Neverland is an evolutionally conserved Rieske-domain protein that is essential for ecdysone synthesis and insect growth

Steroid hormones mediate a wide variety of developmental and physiological events in multicellular organisms. During larval and pupal stages of insects, the principal steroid hormone is ecdysone, which is synthesized in the prothoracic gland (PG) and plays a central role in the control of development. Although many studies have revealed the biochemical features of ecdysone synthesis in the PG, many aspects of this pathway have remained unclear at the molecular level. We describe the neverland (nvd) gene, which encodes an oxygenase-like protein with a Rieske electron carrier domain, from the silkworm Bombyx mori and the fruitfly Drosophila melanogaster. nvd is expressed specifically in tissues that synthesize ecdysone, such as the PG. We also show that loss of nvd function in the PG causes arrest of both molting and growth during Drosophila development. Furthermore, the phenotype is rescued by application of 20-hydroxyecdysone or the precursor 7-dehydrocholesterol. Given that the nvd family is evolutionally conserved, these results suggest that Nvd is an essential regulator of cholesterol metabolism or trafficking in steroid synthesis across animal phyla.     

Application of 2-amino-6-vinylpurine as an efficient agent for conjugation of oligonucleotides

Attempts have been made to conjugate a variety of molecules with oligonucleotides to achieve useful functions. In this study, we have established a new efficient method for post-synthetic conjugation of oligonucleotides with the use of the 2-amino-6-vinylpurine nucleoside. Amino nucleophiles form the corresponding conjugates under acidic conditions, whereas thiol nucleophiles reacted efficiently under alkaline conditions. Thus, glutathione and HS-Cys-(Arg)8 without protecting groups were efficiently conjugated to the 2-amino-6-vinylpurine-bearing ODN under alkaline conditions. The use of 2-amino-6-vinylpurine as an agent for conjugation is advantageous in that it is stable during the reaction and may be applied to conjugation of ODNs with multiple functional molecules.     

Photobiocatalyzed asymmetric reduction of ketones using Chlorella sp. MK201

Aromatic ketones were reduced using suspension culture of Chlorella sp. MK201 under fluorescent light illumination producing the corresponding chiral alcohols in high yields with excellent enantiomeric excess (ee). For example, 2′,3′,4′,5′,6′-pentafluoroacetophenone at 0.25?mg/ml was converted to the corresponding (S)-alcohol in 80?% yield with?>99?% ee by 1?mg dry wt of Chlorella/ml in 12?h illumination (2,000 lux).     

Differential roles for parietal and occipital cortices in visual working memory

Visual working memory (VWM) is known as a highly capacity-limited cognitive system that can hold 3-4 items. Recent studies have demonstrated that activity in the intraparietal sulcus (IPS) and occipital cortices correlates with the number of representations held in VWM. However, differences among those regions are poorly understood, particularly when task-irrelevant items are to be ignored. The present fMRI-based study investigated whether memory load-sensitive regions such as the IPS and occipital cortices respond differently to task-relevant information. Using a change detection task in which participants are required to remember pre-specified targets, here we show that while the IPS exhibited comparable responses to both targets and distractors, the dorsal occipital cortex manifested significantly weaker responses to an array containing distractors than to an array containing only targets, despite that the number of objects presented was the same for the two arrays. These results suggest that parietal and occipital cortices engage differently in distractor processing and that the dorsal occipital, rather than parietal, activity appears to reflect output of stimulus filtering and selection based on behavioral relevance.     

Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several "moltinism" mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval-larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval-pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH-deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.     

Permanence induced by life-cycle resonances: the periodical cicada problem

Periodical cicadas are known for their unusually long life cycle for insects and their prime periodicity of either 13 or 17 years. One of the explanations for the prime periodicity is that the prime periods are selected to prevent cicadas from resonating with predators with submultiple periods. This paper considers this hypothesis by investigating a population model for periodical predator and prey. The study shows that if the periods of the two periodical species are not coprime, then the predator cannot resist the invasion of the prey. On the other hand, if the periods are coprime, then the predator can resist the invasion of the prey. It is also shown that if the periods are not coprime, then the life-cycle resonance can induce a permanent system, in which no cohorts are missing in both populations. On the other hand, if the periods are coprime, then the system cannot be permanent.     

