Expression screening of 17q12-21 amplicon reveals GRB7 as an ERBB2-dependent oncogene

Gene amplification is a major genetic alteration in human cancers. Amplicons, amplified genomic regions, are believed to contain "driver" genes responsible for tumorigenesis. However, the significance of co-amplified genes has not been extensively studied. We have established an integrated analysis system of amplicons using retrovirus-mediated gene transfer coupled with a human full-length cDNA set. Applying this system to 17q12-21 amplicon observed in breast cancer, we identified GRB7 as a context-dependent oncogene, which modulates the ERBB2 signaling pathway through enhanced phosphorylation of ERBB2 and Akt. Our work provides an insight into the biological significance of gene amplification in human cancers.     

Structural insights into unique substrate selectivity of Thermoplasma acidophilum D-aldohexose dehydrogenase

The d-aldohexose dehydrogenase from the thermoacidophilic archaea Thermoplasma acidophilum (AldT) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and catalyzes the oxidation of several monosaccharides with a preference for NAD⁺ rather than NADP⁺ as a cofactor. It has been found that AldT is a unique enzyme that exhibits the highest dehydrogenase activity against d-mannose. Here, we describe the crystal structures of AldT in ligand-free form, in complex with NADH, and in complex with the substrate d-mannose, at 2.1 Å, 1.65 Å, and 1.6 Å resolution, respectively. The AldT subunit forms a typical SDR fold with an unexpectedly long C-terminal tail and assembles into an intertwined tetramer. The d-mannose complex structure reveals that Glu84 interacts with the axial C2 hydroxyl group of the bound d-mannose. Structural comparison with Bacillus megaterium glucose dehydrogenase (BmGlcDH) suggests that the conformation of the glutamate side-chain is crucial for discrimination between d-mannose and its C2 epimer d-glucose, and the conformation of the glutamate side-chain depends on the spatial arrangement of nearby hydrophobic residues that do not directly interact with the substrate. Elucidation of the d-mannose recognition mechanism of AldT further provides structural insights into the unique substrate selectivity of AldT. Finally, we show that the extended C-terminal tail completely shuts the substrate-binding pocket of the neighboring subunit both in the presence and absence of substrate. The elaborate inter-subunit interactions between the C-terminal tail and the entrance of the substrate-binding pocket imply that the tail may play a pivotal role in the enzyme activity.     

Tetratricopeptide repeat proteins Tom70 and Tom71 mediate yeast mitochondrial morphogenesis

The maintenance of correct mitochondrial shape requires numerous proteins that act on the surface or inside of the organelle. Although the soluble F-box protein Mfb1 was recently found to associate peripherally with mitochondria and to regulate organelle connectivity in budding yeast, how it localizes to mitochondria is unknown. Here, we show that two tetratricopeptide repeat proteins-the general preprotein import receptor Tom70 (a component of translocase of the outer membrane) and its paralogue Tom71-are required for Mfb1 mitochondrial localization. Mitochondria in cells lacking Tom70 and Tom71 form short tubules and aggregates, aberrant morphologies similar to those observed in the mfb1-null mutant. In addition, Mfb1 interacts with Tom71 in vivo, and binds to mitochondria through Tom70 in vitro. Our data indicate an unexpected role for Tom70 in recruitment of soluble proteins to the mitochondrial surface, and indicate that Tom71 has a specialized role in Mfb1-mediated mitochondrial morphogenesis.     

Role of RecJ-like protein with 5'-3' exonuclease activity in oligo(deoxy)nucleotide degradation

RecJ-like proteins belonging to the DHH family have been proposed to function as oligoribonucleases and 3'-phosphoadenosine 5'-phosphate (pAp) phosphatases in bacteria and archaea, which do not have Orn (oligoribonuclease) and CysQ (pAp phosphatase) homologs. In this study, we analyzed the biochemical and physiological characterization of the RecJ-like protein TTHA0118 from Thermus thermophilus HB8. TTHA0118 had high enzymatic activity as an oligodeoxyribonucleotide- and oligoribonucleotide-specific exonuclease and as pAp phosphatase. The polarity of degradation was 5' to 3', in contrast to previous reports about Bacillus subtilis NrnA, a RecJ-like protein. TTHA0118 preferentially hydrolyzed short oligodeoxyribonucleotides and oligoribonucleotides, whereas the RecJ exonuclease from T. thermophilus HB8 showed no such length dependence on oligodeoxyribonucleotide substrates. An insertion mutation of the ttha0118 gene led to growth reduction in minimum essential medium. Added 5'-mononucleotides, nucleosides, and cysteine increased growth of the ttha0118 mutant in minimum essential medium. The RecJ-like protein Mpn140 from Mycoplasma pneumoniae M129, which cannot synthesize nucleic acid precursors de novo, showed similar biochemical features to TTHA0118. Furthermore, B. subtilis NrnA also hydrolyzed oligo(deoxy)ribonucleotides in a 5'-3' direction. These results suggested that these RecJ-like proteins act in recycling short oligonucleotides to mononucleotides and in controlling pAp concentrations in vivo.     

Metabolic Quotient Measured by Free-Water Method in Six Enclosures with Different Silver Carp Densities

Quasi-continuous DO and pH measurements (total 47 days) were conducted during enclosure experiments (6 enclosures; 5 × 5 × 2.5 m), in which a biomass gradient of silver carp was created. After subtracting the air–water exchanges of O₂ and CO₂, the chemical and biochemical changes in DO (dissolved oxygen) and DIC (dissolved inorganic carbon) were estimated in order to evaluate MQ (metabolic quotient: DO change divided by DIC change) at intervals of 1 hour. By removing small absolute changes below the threshold value (0.01 mM h^?1), the averaged values of the 24 MQ means for the respective 1-hour periods ranged from 0.96 to 1.20 in the six enclosures. Because the MQs in the daytime inversely correlated well with the ratio of NH⁺ ₄–N to (NH⁺ ₄–N + NO^? ₃–N), not the ecosystems, i.e., density of fish, community structure of zooplankton and phytoplankton, but the form of nitrogen uptaken for primary production principally determined the MQs. The higher MQs observed in the daytime compared with the nighttime (from 14% to 21% except 3% for one enclosure) could not be explained by the denitrification and/or dissolution of CaCO₃ in the sediments, therefore suggesting the selectively faster decomposition of part of the organic matter provided through primary production, in other words, an accumulation of another part of the organic matter in the diurnal and/or daily time scale.     

Distinct tRNA modifications in the thermo-acidophilic archaeon, Thermoplasma acidophilum

Thermoplasma acidophilum is a thermo-acidophilic archaeon. We purified tRNA^Leu (UAG) from T. acidophilum using a solid-phase DNA probe method and determined the RNA sequence after determining via nucleoside analysis and m⁷G-specific aniline cleavage because it has been reported that T. acidophilum tRNA contains m⁷G, which is generally not found in archaeal tRNAs. RNA sequencing and liquid chromatography–mass spectrometry revealed that the m⁷G modification exists at a novel position 49. Furthermore, we found several distinct modifications, which have not previously been found in archaeal tRNA, such as 4-thiouridine9, archaeosine13 and 5-carbamoylmethyuridine34. The related tRNA modification enzymes and their genes are discussed.