Ancestral functions of Delta/Notch signaling in the formation of body and leg segments in the cricket Gryllus bimaculatus

Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, we analyzed the function of the Delta (Gb'Delta) and Notch (Gb'Notch) genes in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. We found that Gb'Delta and Gb'Notch were expressed in developing legs, and that RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. Our results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, we found that Gb'Delta was expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries.     

Peptidomics-based discovery of an antimicrobial peptide derived from insulin-like growth factor-binding protein 5

Antimicrobial peptides (AMPs) are effector molecules that are able to kill or inactivate microbial pathogens. However, most AMPs harbor multiple basic amino acids that hamper current proteomic identification. In our peptidomic survey of endogenous peptides, we identified a novel intramolecular disulfide-linked 22-residue amidated peptide. This peptide, designated AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5), showed antimicrobial activity against six of the eight microorganisms tested at concentrations comparable to or lower than those for well-characterized AMPs cathelicidin and β-defensin-2. AMP-IBP5 is identical at the amino acid level between human, mouse, rat, pig, and cow. Natural occurrence of this peptide as the originally isolated form was demonstrated in the rat brain and intestine, using mass spectrometric characterization of major immunoreactivity. The peptide is flanked N-terminally by a single arginine and C-terminally by a common amidation signal, indicating that insulin-like growth factor-binding protein 5 (IGFBP-5) undergoes specific cleavage by a defined set of processing proteases. Furthermore, the intramolecular linkage C199-C210 reveals itself as a correct disulfide pairing in the precursor protein, the finding not inferred from closely related family members IGFBP-4 and -6. In principle, neither conventional proteomics nor bioinformatics would achieve the identification of this AMP. Our study exemplifies the impact of peptidomics to study naturally occurring peptides.     

Docking study and binding free energy calculation of poly (ADP-ribose) polymerase inhibitors

Recently, the massively parallel computation of absolute binding free energy with a well-equilibrated system (MP-CAFEE) has been developed. The present study aimed to determine whether the MP-CAFEE method is useful for drug discovery research. In the drug discovery process, it is important for computational chemists to predict the binding affinity accurately without detailed structural information for protein / ligand complex. We investigated the absolute binding free energies for Poly (ADP-ribose) polymerase-1 (PARP-1) / inhibitor complexes, using the MP-CAFEE method. Although each docking model was used as an input structure, it was found that the absolute binding free energies calculated by MP-CAFEE are well consistent with the experimental ones. The accuracy of this method is much higher than that using molecular mechanics Poisson-Boltzmann / surface area (MM / PBSA). Although the simulation time is quite extensive, the reliable predictor of binding free energies would be a useful tool for drug discovery projects.     

Clonal spread of antimicrobial-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates among pups in two kennels

Although the dog breeding industry is common in many countries, the presence of antimicrobial resistant bacteria among pups in kennels has been infrequently investigated. This study was conducted to better understand the epidemiology of antimicrobial-resistant Escherichia coli isolates from kennel pups not treated with antimicrobials. We investigated susceptibilities to 11 antimicrobials, and prevalence of extended-spectrum β-lactamase (ESBL) in 86 faecal E. coli isolates from 43 pups in two kennels. Genetic relatedness among all isolates was assessed using pulsed-field gel electrophoresis (PFGE). Susceptibility tests revealed that 76% of the isolates were resistant to one or more of tested antimicrobials, with resistance to dihydrostreptomycin most frequently encountered (66.3%) followed by ampicillin (60.5%), trimethoprim-sulfamethoxazole (41.9%), oxytetracycline (26.7%), and chloramphenicol (26.7%). Multidrug resistance, defined as resistance against two or more classes of antimicrobials, was observed in 52 (60.5%) isolates. Three pups in one kennel harboured SHV-12 ESBL-producing isolates. A comparison between the two kennels showed that frequencies of resistance against seven antimicrobials and the variation in resistant phenotypes differed significantly. Analysis by PFGE revealed that clone sharing rates among pups of the same litters were not significantly different in both kennels (64.0% vs. 88.9%), whereas the rates among pups from different litters were significantly different between the two kennels (72.0% vs. 33.3%, P < 0.05). The pups in the two kennels had antimicrobial-resistant E. coli clones, including multidrug-resistant and ESBL-producing clones. It is likely that resistant and susceptible bacteria can clonally spread among the same and/or different litters thus affecting the resistance prevalence.     