Nuclear marginalization of host cell chromatin associated with expansion of two discrete virus-induced subnuclear compartments during baculovirus infection

Chromatin structure is strictly regulated during the cell cycle. DNA viruses occasionally disturb the spatial organization of the host cell chromatin due to formation of the viral DNA replication compartment. To examine chromatin behavior in baculovirus-infected cells, we constructed recombinant plasmids expressing fluorescent protein-tagged histone H4 molecules and visualized the intracellular localization of chromatin by their transient expression in live infected cells. Similar to other DNA viruses, the baculovirus Bombyx mori nucleopolyhedrovirus induced marginal relocation of chromatin within the nuclei of BmN cells, simultaneously with expansion of the viral DNA replication compartment, the virogenic stroma (VS). In the late stage of infection, however, the peristromal region (PR), another virus-induced subnuclear compartment, was also excluded from the chromatin-localizing area. Provided that late-gene products such as PR proteins (e.g., envelope proteins of the occlusion-derived virus) were expressed, blockage of viral DNA synthesis failed to inhibit chromatin relocation, despite abrogation of VS expansion. Instead, chromatin became marginalized concomitantly with PR expansion, suggesting that the PR contributes directly to chromatin replacement. In addition, chromatin was excluded from relatively large subnuclear structures that were induced in uninfected cells by cotransfection with four baculovirus genes, ie1, lef3, p143, and hr. Omission of any of the four genes, however, failed to result in formation of the large structures or chromatin exclusion. This correlation between compartmentalization and chromatin exclusion suggests the possibility that a chromatin-exclusive property of viral molecules, at least in part, supports nuclear compartmentalization of virus-infected cells.     

p66shc inhibits pro-survival epidermal growth factor receptor/ERK signaling during severe oxidative stress in mouse renal proximal tubule cells

The fully executed epidermal growth factor receptor (EGFR)/Ras/MEK/ERK pathway serves a pro-survival role in renal epithelia under moderate oxidative stress. We and others have demonstrated that during severe oxidative stress, however, the activated EGFR is disconnected from ERK activation in cultured renal proximal tubule cells and also in renal proximal tubules after ischemia/reperfusion injury, resulting in necrotic death. Studies have shown that the tyrosine-phosphorylated p46/52 isoforms of the ShcA family of adaptor proteins connect the activated EGFR to activation of Ras and ERK, whereas the p66(shc) isoform can inhibit this p46/52(shc) function. Here, we determined that severe oxidative stress (after a brief period of activation) terminates activation of the Ras/MEK/ERK pathway, which coincides with ERK/JNK-dependent Ser(36) phosphorylation of p66(shc). Isoform-specific knockdown of p66(shc) or mutation of Ser(36) to Ala, but not to Asp, attenuated severe oxidative stress-mediated ERK inhibition and cell death in vitro. Also, severe oxidative stress (unlike ligand stimulation and moderate oxidative stress, both of which support survival) increased binding of p66(shc) to the activated EGFR and Grb2. This binding dissociated the SOS1 adaptor protein from the EGFR-recruited signaling complex, leading to termination of Ras/MEK/ERK activation. Notably, Ser(36) phosphorylation of p66(shc) and its increased binding to the EGFR also occurred in the kidney after ischemia/reperfusion injury in vivo. At the same time, SOS1 binding to the EGFR declined, similar to the in vitro findings. Thus, the mechanism we propose in vitro offers a means to ameliorate oxidative stress-induced cell injury by either inhibiting Ser(36) phosphorylation of p66(shc) or knocking down p66(shc) expression in vivo.     

Intracellular changes of metal elements by fucoidan extracted from brown seaweed (Cladosiphon okamuranus)

This study was undertaken to elucidate the intracellular changes of metal elements after the administration of fucoidan extracted from Cladosiphon okamuranus. TRL1215 cells (normal rat liver cell line) were treated with 0, 0.1, or 1.0 mg/ml fucoidan and incubated in 5% CO2 at 37 degrees C. The cellular levels of Mg, Al, Fe, and Zn were significantly increased in the 1.0 mg/ml fucoidan-treated cells compared to those of the 0.1 mg/ml fucoidan-treated cells and the control. Next, TRL1215 cells were cultured on Mylar film overnight. At 24 h after 5-bromo-2'-deoxyuridine dosing, 0, 0.1, or 1.0 mg/ml fucoidan was treated for 9 h. The cellular distribution of elements was analyzed using in-air micro-micro-particle induced X-ray emission. The X-ray spectra showed that yields of Al, Mg, and Zn were high in order of the 1.0 mg/ml fucoidan-treated sample, the 0.1 mg/ml fucoidan-treated sample, and the control. Fe yield was mildly increased by fucoidan administration. In fucoidan-treated cells, the focal accumulation of Br was correlated spatially with phosphorous-rich region, suggesting that Br was localized within the nucleus. Al distribution provided a spatial association with Br map. These data suggest that fucoidan increases the accumulations of Al, Mg, Fe, and Zn in normal rat hepatocytes, and fucoidan-binding Al is postulated to be transferred into the nucleus.