Experimental febrile seizures increase dendritic complexity of newborn dentate granule cells.

OBJECTIVE: Febrile seizures (FS) are fever-associated convulsions, being the most common seizure disorder in early childhood. A subgroup of these children later develops epilepsy characterized by a hyperexcitable neuronal network in the hippocampus. Hippocampal excitability is regulated by the hippocampal dentate gyrus (DG) where postnatal neurogenesis occurs. Experimental FS increase the survival of newborn hippocampal dentate granule cells (DGCs), yet the significance of this neuronal subpopulation to the hippocampal network remains unclear. In the current study, we characterized the temporal maturation and structural integration of these post-FS born DGCs in the DG. METHODS: Experimental FS were induced in 10-day-old rat pups. The next day, retroviral particles coding for enhanced green fluorescent protein (eGFP) were stereotactically injected in the DG to label newborn cells. Histochemical analyses of eGFP expressing DGCs were performed one, 4, and 8 weeks later and consisted of the following: (1) colocalization with neurodevelopmental markers doublecortin, calretinin, and the mature neuronal marker NeuN; (2) quantification of dendritic complexity; and (3) quantification of spine density and morphology. RESULTS: At neither time point were neurodevelopmental markers differently expressed between FS animals and normothermia (NT) controls. One week after treatment, DGCs from FS animals showed dendrites that were 66% longer than those from NT controls. At 4 and 8 weeks, Sholl analysis of the outer 83% of the molecular layer showed 20-25% more intersections in FS animals than in NT controls (p < 0.01). Although overall spine density was not affected, an increase in mushroom-type spines was observed after 8 weeks. SIGNIFICANCE: Experimental FS increase dendritic complexity and the number of mushroom-type spines in post-FS born DGCs, demonstrating a more mature phenotype and suggesting increased incoming excitatory information. The consequences of this hyperconnectivity to signal processing in the DG and the output of the hippocampus remain to be studied.

Origin and functional diversification of an amphibian defense peptide arsenal.

The skin secretion of many amphibians contains an arsenal of bioactive molecules, including hormone-like peptides (HLPs) acting as defense toxins against predators, and antimicrobial peptides (AMPs) providing protection against infectious microorganisms. Several amphibian taxa seem to have independently acquired the genes to produce skin-secreted peptide arsenals, but it remains unknown how these originated from a non-defensive ancestral gene and evolved diverse defense functions against predators and pathogens. We conducted transcriptome, genome, peptidome and phylogenetic analyses to chart the full gene repertoire underlying the defense peptide arsenal of the frog Silurana tropicalis and reconstruct its evolutionary history. Our study uncovers a cluster of 13 transcriptionally active genes, together encoding up to 19 peptides, including diverse HLP homologues and AMPs. This gene cluster arose from a duplicated gastrointestinal hormone gene that attained a HLP-like defense function after major remodeling of its promoter region. Instead, new defense functions, including antimicrobial activity, arose by mutation of the precursor proteins, resulting in the proteolytic processing of secondary peptides alongside the original ones. Although gene duplication did not trigger functional innovation, it may have subsequently facilitated the convergent loss of the original function in multiple gene lineages (subfunctionalization), completing their transformation from HLP gene to AMP gene. The processing of multiple peptides from a single precursor entails a mechanism through which peptide-encoding genes may establish new functions without the need for gene duplication to avoid adaptive conflicts with older ones.

Molecular diversity of the telson and venom components from Pandinus cavimanus (Scorpionidae Latreille 1802): transcriptome, venomics and function.

