Anatomical and functional characterisation of the stomatogastric nervous system of blowfly (Calliphora vicina) larvae

The anatomy and functionality of the stomatogastric nervous system (SNS) of third-instar larvae of Calliphora vicina was characterised. As in other insects, the Calliphora SNS consists of several peripheral ganglia involved in foregut movement regulation. The frontal ganglion gives rise to the frontal nerve and is connected to the brain via the frontal connectives and antennal nerves (ANs). The recurrent nerve connects the frontal- to the hypocerebral ganglion from which the proventricular nerve runs to the proventricular ganglion. Foregut movements include rhythmic contractions of the cibarial dilator muscles (CDM), wavelike movements of crop and oesophagus and contractions of the proventriculus. Transections of SNS nerves indicate mostly myogenic crop and oesophagus movements and suggest modulatory function of the associated nerves. Neural activity in the ANs, correlating with postsynaptic potentials on the CDM, demonstrates a motor pathway from the brain to CDM. Crop volume is monitored by putative stretch receptors. The respective sensory pathway includes the recurrent nerve and the proventricular nerve. The dorsal organs (DOs) are directly connected to the SNS. Mechanical stimulation of the DOs evokes sensory activity in the AN. This suggests the DOs can provide sensory input for temporal coordination of feeding behaviour.     

Parameters of Metabolic Syndrome and Its Relationship with Zincemia and Activities of Superoxide Dismutase and Glutathione Peroxidase in Obese Women

Alterations in antioxidant defense in obese people with metabolic syndrome can contribute to oxidative stress. This study assessed the relationship between the parameters of metabolic syndrome and the zincemia, activity of superoxide dismutase, and glutathione peroxidase enzymes in obese women. Seventy-three premenopausal women, aged between 20 and 50 years, were divided into two groups: case group, composed of obese (n = 37), and control group, composed of no obese (n = 36). Analyses of zinc intake, parameters of metabolic syndrome, plasma, and erythrocyte zinc, and activities of superoxide dismutase and glutathione peroxidase were carried out. The mean values of body mass index of obese women and control group were 34.5 ± 3.4 and 21.7 ± 1.9 kg/m², respectively (p < 0.05). In the study, body mass index, waist circumference, and zinc intake were higher in obese women than control group (p < 0.05). The plasma zinc and activity of superoxide dismutase did not show significant differences between obese and controls (p > 0.05). The values of erythrocyte zinc was 36.4 ± 15.0 μg/gHb and 45.4 ± 14.3 μg/gHb and of glutathione peroxidase was 46.4 ± 19.4 U/gHb and 36.7 ± 13.6 U/gHb in obese women and controls, respectively (p < 0.05). The study shows that there are alterations in biochemical parameters of zinc in obese women, with low zinc concentrations in erythrocytes. Regression analysis demonstrates that the erythrocyte zinc and activity of superoxide dismutase enzyme is influenced by components of the metabolic syndrome, and the plasmatic glucose, body mass index, and waist circumference have a negative correlation with this enzyme.     

Endogenous biotin‐binding proteins: an overlooked factor causing false positives in streptavidin‐based protein detection

Biotinylation is widely used in DNA, RNA and protein probing assays as this molecule has generally no impact on the biological activity of its substrate. During the streptavidin‐based detection of glycoproteins in Lactobacillus rhamnosus GG with biotinylated lectin probes, a strong positive band of approximately 125 kDa was observed, present in different cellular fractions. This potential glycoprotein reacted heavily with concanavalin A (ConA), a lectin that specifically binds glucose and mannose residues. Surprisingly, this protein of 125 kDa could not be purified using a ConA affinity column. Edman degradation of the protein, isolated via cation and anion exchange chromatography, lead to the identification of the band as pyruvate carboxylase, an enzyme of 125 kDa that binds biotin as a cofactor. Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin‐associated proteins. Indeed, biotin is a known cofactor of numerous carboxylases. The potential occurence of false positive bands with biotinylated protein probes should thus be considered when using streptavidin‐based detection, e.g. by developing a blot using only the streptavidin conjugate. To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used.     