Effects of orexins/hypocretins on neuronal activity in the paraventricular nucleus of the thalamus in rats in vitro

Orexin-A (ORX-A) and orexin-B (ORX-B), also called hypocretin-1 and hypocretin-2, respectively, act upon orexin 1 (OX1R) and orexin 2 (OX2R) receptors, and are involved in the regulation of sleep-wakefulness and energy homeostasis. Orexin neurons in the lateral hypothalamic perifornical region project heavily to the paraventricular nucleus of the thalamus (PVT), which is deeply involved in the control of motivated behaviors. In the present study, electrophysiological and cytosolic Ca2+ concentration ([Ca2+]i) imaging studies on the effects of ORX-A and ORX-B on neurons in the PVT were carried out in rat brain slice preparations. ORX-A and/or ORX-B were applied extracellularly in the perfusate. Extracellular recordings showed that about 80% of the PVT neurons were excited dose-dependently by both ORX-A and ORX-B at concentrations of 10(-8) to 10(-6)M, and the increase in firing rate was about three times larger for ORX-B than for ORX-A at 10(-7)M. When both ORX-A and ORX-B were applied simultaneously at 10(-7)M, the increase in firing rate was almost equal to that of ORX-B at 10(-7)M, suggesting that the PVT neurons do not show a high affinity to ORX-A which is expected if they have OX1R receptors. The excitatory effect of ORX-B was seen in low Ca2+ and high Mg2+ ACSF as well as in normal ACSF, and the increase in firing rate was greater in low Ca2+ and high Mg2+ ACSF than in normal ACSF. [Ca2+]i imaging studies demonstrated that [Ca2+]i was increased in about 50% of the PVT neurons by both 10(-7)M ORX-A and ORX-B with a stronger effect for ORX-B, and the increase in [Ca2+]i induced by ORX-B was abolished in Ca2+-free ACSF, suggesting that ORX-B does not release Ca2+ from intracellular Ca2+ stores. Subsequent whole cell patch clamp recordings revealed that an after hyperpolarization seen following each action potential in normal ACSF disappeared in Ca2+-free ACSF, and the mean magnitude of the depolarization induced by ORX-B was same in normal, Ca2+-free and TTX-containing Ca2+-free ACSFs. Furthermore, ORX-B-induced depolarization was reversed to hyperpolarization when membrane potential was lowered to about -97 mV, and an increase of extracellular K+ concentration from 4.25 to 13.25 mM abolished the ORX-B-induced depolarization, indicating that the ORX-B-induced depolarization is associated with an increase in the membrane resistance resulting from a closure of K+ channels. These results suggest that orexins depolarize and excite post-synaptically PVT neurons via OX2R receptors, and that orexin-activated PVT neurons play a role in the integration of sleep-wakefulness and energy homeostasis, and in the control of motivated behaviors.     

Polysialic acid facilitates tumor invasion by glioma cells

Polysialic acid (PSA) is thought to attenuate neural cell adhesion molecule (NCAM) adhesion, thereby facilitating neural cell migration and regeneration. Although the expression of PSA has been shown to correlate with the progression of certain tumors such as small cell lung carcinoma, there have been no studies to determine the roles of PSA in gliomas, the most common type of primary brain tumor in humans. In this study, we first revealed that among patients with glioma, PSA was detected more frequently in diffuse astrocytoma cells, which spread extensively. To determine directly the role of PSA in glioma cell invasion, we transfected C6 glioma cells with polysialyltransferases to express PSA. In those transfected cells, PSA is attached mainly to NCAM-140, whereas the mock-transfected C6 cells express equivalent amounts of PSA-free NCAM-140. Both PSA negative and positive C6 cell lines exhibited almost identical growth rates measured in vitro. However, PSA positive C6 cells exhibited increased invasion to the corpus callosum, where the mock-transfected C6 glioma cells rarely invaded when inoculated into the brain. By contrast, the invasion to the corpus callosum by both the mock-transfected and PSA positive C6 cells was observed in NCAM-deficient mice. These results combined indicate that PSA facilitates tumor invasion of glioma in the brain, and that NCAM-NCAM interaction is likely attenuated in the PSA-mediated tumor invasion.     

Black tea theaflavins suppress dioxin-induced transformation of the aryl hydrocarbon receptor

Dioxins cause various adverse effects through transformation of aryl hydrocarbon receptor (AhR). In this study, we investigated whether black tea extract and its components, theaflavins, suppress AhR transformation in vitro. First, we confirmed that black tea extract strongly suppressed AhR transformation compared to green and oolong tea, although the catechin contents did not change significantly among the extracts. Then we isolated four theaflavins as active compounds from black tea leaves. They suppressed 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation in a dose-dependent manner. The IC(50) values of theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate (Tfdg) were 4.5, 2.3, 2.2, and 0.7 muM, respectively. The suppressive effect of Tfdg was observed not only by pre-treatment but also by post-treatment. This suggests that theaflavins inhibit the binding of TCDD to the AhR and also the binding of the transformed AhR to the specific DNA-binding site as putative mechanisms.     

Effects of serum deprivation on expression of proteolytic-related genes in chick myotube cultures

We previously reported that serum deprivation stimulates myofibrillar proteolysis in chick myotubes. In the present study, we examined the effect of serum deprivation on expression of the proteolytic-related genes (ubiquitin, proteasome, calpains, and cathepsin B) by real-time PCR of cDNA in chick myotubes. Myotubes were incubated with serum-free medium for 24 h. Ubiquitin and proteasome subunits (C1 and C2) and calpains (m-, mu-, and p94/calpain-3) but not cathepsin B mRNA expression were increased by serum deprivation. These results indicate that serum deprivation stimulates ubiquitin-proteasome and calpain proteolytic pathways, resulting in an increase in myofibrillar proteolysis in chick myotubes.