Venom from the scorpion Pandinus cavimanus was obtained by electrical stimulation of the telson (stinger). Total venom was toxic to crickets at 7-30 µg and a paralysis or lethal effect was observed at 30 µg of venom (death at 1.5 µg/mg of cricket). Electrophysiological analyses showed cytolytic activity of total venom on oocytes at 7 µg. HPLC allowed separation of the venom components. A total of 38 fractions from total venom were tested on voltage-gated Na(+) and K(+) channels. Some fractions block K(+) currents in different degrees. By using MS analysis, we obtained more than 700 different molecular masses from telson and venom fractions (by LC-MS/MS and MALDI-TOF MS analyses). The number of disulfide bridges of the telson components was determined. A cDNA library from P. cavimanus scorpion was constructed and a random sequencing screening of transcripts was conducted. Different clones were obtained and were analyzed by bioinformatics tools. Our results reveal information about new genes related to some cellular processes and genes involved in venom gland functions (toxins, phospholipases and antimicrobial peptides). Expressed sequence tags from venom glands provide complementary information to MS and reveal undescribed components related to the biological activity of the venom.

Proteome changes in auricular lymph nodes and serum after dermal sensitization to toluene diisocyanate in mice.

Some reactive chemicals, such as diisocyanates, are capable of initiating an allergic response, which can lead to occupational asthma after a latency period. Clinical symptoms such as cough, wheezing, and dyspnea occur only late, making it difficult to intervene at an early stage. So far, most studies using proteomics in lung research have focused on comparisons of healthy versus diseased subjects. Here, using 2D-DIGE, we explored proteome changes in the local draining lymph nodes and serum of mice dermally sensitized once or twice with toluene-2,4-diisocyanate (TDI) before asthma is induced. In the lymph nodes, we found 38 and 58 differentially expressed proteins after one and two treatments, respectively, between TDI-treated and vehicle-treated mice. In serum, seven and 16 differentially expressed proteins were detected after one and two treatments, respectively. We identified 80-85% of the differentially expressed proteins by MS. Among them, lymphocyte-specific protein-1, coronin 1a, and hemopexin were verified by Western blotting or ELISA in an independent group of mice. This study revealed alterations in the proteomes early during sensitization in a mouse model before the onset of chemical-induced asthma. If validated in humans, these changes could lead to earlier diagnosis of TDI-exposed workers.

Experimental early-life febrile seizures cause a sustained increase in excitatory neurotransmission in newborn dentate granule cells.

Prolonged febrile seizures (FS) are a risk factor for the development of hippocampal-associated temporal lobe epilepsy. The dentate gyrus is the major gateway to the hippocampal network and one of the sites in the brain where neurogenesis continues postnatally. Previously, we found that experimental FS increase the survival rate and structural integration of newborn dentate granule cells (DGCs). In addition, mature post-FS born DGCs express an altered receptor panel. Here, we aimed to study if these molecular and structural changes are accompanied by an altered cellular functioning. Experimental FS were induced by hyperthermia in 10-days-old Sprague-Dawley rats. Proliferating progenitor cells were labeled the next day by injecting green fluorescent protein expressing retroviral particles bilaterally in the dentate gyri. Eight weeks later, spontaneous excitatory and inhibitory postsynaptic events (sEPSCs and sIPSCs, respectively) were recorded from labeled DGCs using the whole-cell patch-clamp technique. Experimental FS resulted in a robust decrease of the inter event interval (p < .0001) and a small decrease of the amplitude of sEPSCs (p < .001). Collectively the spontaneous excitatory charge transfer increased (p < .01). Experimental FS also slightly increased the frequency of sIPSCs (p < .05), while the amplitude of these events decreased strongly (p < .0001). The net inhibitory charge transfer remained unchanged. Experimental, early-life FS have a long-term effect on post-FS born DGCs, as they display an increased spontaneous excitatory input when matured. It remains to be established if this presents a mechanism for FS-induced epileptogenesis.

Conservation of fruitless' role as master regulator of male courtship behaviour from cockroaches to flies.