Histochemical and immunocytochemical study of the migration of neurons from the rat olfactory placode

Immunocytochemical and histochemical methods have been used to describe the neuronal population migrating from the rat olfactory placode and to analyze the spatio-temporal evolution of this neuronal migration during development. Several neuronal markers, such as binding to the lectin Ulex europaeus (UEA I) and the presence of neuron-specific enolase (NSE), olfactory marker protein (OMP), and luteinizing hormone-releasing hormone (LHRH), have been tested in order to determine whether migrating neurons originate from both the medial and the lateral parts of the placode and whether they all express LHRH. Our data show that a large population of differentiated migrating neurons can be identified with an antibody against NSE from the 14th day of gestation and with UEA I one day later. Migrating neurons are closely associated with both the vomeronasal axon fascicles emerging from the medial pit and the olfactory axons originating from the lateral pit. However, the neuron migration from the lateral pit appears to be more discrete than that from the medial pit. No LHRH immunoreactivity has been detected among neurons migrating from the lateral pit. Some neurons accompanying the olfactory axon fascicles exhibit a high level of maturation as shown by their OMP-positivity. Numerous neurons positive for both NSE and UEA I have also been observed within the presumptive olfactory nerve layer in early embryonic stages.     

Identification of Ruminococcus flavefaciens as the Predominant Cellulolytic Bacterial Species of the Equine Cecum

Detection and quantification of cellulolytic bacteria with oligonucleotide probes showed that Ruminococcus flavefaciens was the predominant species in the pony and donkey cecum. Fibrobacter succinogenes and Ruminococcus albus were present at low levels. Four isolates, morphologically resembling R. flavefaciens, differed from ruminal strains by their carbohydrate utilization and their end products of cellobiose fermentation.     

Binding of human centrin 2 to the centrosomal protein hSfi1

hSfi1, a human centrosomal protein with homologs in other eukaryotic organisms, includes 23 repeats, each of 23 amino acids, separated by 10 residue linkers. The main molecular partner in the centrosome is a small, calcium-binding EF-hand protein, the human centrin 2. Using isothermal titration calorimetry experiments, we characterized the centrin-binding capacity of three isolated hSfi1 repeats, two exhibiting the general consensus motif and the third being the unique Pro-containing human repeat. The two standard peptides bind human centrin 2 and its isolated C-terminal domain with high affinity (approximately 10(7) M(-1)) by an enthalpy-driven mechanism, with a moderate Ca2+ dependence. The Pro-containing repeat shows a binding affinity that is two orders of magnitude lower. The target binding site is localized within the C-terminal domain of human centrin 2. Fluorescence titration and NMR spectroscopy show that the well-conserved Trp residue situated in the C-terminus of each repeat is deeply embedded in a protein hydrophobic cavity, indicating that the peptide direction is reversed relative to previously studied centrin targets. The present results suggest that almost all of the repeats of the Sfi1 protein may independently bind centrin molecules. On the basis of this hypothesis and previous studies on centrin self-assembly, we propose a working model for the role of centrin-Sfi1 interactions in the dynamic structure of centrosome-associated contractile fibers.     

Effect of Bacillus thuringiensis Cry1 Toxins in Insect Hemolymph and Their Neurotoxicity in Brain Cells of Lymantria dispar

