ALBERT

Description: Bacteriophage DNA packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage 29 has a unique ring of RNA (pRNA) that is an essential component of its motor, serving as a scaffold for the packaging ATPase. Previously, deletion of a three-base bulge (18-CCA-20) in the pRNA A-helix was shown to abolish packaging activity. Here, we solved the structure of this crucial bulge by nuclear magnetic resonance (NMR) using a 27mer RNA fragment containing the bulge (27b). The bulge actually involves five nucleotides (17-UCCA-20 and A100), as U17 and A100 are not base paired as predicted. Mutational analysis showed these newly identified bulge residues are important for DNA packaging. The bulge introduces a 33–35° bend in the helical axis, and inter-helical motion around this bend appears to be restricted. A model of the functional 120b pRNA was generated using a 27b NMR structure and the crystal structure of the 66b prohead-binding domain. Fitting this model into a cryo-EM map generated a pentameric pRNA structure; five helices projecting from the pRNA ring resemble an RNA claw. Biochemical analysis suggested that this shape is important for coordinated motor action required for DNA translocation.

Description: GPR34 is a G protein-coupled receptor belonging to the P2Y family. Here, we attempted to resolve conflicting reports about whether it is a functional lysophosphatidylserine (LysoPS) receptor. In HEK293 cells expressing human, mouse or rat GPR34 and Gα chimera between Gαq and Gαi1(Gq/i1), LysoPS quickly elevated intracellular Ca 2+ ion levels ([Ca 2+ ] i ). LysoPS also stimulated alkaline phosphatase (AP)-tagged TGFα (AP-TGFα) release in GPR34-expressing HEK293 cells and induced the migration of CHO-K1 cells expressing GPR34. Other lysophospholipids did not induce these actions. Replacement of the serine residue of LysoPS abolished the reactivity of LysoPS with GPR34, indicating that GPR34 strictly recognizes the serine head group of LysoPS. Recombinant phosphatidylserine-specific phospholipase A 1 (PS-PLA 1 ) that deacylates fatty acid at the sn -1 position of PS and produces 2-acyl-LysoPS, but not catalytically inactive mutant PS-PLA 1 , stimulated the release of AP-TGFα from GPR34-expressing cells. Consistent with the result, LysoPS was detected in the cells treated with wild-type PS-PLA 1 but not with the mutant PS-PLA 1 . PS treated with PLA 1 was much more effective at stimulating AP-TGFα release than PS treated with PLA 2 . In addition, migration-resistant 2-acyl-1-deoxy-LysoPS, a 2-acyl-LysoPS analogue, was much more potent than 1-acyl-2-deoxy-LysoPS. The present studies confirm that GPR34 is a cellular receptor for LysoPS, especially with a fatty acid at the sn -2 position.

Description: Calpain belongs to the superfamily of Ca 2+ -regulated cysteine proteases, which are indispensable to the regulation of various cellular functions. Of the 15 mammalian calpain isoforms, µ- and m-calpains are the best characterized. Both µ- and m-calpain are ubiquitously expressed and exist as heterodimers, containing a distinct 80-kDa catalytic subunit (CAPN1 and CAPN2, respectively) and the common, 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for use in structural and physiological studies, however Escherichia coli systems have proven incompatible with large-scale preparation of calpain, with the exception of rat m-calpain. Here, we have established a highly efficient method to purify active recombinant human m-calpain using an E. coli expression system at low temperature (22°C). This was achieved by co-expressing CAPN2 with a C-terminal histidine-tag, and CAPNS1, lacking the first Gly-repeated region at the N-terminal. After three sequential passes through a chromatographic column, ~5 mg of human m-calpain was homogenously purified from 1 l of E. coli culture. Proteins were stable for several months. This is the first report of efficient, large-scale purification of recombinant human m-calpain using an E. coli expression system.

