, there are no reliable circulating markers predicting susceptibility and internal organ complications. This study has exploited a proteome-wide profiling method with the aim to identify new markers to identify SSc phenotype. METHOD: 40 SSc patients were included for proteomic identification. Patients were stratified as having diffuse cutaneous SSc (dcSSc) (n=19) or limited cutaneous SSc (lcSSc) (n=21) according to the extent of skin involvement. As controls 19 healthy donors were included. Blood was drawn and plasma was stored before analysing with the SELDI-TOF-MS. For replication in serum, the cohort was extended with 60 SSc patients. RESULTS: Proteomic analysis revealed a list of 25 masspeaks that were differentially expressed between SSc patients and healthy controls. One of the peaks was suggestive for S100A8, a masspeak we previously found in supernatant of plasmacytoid dendritic cells from SSc patients. Increased expression of S100A8/A9 in SSc patients was confirmed in replication cohort compared with controls. Intriguingly, S100A8/A9 was highest in patients with limited cutaneous SSc having lung fibrosis. CONCLUSIONS: S100A8/A9 was robustly found to be elevated in the circulation of SSc patients, suggesting its use as a biomarker for SSc lung disease and the need to further explore the role of TLR in SSc.

Retrospective evaluation of bleeding tendency and simultaneous thrombin and plasmin generation in patients with rare bleeding disorders.

Rare bleeding disorders (RBDs) are a heterogeneous group of diseases with varying bleeding tendency, only partially explained by their laboratory phenotype. We analysed the separate groups of RBD abnormalities, and we investigated retrospectively whether the novel haemostasis assay (NHA) was able to differentiate between bleeding tendency. We have performed simultaneous thrombin generation (TG) and plasmin generation (PG) measurements in 41 patients affected with deficiencies in prothrombin, factor (F) V, FVII, FX, FXIII and fibrinogen. Parameters of the NHA were classified based on (major or minor) bleeding tendency. Patients with deficiencies in coagulation propagation (FII, FV and FX) and major type of bleedings had diminished TG (expressed as AUC) below 20% of control. FVII deficient patients only had prolonged thrombin lag-time ratio of 1.6 ± 0.2 (P < 0.05) and normal AUC (92-125%). Afibrinogenemic patients demonstrated PG of 2-29% of normal and appeared to correlate with the type of mutation. Thrombin peak-height (57 ± 16%) was reduced (not significant) in these patients and AUC was comparable to the reference (102 ± 27%). FXIII-deficient plasmas resulted in a reduced thrombin peak-height of 59 ± 13% (P < 0.05) and normal AUC (90 ± 14%). Thrombin peak-height (P < 0.01) and plasmin potential (P < 0.05) were lower in the major bleeders compared with the minor bleeders. These results provided distinct TG and PG curves for each individual abnormality and differentiation of bleeding tendency was observed for thrombin and PG parameters. Prospective studies are warranted to confirm these retrospective results.

Alterations of coagulation and fibrinolysis in patients with angioedema due to C1-inhibitor deficiency.

Patients with functional deficiency of C1-inhibitor (C1-INH) suffer from recurrent acute attacks (AA) of localized oedema associated with activation of the contact system, complement and fibrinolysis. To unravel further the role of coagulation and fibrinolysis in the pathophysiology of C1-INH deficiency, we performed simultaneous thrombin and plasmin generation measurements in plasma from patients with hereditary angioedema (HAE) due to C1-INH deficiency during AA (n = 23), in remission (R) (n = 20) and in controls (n = 20). During AA thrombin generation after in-vitro activation of plasma was higher than in controls, as demonstrated by shorter thrombin peak-time (P < 0·05), higher thrombin peak-height (P < 0·001) and increased area under the curve (AUC) (P < 0·05). Additionally, elevated levels of prothrombin fragment 1+2 (P < 0·0001) were observed in non-activated plasma from the same patients. In contrast, in activated plasma from patients during AA plasmin generation estimated as plasmin peak-height (P < 0·05) and plasmin potential (P < 0·05) was reduced, but non-activated plasma of the same patients showed elevated plasmin-anti-plasmin (PAP) complexes (P < 0·001). This apparent discrepancy can be reconciled by elevated soluble thrombomodulin (sTM) (P < 0·01) and thrombin activatable fibrinolysis inhibitor (TAFI) in patients during AA providing possible evidence for a regulatory effect on fibrinolysis. Plasminogen activator inhibitor-1 (PAI-1) was reduced in patients during AA indicating, together with the observed reduction of plasmin generation, the consumption of fibrinolytic factors. In conclusion, our results support the involvement of coagulation and fibrinolysis in the pathophysiology of HAE and show the possible application of simultaneous measurement of thrombin and plasmin generation to evaluate different clinical conditions in HAE patients.