Little information is available on the systemic effects of Bacillus thuringiensis toxins in the hemocoel of insects. In order to test whether B. thuringiensis-activated toxins elicit a toxic response in the hemocoel, we measured the effect of intrahemocoelic injections of several Cry1 toxins on the food intake, growth, and survival of Lymantria dispar (Lepidoptera) and Neobellieria bullata (Diptera) larvae. Injection of Cry1C was highly toxic to the Lymantria larvae and resulted in the complete inhibition of food intake, growth arrest, and death in a dose-dependent manner. Cry1Aa and Cry1Ab (5 μg/0.2 g [fresh weight] [g fresh wt]) also affected growth and food intake but were less toxic than Cry1C (0.5 μg/0.2 g fresh wt). Cry1E and Cry1Ac (5 μg/0.2 g fresh wt) had no toxic effect upon injection. Cry1C was also highly toxic to N. bullata larvae upon injection. Injection of 5 μg/0.2 g fresh wt resulted in rapid paralysis, followed by hemocytic melanization and death. Lower concentrations delayed pupariation or gave rise to malformation of the puparium. Finally, Cry1C was toxic to brain cells of Lymantria in vitro. The addition of Cry1C (20 μg/ml) to primary cultures of Lymantria brain cells resulted in rapid lysis of the cultured neurons.     

AMP-activated protein kinase phosphorylates and inactivates liver glycogen synthase

Recombinant muscle GYS1 (glycogen synthase 1) and recombinant liver GYS2 were phosphorylated by recombinant AMPK (AMP-activated protein kinase) in a time-dependent manner and to a similar stoichiometry. The phosphorylation site in GYS2 was identified as Ser7, which lies in a favourable consensus for phosphorylation by AMPK. Phosphorylation of GYS1 or GYS2 by AMPK led to enzyme inactivation by decreasing the affinity for both UDP-Glc (UDP-glucose) [assayed in the absence of Glc-6-P (glucose-6-phosphate)] and Glc-6-P (assayed at low UDP-Glc concentrations). Incubation of freshly isolated rat hepatocytes with the pharmacological AMPK activators AICA riboside (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) or A769662 led to persistent GYS inactivation and Ser7 phosphorylation, whereas inactivation by glucagon treatment was transient. In hepatocytes from mice harbouring a liver-specific deletion of the AMPK catalytic α1/α2 subunits, GYS2 inactivation by AICA riboside and A769662 was blunted, whereas inactivation by glucagon was unaffected. The results suggest that GYS inactivation by AMPK activators in hepatocytes is due to GYS2 Ser7 phosphorylation.     

Proteomics analysis of cytokine-induced dysfunction and death in insulin-producing INS-1E cells: new insights into the pathways involved

Cytokines released by islet-infiltrating immune cells play a crucial role in beta-cell dysfunction and apoptotic cell death in the pathogenesis of type 1 diabetes and after islet transplantation. RNA studies revealed complex pathways of genes being activated or suppressed during this beta-cell attack. The aim of the present study was to analyze protein changes in insulin-producing INS-1E cells exposed to inflammatory cytokines in vitro using two-dimensional DIGE. Within two different pH ranges we observed 2214 +/- 164 (pH 4-7) and 1641 +/- 73 (pH 6-9) spots. Analysis at three different time points (1, 4, and 24 h of cytokine exposure) revealed that the major changes were taking place only after 24 h. At this time point 158 proteins were altered in expression (4.1%, n = 4, p < or = 0.01) by a combination of interleukin-1beta and interferon-gamma, whereas only 42 and 23 proteins were altered by either of the cytokines alone, giving rise to 199 distinct differentially expressed spots. Identification of 141 of these by MALDI-TOF/TOF revealed proteins playing a role in insulin secretion, cytoskeleton organization, and protein and RNA metabolism as well as proteins associated with endoplasmic reticulum and oxidative stress/defense. We investigated the interactions of these proteins and discovered a significant interaction network (p < 1.27e-05) containing 42 of the identified proteins. This network analysis suggests that proteins of different pathways act coordinately in a beta-cell dysfunction/apoptotic beta-cell death interactome. In addition the data suggest a central role for chaperones and proteins playing a role in RNA metabolism. As many of these identified proteins are regulated at the protein level or undergo post-translational modifications, a proteomics approach, as performed in this study, is required to provide adequate insight into the mechanisms leading to beta-cell dysfunction and apoptosis. The present findings may open new avenues for the understanding and prevention of beta-cell loss in type 1 diabetes.