Description: Intellectual disability (ID) is a highly prevalent disorder that affects 1–3% of the population. The Aristaless-related homeobox gene ( ARX ) is a frequently mutated X-linked ID gene and encodes a transcription factor indispensable for proper forebrain, testis and pancreas development. Polyalanine expansions account for over half of all mutations in ARX and clinically give rise to a spectrum of ID and seizures. To understand how the polyalanine expansions cause the clinical phenotype, we studied mouse models of the two most frequent polyalanine expansion mutations ( Arx (GCG)7 and Arx 432-455dup24 ). Neither model showed evidence of protein aggregates; however, a marked reduction of Arx protein abundance within the developing forebrain was striking. Examining the expression of known Arx target genes, we found a more prominent loss of Lmo1 repression in Arx (GCG7)/Y compared with Arx 432-455dup24/Y mice at 12.5 and 14.5 dpc, stages of peak neural proliferation and neurogenesis, respectively. Once neurogenesis concludes both mutant mouse models showed similar loss of Lmo1 repression. We propose that this temporal difference in the loss of Lmo1 repression may be one of the causes accounting for the phenotypic differences identified between the Arx (GCG)7 and Arx 432-455dup24 mouse models. It is yet to be determined what effect these mutations have on ARX protein in affected males in the human setting.

Description: In most bacteria, two tRNAs decode the four arginine CG N codons. One tRNA harboring a wobble inosine (tRNA Arg I CG ) reads the CG U , CG C and CG A codons, whereas a second tRNA harboring a wobble cytidine (tRNA Arg C CG ) reads the remaining CG G codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNA Arg C CG . This raises the question of how these organisms decode CG G codons. Examination of 36 Mollicute genomes for genes encoding tRNA Arg and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNA Arg A CG , capable of reading all CG N codons. This hypothesis was verified in Mycoplasma capricolum , which contains a small fraction of tRNA Arg A CG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNA Arg C CG and tadA , and then acquired a new tRNA Arg U CG. This permitted further tRNA Arg A CG mutations with tRNA Arg G CG or its disappearance, leaving a single tRNA Arg U CG to decode the four CG N codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy.

Description: Farnesoid X receptor (FXR), a pivotal factor maintaining bile acid homeostasis, has been recently shown to be a critical factor required for liver regeneration. The elucidation of the mechanism how FXR controls the proliferation of hepatocellular carcinoma cells is useful to establish the therapy for liver cancer. Here, we show that FXR plays a crucial role in the proliferation of human hepatocellular carcinoma cell line, HepG2, Huh7 and HLE. The treatment of HepG2 with FXR siRNA elevates the level of p16/INK4a expression resulting in the inhibition of cell proliferation. By contrast, FXR activation reduces p16/INK4a expression and stimulates the cell proliferation. The ectopic expression of the active form of Ras that causes strong activation of extracellular signal-regulated kinase (ERK) leads to the decrease in FXR expression, suggesting that FXR expression is negatively regulated via Ras/ERK pathway. The elevation of p16/INK4a expression and the inhibition of cell proliferation by FXR knockdown are also observed in Huh7 and HLE. In this study, we have suggested a novel mechanism by which hepatocellular carcinoma cell proliferation is regulated: FXR stimulates cell proliferation by suppressing the p16/INK4a expression, whereas Ras/ERK pathway down-regulates the FXR expression, leading to the suppressed cell proliferation in hepatocellular carcinoma cell lines.

Description: Recent progress in high-speed sequencing technology has revealed that tumors harbor novel mutations in a variety of genes including those for molecules involved in epigenetics and splicing, some of which were not categorized to previously thought malignancy-related genes. However, despite thorough identification of mutations in solid tumors and hematological malignancies, how these mutations induce cell transformation still remains elusive. In addition, each tumor usually contains multiple mutations or sometimes consists of multiple clones, which makes functional analysis difficult. Fifteen years ago, it was proposed that combination of two types of mutations induce acute leukemia; Class I mutations induce cell growth or inhibit apoptosis while class II mutations block differentiation, co-operating in inducing acute leukemia. This notion has been proven using a variety of mouse models, however most of recently found mutations are not typical class I/II mutations. Although some novel mutations have been found to functionally work as class I or II mutation in leukemogenesis, the classical class I/II theory seems to be too simple to explain the whole story. We here overview the molecular basis of hematological malignancies based on clinical and experimental results, and propose a new working hypothesis for leukemogenesis.

