Knockout of a transgene by transcription activator-like effector nucleases (TALENs) in the sawfly, Athalia rosae (Hymenoptera) and the ladybird beetle, Harmonia axyridis (Coleoptera).

Transcription activator-like effector nucleases (TALENs) are efficient tools for targeted genome editing and have been utilized in a number of insects. Here, we demonstrate the gene disruption (knockout) caused by TALENs targeting a transgene, 3xP3-driven enhanced green fluorescence protein (EGFP), that is integrated in the genome of two species, the sawfly Athalia rosae (Hymenoptera) and the ladybird beetle Harmonia axyridis (Coleoptera). Messenger RNAs of TALENs targeting the sequences adjacent to the chromophore region were microinjected into the eggs/embryos of each species. In At. rosae, when microinjection was performed at the posterior end of eggs, 15% of G(0) individuals showed a somatic mosaic phenotype for eye EGFP fluorescence. Three-quarters of the somatic mosaics produced EGFP-negative G(1) progeny. When eggs were injected at the anterior end, 63% of the G(0) individuals showed somatic mosaicism, and 17% of them produced EGFP-negative G(1) progeny. In H. axyridis, 25% of posterior-injected and 8% of anterior-injected G(0) individuals produced EGFP-negative G(1) progeny. In both species, the EGFP-negative progeny retained the EGFP gene, and various deletions were detected in the target sequences, indicating that gene disruption was successfully induced. Finally, for both species, 18-21% of G(0) founders produced gene knockout progeny sufficient for establishing knockout strains.

Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis.

Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to ~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects.

Biosynthesis of glycosaminoglycans in peritoneal macrophages from the guinea pig.

The majority of glycosaminoglycans synthesized in peritoneal macrophages from the guinea pig in vitro were secreted into culture medium. The secreted glycosaminoglycans were reduced in size with alkali treatment indicating that the glycosaminoglycans existed in the form of proteoglycans. After the glycosaminoglycans were digested with chondroitinase AC and ABC, the high voltage paper electrophoretic analysis and the descending paper chromatographic analysis indicated the presence of a considerable amount of unsaturated disulfated disaccharides. Based on the enzymatic assay with chondro-4- and 6-sulfatase, the positions of sulfation in the disulfated disaccharide have been identified as the 4- and 6-position of N-acetylgalactosamine. Moreover, the results of the ion-exchange chromatography and the chondroitinase AC and ABC digestion indicated that delta Di-diSE derived from dermatan sulfate. This suggests that peritoneal macrophages are capable of synthesizing oversulfated proteodermatan sulfate as main component. The proportion of synthesized oversulfated dermatan sulfate to the total glycosaminoglycans was independent of the incubation time, and the distribution of oversulfated dermatan sulfate in cell and incubation medium also did not change. After exposure of macrophages to Escherichia coli for 15 min, the incorporation of [35S]sulfate and [3H]glucosamine into the glycosaminoglycans was increased by about 40% with no significant change in the proportion of synthesized oversulfated dermatan sulfate, but the release of glycosaminoglycans into the culture medium remains essentially unchanged. The difference of the existence of oversulfated dermatan sulfate is not yet understood.

Off-center rattling and anisotropic expansion of type-I clathrates studied by Raman scattering.

Dynamical motions of the guest ions in type-I clathrates Sr8Ga16Si(30-x)Gex and Ba8Ga16Si(30-x)Gex have been studied by Raman scattering spectroscopy, to clarify the role of guest vibration modes in these systems with unusual thermal transport behaviors. An anomalous decrease of the guest energies with decreasing temperature is observed for both systems. The Ge-doping expands the cage surrounding the 6d site anisotropically for Sr8Ga16Si(30-x)Gex, but isotropically for Ba8Ga16Si(30-x)Gex. Especially for Sr8Ga16Si(30-x)Gex, off-center rattling arises simultaneously with the anisotropic expansion, and it is confirmed that these anomalies play a crucial role to suppress lattice thermal conductivity in these systems.

High-density trapping of cold ytterbium atoms by an optical dipole force.

We have succeeded in trapping a high density of rare-earth atom of ytterbium (Yb) in a crossed far-off resonance trap. The peak density reaches more than 10(14) cm(-3). With a new method of a delayed crossed far-off resonance trap, we have elucidated that the atoms became concentrated into the cross region by atom-atom collisions. We trap fermionic Yb atoms in the same way as bosonic ones.

Photoassociation spectroscopy of laser-cooled ytterbium atoms.

We report the photoassociation spectroscopy of laser-cooled ytterbium atoms in an optical trap. We observed more than 90 photoassociation resonances of vibrational levels in the (1)Sigma(+)(u) state, including 80 consecutive series, up to 490 GHz detuning with respect to the atomic resonance. From the resonance frequencies we derived the atomic radiative lifetime of the (6s6p) 1P1 state to be 5.464+/-0.005 ns, which is about 2 orders of magnitude improvement over previous results. We also observed line broadening of resonances, which is ascribed to the predissociation to the triplet states, and estimated the transition probability to be 0.2. Furthermore, we observed the decrease of the photoassociation signal intensity, from which the scattering length is estimated to be equal to or less than 3 nm.