In Drosophila melanogaster, male courtship behaviour is regulated by the fruitless gene. In D. melanogaster, fruitless encodes a set of putative transcription factors that are sex-specifically spliced. Male-specific variants are necessary and sufficient to elicit male courtship behaviour. Fruitless sequences have been reported in other insect species, but there are no data available on their functional role. In the present work, we cloned and sequenced fruitless in males of the German cockroach, Blattella germanica, and we studied its expression in male brain and testes. B. germanica fruitless encodes a 350-amino acid protein with BTB and Zinc finger domains typical of fruitless sequences. Upon RNAi-mediated knockdown of fruitless in B. germanica, males no longer exhibit courtship behaviour, thus implying that fruitless is necessary for male sexual behaviour in our cockroach model. This suggests that the role of fruitless as master regulator of male sexual behaviour has been conserved along insect evolution, at least from cockroaches to flies.

Proteome analysis of multiple compartments in a mouse model of chemical-induced asthma.

Occupational asthma is the principal cause of work-related respiratory disease in the industrial world. Toluene-2,4-diisocyanate (TDI) is one of the most common respiratory sensitizers leading to occupational asthma. Using a mouse model of chemical-induced asthma, we explored proteome changes in multiple compartments of mice sensitized and challenged with TDI or acetone-olive oil (AOO; vehicle). Airway reactivity to methacholine and a bronchoalveolar lavage (BAL) cell count was assessed in treated and control mice, 1 day after challenge. Subsequently, two-dimensional differential gel electrophoresis (2D-DIGE) was performed on auricular lymph nodes, BAL, and serum comparing TDI-treated and vehicle-treated control mice. The differentially expressed proteins were identified by mass spectrometry and pathway analysis was performed. TDI-treated mice exhibit increased airway reactivity (2.6-fold increase) and a neutrophilic inflammation in the BAL fluid, compared to control mice. 2D-DIGE showed 53, 210, and 40 differentially expressed proteins in the auricular lymph nodes, BAL, and serum of TDI-treated versus vehicle-treated mice, respectively. Several of the identified proteins could be linked with inflammation, neutrophil chemotaxis, and/or oxidative stress. Physiologic and immunologic readouts of the asthmatic phenotype, such as inflammation, were confirmed in three compartments by several of the differentially expressed proteins via 2D-DIGE and computerized pathway analysis.

Neuropeptide biology in Drosophila.

Drosophila melanogaster is since decades the most important invertebrate model. With the publishing of the genome sequence, Drosophila also became a pioneer in (neuro)peptide research. Neuropeptides represent a major group of signaling molecules that outnumber all other types of neurotransmitters/modulators and hormones. By means of bioinformatics 119 (neuro)peptide precursor genes have been predicted from the Drosophila genome. Using the neuropeptidomics technology 46 neuropeptides derived from 19 of these precursors could be biochemically characterized. At the cellular level, neuropeptides usually exert their action by binding to membrane receptors, many of which belong to the family of G-protein coupled receptors or GPCRs. Such receptors are the major target for many contemporary drugs. In this chapter, we will describe the identification, localization and functional characterization of neuropeptide-receptor pairs in Drosophila melanogaster.

A neuromedin-pyrokinin-like neuropeptide signaling system in Caenorhabditis elegans.

Neuromedin U (NMU) in vertebrates is a structurally highly conserved neuropeptide of which highest levels are found in the pituitary and gastrointestinal tract. In Drosophila, two neuropeptide genes encoding pyrokinins (PKs), capability (capa) and hugin, are possible insect homologs of vertebrate NMU. Here, the ligand for an orphan G protein-coupled receptor in the nematode Caenorhabditis elegans (Ce-PK-R) was found using a bioinformatics approach. After cloning and expressing Ce-PK-R in HEK293T cells, we found that it was activated by a neuropeptide from the C. elegans NLP-44 precursor (EC(50)=18nM). This neuropeptide precursor is reminiscent of insect CAPA precursors since it encodes a PK-like peptide and two periviscerokinin-like peptides (PVKs). Analogous to CAPA peptides in insects and NMUs in vertebrates, whole mount immunostaining in C. elegans revealed that the CAPA precursor is expressed in the nervous system. The present data also suggest that the ancestral CAPA precursor was already present in the common ancestor of Protostomians and Deuterostomians and that it might have been duplicated into CAPA and HUGIN in insects. In vertebrates, NMU is the putative homolog of a protostomian CAPA-PK.

