In situ observation of spherical DNA assembly in water and the controlled release of bound dyes

Three strands of 30-mer oligodeoxyribonucleotides (ODNs) were designed to form three-way junctions that possess self-complementary sticky ends. The morphology of self-assembled ODNs in water was observed in situ by confocal laser scanning fluorescence microscopy. The three-way junctions self-assembled into spherical assemblies, in accordance with transmission and scanning electron microscopy. The size of nucleospheres was in the range of several tens of nanometers to micrometers, which varied depending on the concentration of ODNs and added salts. Fluorescence images of spherical ODN assemblies suggested that the nucleospheres possess multiwalled structures. The fluorescence of sodium 1-anilinonaphthalene-8-sulfonate in the presence of nucleospheres revealed that the interior of nucleospheres possesses polarity corresponding to that between methanol and ethanol. A dye-inclusion experiment showed that cationic and even anionic dyes were adsorbed to the interior of the nucleospheres. The dye-included nucleospheres released dyes by thermal dissociation or digestion of the constituent ODNs.     

ELMOD2 is anchored to lipid droplets by palmitoylation and regulates adipocyte triglyceride lipase recruitment

Adipocyte triglyceride lipase (ATGL) is the major enzyme involved in the hydrolysis of triglycerides. The Arf1–coat protein complex I (COPI) machinery is known to be engaged in the recruitment of ATGL to lipid droplets (LDs), but the regulatory mechanism has not been clarified. In the present study, we found that ELMOD2, a putative noncanonical Arf–GTPase activating protein (GAP) localizing in LDs, plays an important role in controlling ATGL transport to LDs. We showed that knockdown of ELMOD2 by RNA interference induced an increase in the amount of ATGL existing in LDs and decreased the total cellular triglycerides. These effects of ELMOD2 knockdown were canceled by transfection of small interfering RNA-resistant cDNA of wild-type ELMOD2 but not by that of mutated ELMOD2 lacking the Arf-GAP activity. ELMOD2 was distributed in the endoplasmic reticulum and mitochondria as well as in LDs, but palmitoylation was required only for distribution to LDs. An ELMOD2 mutant deficient in palmitoylation failed to reconstitute the ATGL transport after the ELMOD2 knockdown, indicating that distribution in LDs is indispensable to the functionality of ELMOD2. These results indicate that ELMOD2 regulates ATGL transport and cellular lipid metabolism by modulating the Arf1-COPI activity in LDs.     

Chemical identification and ethological function of soldier-specific secretion in Japanese subterranean termite Reticulitermes speratus (Rhinotermitidae)

We identified the soldier-specific compounds in the Japanese subterranean termite, Reticulitermes speratus, to clarify their ethological roles. Silica gel column chromatography separated one major soldier-specific compound in the hexane fraction accounting for 70-80% of the total amount of the fraction, while cuticular hydrocarbons constituted the rest. We identified the compound as β-selinene by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Comparative GC analyses of the major exocrine glands detected the compound in the soldier's frontal gland. Both soldiers and workers made aggregation to the hexane fraction, as well as to the crushed heads and head extract of the soldiers. They did not aggregate to cuticular hydrocarbons, making it likely that β-selinene was the aggregation pheromone in this species. The opportunistic predator of this termite, Lasius japonicus, was also attracted to the compounds. The ant workers, therefore, would use the termite aggregation pheromone as a kairomone for hunting them.     

The Effect of the B Subunit of Cholera Toxin on the Action of Nerve Growth Factor on PC12 Cells

Abstract: Exogenous gangliosides, especially ganglioside GM1 (GM1), seem to potentiate the action of nerve growth factor (NGF). We have examined the possible regulation of the NGF signaling pathway in PC12 cells by the B subunit of cholera toxin (CTB), which binds to endogenous GM1 specifically and with a high affinity. CTB treatment (1 μg/ml) enhanced NGF-induced neurite outgrowth from PC12 cells, NGF-induced activation of ribosomal protein S6 kinase, and NGF-induced stimulation of trk phosphorylation. CTB plus NGF also caused a greater inhibition of [³H]-thymidine incorporation into DNA than did NGF alone. These enhancing effects of CTB were blocked by the presence of cytochalasin B in the culture medium but were not affected by the presence of colchicine or by the depletion of Ca²⁺ in the medium. ¹²⁵I-NGF binding experiments revealed that CTB treatment did not affect the specific binding of NGF to the cells. These results strongly suggest that the binding of cell surface GM1 by CTB modulates the pathway of intracellular signaling initiated by NGF and that the association of CTB with a cytoskeletal component is essential for these effects.     

A new type of gene‐disruption cassette with a rescue gene for Pichia pastoris

Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene‐targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene‐disruption system with a rescue gene using an inducible Cre/mutant–loxP system. With only short homology regions, the gene‐disruption cassette of the system replaces its target–gene locus containing a mutation with a compensatory rescue gene. As the cassette contains the AOX1 promoter‐driven Cre gene, when targeted strains are grown on media containing methanol, the DNA fragment, i.e., the marker, rescue and Cre genes, between the mutant‐loxP sequences in the cassette is excised, leaving only the remaining mutant‐loxP sequence in the genome, and consequently a target gene‐disrupted mutant can be isolated. The system was initially validated on ADE2 gene disruption, where the disruption can easily be detected by color‐change of the colonies. Then, the system was applied for knocking‐out URA3 and OCH1 genes, reported to be difficult to accomplish by conventional gene‐targeting methods. All three gene‐disruption cassettes with their rescue genes replaced their target genes, and the Cre/mutant–loxP system worked well to successfully isolate their knock‐out mutants. This study identified a new gene‐disruption system that could be used to effectively and strategically knock out genes of interest, especially whose deletion is detrimental to growth, without using special strains, e.g., deficient in nonhomologous end‐joining, in P. pastoris. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1201–1208, 2017     