Analyses of human colonic mucus obtained by an in vivo sampling technique.

BACKGROUND: The mucus layer is an important dynamic component of the epithelial barrier. It contains mucin glycoproteins and other compounds secreted by the intestinal epithelium, such as secretory IgA. However, a standardized in vivo sampling technique of mucus in humans is not yet available. AIM: To assess the validity and feasibility of mucin and protein determinations in human colonic mucus collected under physiological conditions. SUBJECTS AND METHODS: Triplicate colonic mucus samples were collected in 11 healthy volunteers using cytology brushes during sigmoidoscopy. As an indication of the quantity of collected mucus, total protein and mucin concentrations were determined by measuring oligosaccharide equivalents and monosaccharides. Also secretory IgA and sialic acid concentrations were determined and proteomic analysis was performed using surface enhanced laser desorption/ionization-time of flight-mass spectrometry. RESULTS: Mean values of secretory IgA and sialic acid corrected for the amount of mucus ranged from 0.16 to 1.81 g secretory IgA/mmol oligosaccharide equivalents and from 12.6 to 48.6g sialic acid/mmol oligosaccharide equivalents. Proteomic analysis of mucus is feasible and cluster analysis showed subject specific profiles. CONCLUSION: Using cytology brushes, human colonic mucus can be sampled and under physiological conditions. These samples could give information on the composition and quality of the mucus layer.

Localization of the phase-related 6-kDa peptide (PRP) in different tissues of the desert locust Schistocerca gregaria--immunocytochemical and mass spectrometric approach.

A 6-kDa phase-related peptide (PRP) was recently identified from the hemolymph of the desert locust Schistocerca gregaria. Its presence in much higher concentrations in the crowd-reared (gregarious) phase than in the isolated-reared (solitarious) one suggests a role in phase polyphenism. However, when tested in a variety of classical bioassays, no activity could be found. We hoped that uncovering its site(s) of synthesis might yield hints as to possible functions. An antiserum was raised against the C-terminal 16 aa part of PRP for use in immunocytochemistry. No immunoreactivity was recorded in the fat body, midgut, or Malpighian tubules. The strongest positive immunostaining was observed in the follicle cells of the ovary and in the seminal vesicle tubes of the male accessory gland complex. Also, positive were a pair of large neurosecretory cells in the subesophageal ganglion, the storage part of the corpora cardiaca and some nerve fibers in the brain- and abdominal regions. An additional mass spectrometric analysis was successfully done in combination with a BLAST search to detect possible false positive staining. This confirmed the presence of genuine PRP in most of the immunopositive tissues. Additional experiments are needed to unravel the role of PRP.

Quantitative real-time RT-PCR analysis in desert locusts reveals phase dependent differences in neuroparsin transcript levels.

In different parts of the world, locust swarms cause severe ecological and economic damage. However, the physiological mechanisms underlying this gregarization process remain elusive. In this study, we present a detailed quantitative analysis of two neuroparsin precursor (Scg-NPP1 and Scg-NPP2) transcripts in the brain, fat body, gut, gonads and accessory glands of male and female, gregarious and solitarious desert locusts (Schistocerca gregaria). These transcripts are generally more abundant in solitarious than in gregarious animals. In contrast to their gregarious congeners, solitarious locusts contain detectable Scg-NPP1 and Scg-NPP2 transcript levels in the fat body. Moreover, our data reveal temporal changes of neuroparsin mRNA levels in the brains and fat bodies of adult isolated-reared locusts. This paper provides the first scientific evidence for phase-dependent transcriptional regulation of neuropeptide hormone encoding genes.

Characterization of an RFamide-related peptide orphan GPCR in C. elegans.

We cloned and characterized an orphan FMRFamide-related peptide (FaRP) GPCR in Caenorhabditis elegans. We synthesized numerous structurally different FaRPs that were found in the C. elegans genome by bioinformatic analysis and used them to screen cells expressing the C26F1.6 receptor. Two peptides ending in M(orL)VRFamide elicited a calcium response in receptor-expressing mammalian Chinese hamster ovary cells. The response was dose-dependent and appeared to be very specific; that is, none of the other FaRPs were active, not even closely related peptides also ending in M(orL)VRFamide, which are encoded by the same peptide precursor. Pharmacological profiling with a truncated series of the most active peptide revealed that the full peptide sequence is necessary for receptor activation.

Melatonin-induced neuropeptide release from isolated locust corpora cardiaca.