Cloning of neuropeptide-like precursor 1 in the gray flesh fly and peptide identification and expression.

The neuropeptide-like precursor 1 (NPLP1) was first identified in a peptidomics experiment on Drosophila melanogaster. Limited data on this novel neuropeptide precursor suggest a role in the regulation of ecdysis in holometabolous larvae. In this study, we characterized the NPLP1 precursor in the gray flesh fly, Neobellieria bullata, which is an excellent model for physiological assays and hence to discover the role of the NPLP1 peptides. Antisera against three of the D. melanogaster NPLP1 neuropeptides stained an identical set of neurons in the central nervous system of N. bullata compared to D. melanogaster. A novel approach was applied to identify the N. bullata NPLP1 orthologs. Using a combination of affinity chromatography, mass spectrometry, cDNA cloning and RACE experiments, we obtained almost the complete coding sequence of the NPLP1 mRNA. Three encoded NPLP1 peptides were identified in central nervous system extracts by mass spectrometry. Neither doses of 25-250pmol of synthetic Neb-MGYamide and Neb-PQNamide peptides, nor the NPLP1 antisera did affect the speed of retraction, contraction and tanning in the pupariation bioassay.

Comparative peptidomics of Caenorhabditis elegans versus C. briggsae by LC-MALDI-TOF MS.

Neuropeptides are important signaling molecules that function in cell-cell communication as neurotransmitters or hormones to orchestrate a wide variety of physiological conditions and behaviors. These endogenous peptides can be monitored by high throughput peptidomics technologies from virtually any tissue or organism. The neuropeptide complement of the soil nematode Caenorhabditis elegans has been characterized by on-line two-dimensional liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (2D-nanoLC Q-TOF MS/MS). Here, we use an alternative peptidomics approach combining liquid chromatography (LC) with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to map the peptide content of C. elegans and another Caenorhabditis species, Caenorhabditis briggsae. This study allows a better annotation of neuropeptide-encoding genes from the C. briggsae genome and provides a promising basis for further evolutionary comparisons.

OdK2, a Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae.

The first Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae (OdK2) was purified, sequenced and characterized physiologically. OdK2 consists of 38 amino acids, including six conserved cysteine and a C-terminal lysine residue, as revealed by the unique use of a quadrupole ion cyclotron resonance Fourier-transform mass spectrometer. Based on multiple sequence alignments, OdK2 was classified as alpha-KTX3.11. The pharmacological effects of OdK2 were studied on a panel of eight different cloned K(+) channels (vertebrate Kv1.1-Kv1.6, Shaker IR and hERG) expressed in Xenopus laevis oocytes. Interestingly, OdK2 selectively inhibits the currents through Kv1.3 channels with an IC50 value of 7.2+/-2.7nM.

PEGylation of cholecystokinin prolongs its anorectic effect in rats.

The anorectic compound CCK-9 was coupled to polyethylene glycol 5 kDa, 10 kDa, 20 kDa and 30 kDa, under different reaction conditions. Conjugates were purified by HPLC and characterized by MALDI-TOF MS. A 96% PEGylation yield was obtained in buffer pH 7.5 after 6h reaction at 20 degrees C. The anorectic activity was tested in vivo in rats. A single bolus intra-peritoneal injection of non-modified CCK-9 resulted in a significant initial food intake reduction 30 min after food presentation (87% compared to paired control group). When PEG-CCK-9 conjugates modified with polymers of molecular weight up to 20 kDa were injected, lower but statistically significant initial food intake reductions were obtained (76% for PEG 10 kDa-CCK-9 conjugate compared to control group). The cumulative food intake reduction of non-modified CCK-9 is normalized within 1-2h, whereas the PEG-CCK-9 molecules showed a prolonged anorectic activity lasting for 6h for PEG 5 kDa-CCK-9; 23 h for PEG 10 kDa-CCK-9 and between 8h and 23 h for PEG 20 kDa-CCK-9. For PEG 30 kDa-CCK-9 conjugate, neither an initial nor a cumulative FI reduction was observed. PEG-CCK-9 conjugates show a significantly prolonged anorectic activity in comparison to the non-modified peptide. This effect is most evident for the PEG 10 kDa-CCK-9 conjugate.