Voltage-Controlled Fluorometry of the Transfer of Nonfluorescent Ions across the 1,2-Dichloroethane/Water Interface Using Fluorescent Ionophores.

The voltage-driven transfer of alkali and alkaline-earth metal ions across the polarized 1,2-dichloroethane (DCE)/water interface has been measured fluorometrically by using two fluorescent ionophores, coumarin 153-linked monoaza-15-crown-5 (C153-crown(O(4))) and two-coumarin 153-linked diaza-18-crown-6 ((C153)(2)-K(22)). The 1:1 complex formation prevails for both monovalent and divalent ion transfer, when the concentration of C153-crown(O(4)) in DCE is at the micromolar level. In the Mg(2+) ion transfer, the formation of the 1:2 (metal-ligand) complex becomes significant at the millimolar level of C153-crown(O(4)). C153-crown(O(4)) seems to adsorb at the interface. The complexation of Ba(2+) with (C153)(2)-K(22) is significantly slower than the complexation with C153-crown(O(4)).

Effects of nefiracetam on deficits in active avoidance response and hippocampal cholinergic and monoaminergic dysfunctions induced by AF64A in mice.

The effects of nefiracetam [DM-9384; N-(2,6-dimethyl-phenyl)-2-(2-oxo-pyrrolidinyl)acetamide] and of phosphatidylcholine on a step-up active avoidance response, locomotor activities and regional brain cholinergic and monoaminergic neurotransmitters in AF64A-treated mice were investigated. Intracerebroventricular (i.c.v.) injection of AF64A (ethylcholine mustard aziridinium ion; 8 nmol/ventricle) impaired acquisition and retention of the avoidance task, and increased vertical and horizontal locomotor activities. Regional levels of acetylcholine, noradrenaline, 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were significantly decreased and homovanillic acid (HVA) levels were increased in the hippocampus but not in the septum, cerebral cortex or striatum of AF64A-treated animals. Administration of nefiracetam (3 mg/kg, p.o.) twice daily for 9 days to AF64A-treated animals ameliorated the deficit in active avoidance response in addition to attenuating the increase in locomotor activities. In parallel with these behavioural effects, nefiracetam reversed AF64A-induced alterations in the hippocampal profiles of cholinergic and monoaminergic neurotransmitters and their metabolites. In contrast, administration of phosphatidylcholine (30 mg/kg, p.o.) twice daily for 9 days had no significant effect on the deficit in active avoidance response, despite significantly reversing the decrease in acetylcholine levels in the hippocampus. These results indicate that the effects of nefiracetam on AF64A-induced behavioural deficits are probably due to its ability to facilitate both cholinergic and monoaminergic neurotransmitter systems.

Effects of DM-9384, a cyclic derivative of GABA, on amnesia and decreases in GABAA and muscarinic receptors induced by cycloheximide.

The effects of N-(2,6-dimethyl-phenyl)-2-(2-oxo-1-pyrrolidinyl)-acetamide [DM-9384], a cyclic derivative of GABA, were investigated in the cycloheximide (CXM)-induced amnesia animal model using the passive avoidance task. Pre- and post-training and pre-retention test administration of DM-9384 attenuated the CXM-induced amnesia as indicated by prolongation of step-down latency. Aniracetam, another cyclic derivative of GABA, also showed antiamnesic effects. Scopolamine, a muscarinic ACh receptor antagonist, and the GABA antagonists, picrotoxin and bicuculline, all antagonized the antiamnesic effects of DM-9384. CXM decreased the number of GABAA and muscarinic ACh receptor binding sites. DM-9384 not only inhibited this effect but actually increased the latter. These results suggest that DM-9384 attenuates CXM-induced amnesia by interacting with GA-BAergic and AChergic neuronal systems and enhancing protein synthesis in the brain.

Effects of nefiracetam, DM-9384 on amnesia and decrease in choline acetyltransferase activity induced by cycloheximide.

The effects of nefiracetam, [N-(2,6-dimethyl-phenyl)-2-(2-oxo-pyrrolidinyl)acetamide, DM-9384], a cyclic derivative of GABA, were investigated in the cycloheximide (CXM)-induced amnesia animal model using the passive avoidance task. Pre-training administration of DM-9384 attenuated the CXM-induced amnesia as indicated by prolongation of step-down latency. It protected against CXM-induced inhibition of choline acetyltransferase activity in the cerebral cortex. These results suggest that DM-9384 attenuates CXM-induced amnesia by interacting with AChergic neuronal system and enhancing protein synthesis in the brain.