A method, based on a combination of mass spectrometry and liquid chromatography, was developed to investigate the release of neuropeptides from isolated locust corpora cardiaca. Melatonin, octopamine, trehalose and forskolin were administered to the perifused glands. The neuropeptides present in the releasates (spontaneous versus induced) were visualized by either conventional or capillary HPLC. Identification was achieved by means of MALDI-TOF MS and/or nanoflow-LC-Q-TOF MS. The observed effects of these chemicals regarding AKH release were in line with previous studies and validate the method. The most important finding of this study was that administration of melatonin stimulated the release of adipokinetic hormone precursor related peptides (APRP 1 and APRP 2), neuroparsins (NP A1, NP A2 and NP B) and diuretic peptide.

Neuroendocrinological and molecular aspects of insect reproduction.

This review summarizes recent advances and novel concepts in the area of insect reproductive neuroendocrinology. The role of 'classic' hormones, such as ecdysteroids and juvenoids, to control reproduction is well documented in a large variety of insect species. In adult gonads, ecdysteroids appear to induce a cascade of transcription factors, many of which also occur during the larval molting response. Recent molecular and functional data have created opportunities to study an additional level of regulation, that of neuropeptides, growth factors and their respective receptors. As a result, many homologs of factors playing a role in vertebrate reproductive physiology have been discovered in insects. This review highlights several neuropeptides controlling the biosynthesis and release of the 'classic' insect hormones, as well as various peptides and biogenic amines that regulate behavioural aspects of the reproduction process. In addition, hormone metabolizing enzymes and second messenger pathways are discussed with respect to their role in reproductive tissues. Finally, we speculate on future prospects for insect neuroendocrinological research as a consequence of the recent 'Genomics Revolution'.

Glucocorticoid-inducible gene expression vectors for use in Drosophila melanogaster Schneider 2 cells.

Inducible, vector-based, expression systems that allow fine control of transgene expression are gaining more and more use in fundamental research as well as in therapeutic applications. In an effort to develop a tightly regulated heterologous expression system for Drosophila Schneider 2 cells, three different inducible reporter constructs were compared. These comprised six copies of the glucocorticoid response element fused to one of three distinct types of Drosophila gene promoters: (1) a TATA-box containing, (2) a TATA-less and (3) a bidirectional core sequence. These were fused to a luciferase reporter gene. The promoter constructs displayed different basal as well as agonist-induced activities. The implications of the observations made are discussed in the context of promoter properties and of induction of genes that may be studied in Drosophila.

cDNA cloning and transcript distribution of two novel members of the neuroparsin family in the desert locust, Schistocerca gregaria.

This study describes the identification and distribution of two novel neuroparsin precursor transcripts (Scg-NPP3/Scg-NPP4) in the desert locust, Schistocerca gregaria. Unlike Scg-NPP1 and Scg-NPP2, both transcripts were not only detected in the brain, but also in various other tissues, such as fat body, ventral nerve cord, testis and male accessory glands. Northern analysis showed that the levels of these transcripts are regulated during larval development, as well as during moulting and reproductive cycles. A significant increase in both mRNAs was observed during the period that just precedes the initial sexual activity of adult females and males. In silico analysis of sequence databases revealed the existence of several other neuroparsin-like peptides in a variety of arthropod species, including crustaceans and chelicerates. Neuroparsins also display similarities with vertebrate IGFBP.

Molecular evidence for the expression of angiotensin converting enzyme in hemocytes of Locusta migratoria: stimulation by bacterial lipopolysaccharide challenge.

The presence of angiotensin converting enzyme (ACE) in insects has been reported many times, but numerous questions about the functional role of this enzyme in insects remain. Here we show by RT-PCR experiments that ACE has a wide tissue distribution in Locusta migratoria, suggesting diverse roles for this enzyme in the locust. Immune challenge through injection of bacterial lipopolysaccharides resulted in a tenfold increase of ACE gene transcripts in the hemocytes and is suggestive for a role of ACE in the cellular defense of the locust. However, phenotypic knockout experiments with the ACE inhibitor captopril showed that ACE is not essential for the efficient clearance of injected E. coli bacteria.

Search for phase specific genes in the brain of desert locust, Schistocerca gregaria (Orthoptera: Acrididae) by differential display polymerase chain reaction.

Differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) in combination with semi-quantitative RT-PCR was used to compare differences in gene expression between the solitary and gregarious phase of Schistocerca gregaria. Twenty-six primer combinations were used, which produced 8 differential bands. Two out of the 8 differentials, one typical for the solitary and one for the gregarious phase, were further analyzed by semi-quantitative RT-PCR. The expression level of the solitary phase specific gene (SSG) was 2 times higher in solitary animals as compared to gregarious ones, while the gregarious specific gene (GSG) gave a 4-fold higher expression level in gregarious animals than in solitaries. Sequence analysis demonstrated that SSG does not belong to a known gene family, while the GSG belongs to the SPARC protein family.

An endothelin-converting enzyme homologue in the locust, Locusta migratoria: functional activity, molecular cloning and tissue distribution.