Purification and thermal and high-pressure inactivation of pectinmethylesterase isoenzymes from tomatoes (Lycopersicon esculentum): a novel pressure labile isoenzyme.

Tomato pectinmethylesterase (PME) was successfully purified by a two-step method consisting of affinity chromatography followed by cation exchange chromatography. According to this procedure, four different isoenzymes were identified representing molar masses around 34.5-35.0 kDa. Thermal and high-pressure inactivation kinetics of the two major isoenzymes of tomato PME were studied. A striking difference between their process stability was found. The thermostable isoenzyme was completely inactivated after 5.0 min at 70 degrees C, whereas for the thermolabile isoenzyme, temperatures at around 60 degrees C were sufficient for complete inactivation. The thermostable isoenzyme was also found to be pressure stable since no inactivation was observed after 5.0 min of treatment at 800 MPa and 20 or 40 degrees C. The thermolabile isoenzyme appeared to be pressure labile since it could be completely inactivated after 5.0 min of treatment at 700 MPa and 20 degrees C or 650 MPa and 40 degrees C. Inactivation kinetics at pH 6.0 could be accurately described by a first-order model.

Annotation of novel neuropeptide precursors in the migratory locust based on transcript screening of a public EST database and mass spectrometry.

BACKGROUND: For holometabolous insects there has been an explosion of proteomic and peptidomic information thanks to large genome sequencing projects. Heterometabolous insects, although comprising many important species, have been far less studied. The migratory locust Locusta migratoria, a heterometabolous insect, is one of the most infamous agricultural pests. They undergo a well-known and profound phase transition from the relatively harmless solitary form to a ferocious gregarious form. The underlying regulatory mechanisms of this phase transition are not fully understood, but it is undoubtedly that neuropeptides are involved. However, neuropeptide research in locusts is hampered by the absence of genomic information. RESULTS: Recently, EST (Expressed Sequence Tag) databases from Locusta migratoria were constructed. Using bioinformatical tools, we searched these EST databases specifically for neuropeptide precursors. Based on known locust neuropeptide sequences, we confirmed the sequence of several previously identified neuropeptide precursors (i.e. pacifastin-related peptides), which consolidated our method. In addition, we found two novel neuroparsin precursors and annotated the hitherto unknown tachykinin precursor. Besides one of the known tachykinin peptides, this EST contained an additional tachykinin-like sequence. Using neuropeptide precursors from Drosophila melanogaster as a query, we succeeded in annotating the Locusta neuropeptide F, allatostatin-C and ecdysis-triggering hormone precursor, which until now had not been identified in locusts or in any other heterometabolous insect. For the tachykinin precursor, the ecdysis-triggering hormone precursor and the allatostatin-C precursor, translation of the predicted neuropeptides in neural tissues was confirmed with mass spectrometric techniques. CONCLUSION: In this study we describe the annotation of 6 novel neuropeptide precursors and the neuropeptides they encode from the migratory locust, Locusta migratoria. By combining the manual annotation of neuropeptides with experimental evidence provided by mass spectrometry, we demonstrate that the genes are not only transcribed but also translated into precursor proteins. In addition, we show which neuropeptides are cleaved from these precursor proteins and how they are post-translationally modified.

The antimicrobial peptide parabutoporin competes with p47(phox) as a PKC-substrate and inhibits NADPH oxidase in human neutrophils.

We investigated parabutoporin (PP), an antimicrobial scorpion peptide, to understand its inhibition on NADPH oxidase in human PMN. We show that PP is a good substrate for all PKC-isotypes, implicated in the activation of NADPH oxidase, and acts as a potent competitive inhibitor of in vitro p47(phox)-phosphorylation by PKC-alpha, -betaI, -betaII and -delta, but not PKC-zeta. In PMN, PP also inhibits the PMA-stimulated phosphorylation of p47(phox) and its subsequent translocation. In contrast, PP affects the PKC-independent activation to a much lesser degree. This indicates that PP inhibits the activation of NADPH oxidase at submicromolar concentrations in a strongly PKC-dependent manner.

