Characterization of thermostable FMN-dependent NADH azoreductase from the moderate thermophile Geobacillus stearothermophilus

The gene encoding an FMN-dependent NADH azoreductase, AzrG, from thermophilic Geobacillus stearothermophilus was cloned and functionally expressed in recombinant Escherichia coli. Purified recombinant AzrG is a homodimer of 23 kDa and bore FMN as a flavin cofactor. The optimal temperature of AzrG was 85 °C for the degradation of Methyl Red (MR). AzrG remained active for 1 h at 65 °C and for 1 month at 30 °C, demonstrating both superior thermostability and long-term stability of the enzyme. AzrG efficiently decolorized MR, Ethyl Red at 30 °C. Furthermore, the enzyme exhibited a wide-range of degrading activity towards several tenacious azo dyes, such as Acid Red 88, Orange I, and Congo Red. These results suggested the sustainable utilization of G. stearothermophilus as an azo-degrading strain for AzrG carrying whole-cell wastewater treatments for azo pollutants under high temperature conditions.     

Gonadotropic regulation of circadian clockwork in rat granulosa cells

The circadian clock is responsible for the generation of circadian rhythms in hormonal secretion and metabolism. These peripheral clocks could be reset by various cues in order to adapt to environmental variations. The ovary can be characterized as having highly dynamic physiology regulated by gonadotropins. Here, we aimed to address the status of circadian clock in the ovary, and to explore how gonadotropins could regulate clockwork in granulosa cells (GCs). To this end, we mainly utilized the immunohistochemistry, RT-PCR, and real-time monitoring of gene expression methods. PER1 protein was constantly abundant across the daily cycle in the GCs of immature ovaries. In contrast, PER1 protein level was obviously cyclic through the circadian cycle in the luteal cells of pubertal ovaries. In addition, both FSH and LH induced Per1 expression in cultured immature and mature GCs, respectively. The promoter analysis revealed that the Per1 expression was mediated by the cAMP response element binding protein. In cultured transgenic GCs, both FSH and LH also induced the circadian oscillation of Per2. However, the Per2 oscillation promoted by FSH quickly dampened within only one cycle, whereas the Per2 oscillation promoted by LH was persistently maintained. Collectively, these findings strongly suggest that both FSH and LH play an important role in regulating circadian clock in the ovary; however, they might exert differential actions on the clockwork in vivo due to each specific role within ovarian physiology.     

Improvement of base selectivity and binding affinity by controlling hydrogen bonding motifs between nucleobases and isoxanthopterin: application to the detection of T/C mutation

At an abasic site in an oligo-DNA duplex, isoxanthopterin (IX)(dagger) can bind to thymine (T) and cytosine (C) with strong affinity compared to adenine and guanine, but the base selectivity for T against C is moderate. In order to improve both binding affinity and base selectivity for T against C, a methyl group is introduced to IX, which is known as 3-methyl isoxanthopterin (3-MIX),(dagger) by which binding affinity for C is expected to decrease. Indeed, 3-MIX specifically binds to T more strongly than IX and loses its binding affinity for C. The improved binding ability of 3-MIX for T would be suitable for the practical use in SNP typing related to T.     

Expression of antimicrobial peptide genes encoding Enbocin and Gloverin isoforms in the silkworm, Bombyx mori

Antimicrobial peptides, Enbocin and Gloverin isoforms from the silkworm Bombyx mori, were analyzed for expression of these peptide genes. Tissue-specific expression of Enbocin and Bmgloverin isoform genes was observed mainly in the fat body upon injection of Escherichia coli. Peptidoglycan and lipopolysaccharide triggered expression of these genes in vivo. On the other hand, lipid A activated Bmgloverin isoform genes but not Enbocin isoform genes. These results illustrate the fact that expression of Enbocin and Bmgloverin isoform genes is inducible by bacteria and that the effects of bacterial cell wall components on the activation of these peptide genes are not necessarily the same. In addition, selective activation of the Enbocin2, Bmgloverin2, and Bmgloverin4 genes by BmRelB rather than BmRelA was observed, providing additional evidence for the occurrence of selective activation of antimicrobial peptide genes by a Rel protein. These results suggest complex regulatory mechanisms in insect antimicrobial peptide genes by bacterial cell wall components.     

NIRF constitutes a nodal point in the cell cycle network and is a candidate tumor suppressor

In biological networks, a small number of “hub” proteins play critical roles in the network integrity and functions. The cell cycle network orchestrates versatile cellular functions through interactions between many signaling modules, whose defects impair diverse cellular processes, often leading to cancer. However, the network architecture and molecular basis that ensure proper coordination between distinct modules are unclear. Here, we show that the ubiquitin ligase NIRF (also known as UHRF2), which induces G₁ arrest, interacts with multiple cell cycle proteins including cyclins (A2, B1, D1 and E1), p53 and pRB, and ubiquitinates cyclins D1 and E1. Consistent with its versatility, a bioinformatic network analysis demonstrated that NIRF is an intermodular hub protein that is responsible for the coordination of multiple network modules. Notably, intermodular hubs are frequently associated with oncogenesis. Indeed, we detected loss of heterozygosity of the NIRF gene in several kinds of tumors. When a cancer outlier profile analysis was applied to the Oncomine database, loss of the NIRF gene was found at statistically significant levels in diverse tumors. Importantly, a recurrent microdeletion targeting NIRF was observed in non-small cell lung carcinoma. Furthermore, NIRF is immediately adjacent to the single nucleotide polymorphism rs719725, which is reportedly associated with the risk of colorectal cancer. These observations suggest that NIRF occupies a prominent position within the cell cycle network, and is a strong candidate for a tumor suppressor whose aberration contributes to the pathogenesis of diverse malignancies.Key words: NIRF, UHRF2, cell cycle network, systems biology, ubiquitin ligase, COPA, tumor suppressor, glioblastoma, non-small cell lung carcinoma, rs719725     

Finding of Torymus koreanus (Hymenoptera: Torymidae) attacking Dryocosmus kuriphilus (Hymenoptera: Cynipidae) in Japan

Torymus koreanus (Hymenoptera: Torymidae) was reared from galls of Dryocosmus kuriphilus (Hymenoptera: Cynipidae) for the first time in Japan. We here report morphological features and partial mtDNA sequencing data of T. koreanus. Torymus koreanus has several common characteristics with species of the cyaneus group defined by Zavada (2003 ). According to the key to species groups defined by Graham and Gijswijt (1998 ), it does not belong to any species group because of the entire posterior margin of metasomal tergum five.