Endothelin-converting enzyme is the key enzyme in the process of endothelin production. Endothelin is a peptide that plays an important role in vasoconstriction and the development of neural crest-derived cells in vertebrates. Activity assays performed on membrane extracts from Locusta migratoria brain revealed the existence of a protease activity responsible for the formation of mature endothelin-1 from its precursor, big endothelin. Cloning experiments led to a cDNA sequence (Lom ECE) with an open reading frame of 727 amino acid residues displaying all the characteristic ECE features. A comparison of ECE activity levels among different tissues of the locust showed a high enzyme activity in the gonads and midgut. RT-PCR experiments showed a wide tissue distribution of Lom ECE mRNA, with transcription being most abundant in brain tissue.

Antimicrobial compounds of low molecular mass are constitutively present in insects: characterisation of beta-alanyl-tyrosine.

The number of bacterial and fungal strains that have developed resistance against the classical antibiotics continues to grow. The intensified search for new antibiotic lead compounds has resulted in the discovery of numerous endogenous peptides with antimicrobial properties in plants, bacteria and animals. Their possible applications as anti-infective agents are often limited by their size, in reference to production costs and susceptibility to proteases. In this article, we report recent isolations of antimicrobial compounds from insects, with molecular masses less than 1 kDa. Experimental approaches are discussed and the first data on the antimicrobial properties of beta-alanyl-tyrosine (252 Da), one of such low molecular mass compounds isolated from the fleshfly Neobellieria bullata, are presented. We also offer evidence for the constitutive presence of antimicrobial compounds in insects of different orders, in addition to the previously identified inducible antimicrobial peptides.

The sex attractant pheromone of the oak processionary, Thaumetopoea processionea a field evaluation.

The sex pheromone of Thaumetopoea processionea has recently been described as a mixture of different isomers with (Z,Z)-11,13-hexadecadien-1-yl acetate as the major active compound. First trials have shown that traps baited with this compound attract male moths in the field. In the present paper we evaluate the use of pheromone traps under practical aspects. The traps can be very well applied to monitor the pest, but the devices have to be installed in the upper crown of the oak trees. It is possible to determine the seasonal flight pattern of the moths. Results of field trials in Germany suggest that the traps can be used to roughly estimate the degree of infestation.

Peptide profiling of a single Locusta migratoria corpus cardiacum by nano-LC tandem mass spectrometry.

The pars intercerebralis-corpora cardiaca complex in insects is the functional equivalent of the vertebrate brain-pituitary axis. During the past few decades more than 40 neuropeptides have been isolated from the locust brain-corpus cardiacum complex. Tedious and time-consuming successive purification rounds of large tissue extracts were necessary to achieve the purification and sequencing of most of these signal molecules. Nowadays, the combination of nanoscale liquid chromatography and the very sensitive tandem mass spectrometry allows us to identify and sequence peptides in very low concentration directly from tissue extracts. In this manuscript, we review previous data on the peptidome analysis of the locust corpora cardiaca, with emphasis on AKH processing. In addition, we report the peptide profiling of a single corpus cardiacum from Locusta migratoria. 23 peptides were isolated and sequenced in a single nano-LC-MS/MS experiment, demonstrating the sensitivity and effectiveness of mass spectrometry in peptide research.

Peptidomics of the locust corpora allata: identification of novel pyrokinins (-FXPRLamides).

The peptidomes of the corpora allata of Locusta migratoria and Schistocerca gregaria were investigated by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography quadrupole time-of-flight tandem mass spectrometry (nanoLC-Q-TOF MSMS). The pyrokinin (-FXPRLamide) family seems to be predominant. In addition to the known pyrokinins, we de novo sequenced four pyrokinins in L. migratoria and five in S. gregaria. In addition, one pyrokinin-like peptide (-PRLamide) was identified in S. gregaria. Besides the -(FX)PRLamides, FLRFamide-1, the allatostatins (A family) and numerous as yet unidentified peptides are also present in the corpora allata.

Corazonin promotes tegumentary pigment migration in the crayfish Procambarus clarkii.

The undecapeptide corazonin (pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-AsnNH(2)) elicits a retraction of erythrophore pigment granules and dispersion of leucophore pigment granules in the crayfish Procambarus clarkii. The effects are dose-dependent from 10(-10) to 10(-5)M. Influence on erythrophores is lower than that of Red Pigment Concentrating Hormone (RPCH), which is inactive on leucophores. Corazonin effects are partly blocked by an anti-corazonin antibody, and even less by an anti-RPCH antibody. Corazonin effects are completely suppressed by the calcium chelator BAPTA. Immunoreactive somata and fibers were identified in various regions of the eyestalk (medulla terminalis, medulla interna and medulla externa) with the anti-corazonin antibody. These results suggest the possible existence of a corazonin-like peptide in crustaceans.