The first potassium channel toxin from the venom of the Iranian scorpion Odonthobuthus doriae.

The very first member of K(+) channels toxins from the venom of the Iranian scorpion Odonthobuthus doriae (OdK1) was purified, sequenced and characterized physiologically. OdK1 has 29 amino acids, six conserved cysteines and a pI value of 4.95. Based on multiple sequence alignments, OdK1 was classified as alpha-KTx 8.5. The pharmacological effects of OdK1 were studied on six different cloned K(+) channels (vertebrate Kv1.1-Kv1.5 and Shaker IR) expressed in Xenopus laevis oocytes. Interestingly, OdK1 selectively inhibited the currents through Kv1.2 channels with an IC50 value of 183+/-3 nM but did not affect any of the other channels.

Effect of temperature and high pressure on the activity and mode of action of fungal pectin methyl esterase.

Pectin was de-esterified with purified recombinant Aspergillus aculeatus pectin methyl esterase (PME) during isothermal-isobaric treatments. By measuring the release of methanol as a function of treatment time, the rate of enzymatic pectin conversion was determined. Elevated temperature and pressure were found to stimulate PME activity. The highest rate of PME-catalyzed pectin de-esterification was obtained when combining pressures in the range 200-300 MPa with temperatures in the range 50-55 degrees C. The mode of pectin de-esterification was investigated by characterizing the pectin reaction products by enzymatic fingerprinting. No significant effect of increasing pressure (300 MPa) and/or temperature (50 degrees C) on the mode of pectin conversion was detected.

OD1, the first toxin isolated from the venom of the scorpion Odonthobuthus doriae active on voltage-gated Na+ channels.

In this study, we isolated and pharmacologically characterized the first alpha-like toxin from the venom of the scarcely studied Iranian scorpion Odonthobuthus doriae. The toxin was termed OD1 and its primary sequence was determined: GVRDAYIADDKNCVYTCASNGYCNTECTKNGAESGYCQWIGRYGNACWCIKLPDEVPIRIPGKCR. Using the two-electrode voltage clamp technique, the pharmacological effects of OD1 were studied on three cloned voltage-gated Na+ channels expressed in Xenopus laevis oocytes (Na(v)1.2/beta1, Na(v)1.5/beta1, para/tipE). The inactivation process of the insect channel, para/tipE, was severely hampered by 200 nM of OD1 (EC50 = 80+/-14 nM) while Na(v)1.2/beta1 still was not affected at concentrations up to 5 microM. Na(v)1.5/beta1 was influenced at micromolar concentrations.

Proteomic Alterations in B Lymphocytes of Sensitized Mice in a Model of Chemical-Induced Asthma.

INTRODUCTION AND AIM: The role of B-lymphocytes in chemical-induced asthma is largely unknown. Recent work demonstrated that transferring B lymphocytes from toluene diisocyanate (TDI)-sensitized mice into naïve mice, B cell KO mice and SCID mice, triggered an asthma-like response in these mice after a subsequent TDI-challenge. We applied two-dimensional difference gel electrophoresis (2D-DIGE) to describe the "sensitized signature" of B lymphocytes comparing TDI-sensitized mice with control mice. RESULTS: Sixteen proteins were identified that were significantly up- or down-regulated in B lymphocytes of sensitized mice. Particularly differences in the expression of cyclophilin A, cofilin 1 and zinc finger containing CCHC domain protein 11 could be correlated to the function of B lymphocytes as initiators of T lymphocyte independent asthma-like responses. CONCLUSION: This study revealed important alterations in the proteome of sensitized B cells in a mouse model of chemical-induced asthma, which will have an important impact on the B cell function.