Preferential paternal origin of microdeletions caused by prezygotic chromosome or chromatid rearrangements in Sotos syndrome

Sotos syndrome (SoS) is characterized by pre- and postnatal overgrowth with advanced bone age; a dysmorphic face with macrocephaly and pointed chin; large hands and feet; mental retardation; and possible susceptibility to tumors. It has been shown that the major cause of SoS is haploinsufficiency of the NSD1 gene at 5q35, because the majority of patients had either a common microdeletion including NSD1 or a truncated type of point mutation in NSD1. In the present study, we traced the parental origin of the microdeletions in 26 patients with SoS by the use of 16 microsatellite markers at or flanking the commonly deleted region. Deletions in 18 of the 20 informative cases occurred in the paternally derived chromosome 5, whereas those in the maternally derived chromosome were found in only two cases. Haplotyping analysis of the marker loci revealed that the paternal deletion in five of seven informative cases and the maternal deletion in one case arose through an intrachromosomal rearrangement, and two other cases of the paternal deletion involved an interchromosomal event, suggesting that the common microdeletion observed in SoS did not occur through a uniform mechanism but preferentially arose prezygotically.     

The relationship between muscle deoxygenation and activation in different muscles of the quadriceps during cycle ramp exercise

The relationship between muscle deoxygenation and activation was examined in three different muscles of the quadriceps during cycling ramp exercise. Seven young male adults (24 ± 3 yr; mean ± SD) pedaled at 60 rpm to exhaustion, with a work rate (WR) increase of 20 W/min. Pulmonary oxygen uptake was measured breath-by-breath, while muscle deoxygenation (HHb) and activity were measured by time-resolved near-infrared spectroscopy (NIRS) and surface electromyography (EMG), respectively, at the vastus lateralis (VL), rectus femoris (RF), and vastus medialis (VM). Muscle deoxygenation was corrected for adipose tissue thickness and normalized to the amplitude of the HHb response, while EMG signals were integrated (iEMG) and normalized to the maximum iEMG determined from maximal voluntary contractions. Muscle deoxygenation and activation were then plotted as a percentage of maximal work rate (%WR(max)). The HHb response for all three muscle groups was fitted by a sigmoid function, which was determined as the best fitting model. The c/d parameter for the sigmoid fit (representing the %WR(max) at 50% of the total amplitude of the HHb response) was similar between VL (47 ± 12% WR(max)) and VM (43 ± 11% WR(max)), yet greater (P < 0.05) for RF (65 ± 13% WR(max)), demonstrating a "right shift" of the HHb response compared with VL and VM. The iEMG also showed that muscle activation of the RF muscle was lower (P < 0.05) compared with VL and VM throughout the majority of the ramp exercise, which may explain the different HHb response in RF. Therefore, these data suggest that the sigmoid function can be used to model the HHb response in different muscles of the quadriceps; however, simultaneous measures of muscle activation are also needed for the HHb response to be properly interpreted during cycle ramp exercise.     

Ventilator-induced lung injury is reduced in transgenic mice that overexpress endothelial nitric oxide synthase

Although mechanical ventilation (MV) is an important supportive strategy for patients with acute respiratory distress syndrome, MV itself can cause a type of acute lung damage termed ventilator-induced lung injury (VILI). Because nitric oxide (NO) has been reported to play roles in the pathogenesis of acute lung injury, the present study explores the effects on VILI of NO derived from chronically overexpressed endothelial nitric oxide synthase (eNOS). Anesthetized eNOS-transgenic (Tg) and wild-type (WT) C57BL/6 mice were ventilated at high or low tidal volume (Vt; 20 or 7 ml/kg, respectively) for 4 h. After MV, lung damage, including neutrophil infiltration, water leakage, and cytokine concentration in bronchoalveolar lavage fluid (BALF) and plasma, was evaluated. Some mice were given N(omega)-nitro-L-arginine methyl ester (L-NAME), a potent NOS inhibitor, via drinking water (1 mg/ml) for 1 wk before MV. Histological analysis revealed that high Vt ventilation caused severe VILI, whereas low Vt ventilation caused minimal VILI. Under high Vt conditions, neutrophil infiltration and lung water content were significantly attenuated in eNOS-Tg mice compared with WT animals. The concentrations of macrophage inflammatory protein-2 in BALF and plasma, as well as plasma tumor necrosis factor-alpha and monocyte chemoattractant protein-1, also were decreased in eNOS-Tg mice. L-NAME abrogated the beneficial effect of eNOS overexpression. In conclusion, chronic eNOS overexpression may protect the lung from VILI by inhibiting the production of inflammatory chemokines and cytokines that are associated with neutrophil infiltration into the air space.     

Synthesis and anti-influenza virus activity of dihydrofuran-fused perhydrophenanthrenes with a benzyloxy-type side-chain

As one of our ongoing research project concerning development of a novel anti-influenza virus agent, dihydrofuran-fused perhydrophenanthrenes were derivatized by means of Williamson ether synthesis and Suzuki–Miyaura cross coupling reactions. Newly synthesized compounds were subjected to evaluation of anti-influenza virus activity using influenza A/Aichi/2/68 (H3N2 subtype) virus strain by a plaque titration method. These investigations revealed that incorporation of benzyl-type ether substituents was effective for exerting the inhibition activity of influenza virus proliferation.