Possibility of Production of Amino Acids by Impact Reaction Using a Light-Gas Gun as a Simulation of Asteroid Impacts

In order to investigate impact production of carbonaceous products by asteroids on Titan and other satellites and planets, simulation experiments were carried out using a 2-stage light gas gun. A small polycarbonate or metal bullet with about 6.5 km/s was injected into a pressurized target chamber filled with 1 atm of nitrogen gas, to collide with a ice + iron target or an iron target or a ice + hexane + iron target. After the impact, black soot including fine particles was deposited on the chamber wall. The soot was carefully collected and analyzed by High Performance Liquid Chromatography (HPLC), Fourier Transform Infrared Spectroscopy (FT-IR), and Laser Desorption Time-of-Flight Mass Spectrometry (LD-ToF-MS). As a result of the HPLC analysis, about 0.04–8 pmol of glycine, and a lesser amount of alanine were found in the samples when the ice + hexane + iron target was used. In case of the ice + iron target and the iron target, less amino acids were produced. The identification of the amino acids was also supported by FTIR and LD-ToF-MS analysis.     

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.