Description: Fin whales ( Balaenoptera physalus ) undergo seasonal migration in the Arctic Sea. Because their migration and distribution is likely affected by changes in global climate, we aimed to examine the migration timing of fin whales, and the relationship with prey availability within the oceanographic environment of the Arctic Sea, using passive and active acoustic monitoring methods. Automatic Underwater Sound Monitoring Systems were deployed in the southern Chukchi Sea from July 2012 to 2014 to determine the acoustic presence of fin whales. Furthermore, water temperature and salinity were recorded by a fixed data logger. An Acoustic Zooplankton Fish Profiler was additionally deployed to estimate prey abundance through backscattering strength. Sea ice concentrations were obtained by remote sensing data. Fin whale calls were automatically detected using a custom-made software, and the per cent of half-hours containing calls were counted. Fin whale calls were detected from 4 August to 20 October 2012 (78 d) and 25 July to 1 November 2013 (100 d). The extended period of acoustic presence of fin whales during 2013 when compared with 2012 is likely related to a longer ice-free period during 2013. Furthermore, generalized linear model analyses showed that half-hour periods containing calls increased with a rise in water temperature and zooplankton abundance during the initial call presence period, while they decreased with a decrease in water temperature and salinity during the end of the call presence period. Our results suggest that the rise in water temperature and zooplankton abundance affect the timing of migration of fin whales in a way that is consistent with the expansion of their suitable habitats and the extension of their presence in the Arctic Sea.

Description: Hydraulic fracturing plays a vital role in the development of unconventional energy resources, such as shale gas/oil and enhanced geothermal systems to increase the permeability of tight rocks. In this study, we conducted hydraulic fracturing experiments in a laboratory using carbonate-rich outcrop samples of Eagle Ford shale from the United States. We used a thermosetting acrylic resin containing a fluorescent compound as a fracturing fluid. Immediately after fracturing, the liquid resin penetrated in the fractured blocks was hardened by applying heat. Then, the crack was viewed under UV irradiation, where the fluorescent resin allowed the induced fracture to be clearly observed, indicating the formation of simple, thin bi-wing planar fractures. We observed the detailed structure of the fractures from microscopy of thin cross-sections, and found that their complexity and width varied with the distance from the wellbore. This likely reflects the change in the stress state around the tip of the growing fracture. The interaction between fractures and constituent grains/other inclusions (e.g. organic substances) seemed to increase the complexity of the fractures, which may contribute to the efficient production of shale gas/oil via hydraulic fracturing. We first detected acoustic emission (AE) signals several seconds before the peak fluid pressure was observed, and the active region gradually migrated along the microscopically observed fracture with increasing magnitude. Immediately after the peak pressure was observed, the fluid pressure dropped suddenly (breakdown) with large seismic waves that were probably radiated by dynamic propagation of the fracture; thereafter, the AE activity stopped. We applied moment tensor inversion for the obtained AE events by carefully correcting the AE sensor characteristics. Almost all of the solutions corresponded to tensile events that had a crack plane along the maximum compression axis, as would be expected based on the conventional theory of hydraulic fracturing. Such domination of tensile events has not been reported in previous studies based on laboratory/in situ experiments, where shear events were often dominant. The extreme domination of the tensile events in the present study is possibly a result of the use of rock samples without any significant pre-existing cracks. Our experiments revealed the fracturing behaviour and accompanying seismic activities of very tight rocks in detail, which will be helpful to our understanding of fracturing behaviour in shale gas/oil resource production.