Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in breast cancer

While the small GTPase Rac1 and its effectors are well-established mediators of mitogenic and motile signaling by tyrosine kinase receptors and have been implicated in breast tumorigenesis, little is known regarding the exchange factors (Rac-GEFs) that mediate ErbB receptor responses. Here, we identify the PIP(3)-Gβγ-dependent Rac-GEF P-Rex1 as an essential mediator of Rac1 activation, motility, cell growth, and tumorigenesis driven by ErbB receptors in breast cancer cells. Notably, activation of P-Rex1 in breast cancer cells requires the convergence of inputs from ErbB receptors and a Gβγ- and PI3Kγ-dependent pathway. Moreover, we identified the GPCR CXCR4 as a crucial mediator of P-Rex1/Rac1 activation in response to ErbB ligands. P-Rex1 is highly overexpressed in human breast cancers and their derived cell lines, particularly those with high ErbB2 and ER expression. In addition to the prognostic and therapeutic implications, our findings reveal an ErbB effector pathway that is crucial for breast cancer progression.     

Role of hydrogen generation by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in the oral cavity

Some gastrointestinal bacteria synthesize hydrogen (H₂) by fermentation. Despite the presence of bactericidal factors in human saliva, a large number of bacteria also live in the oral cavity. It has never been shown that oral bacteria also produce H₂ or what role H₂ might play in the oral cavity. It was found that a significant amount of H₂ is synthesized in the oral cavity of healthy human subjects, and that its generation is enhanced by the presence of glucose but inhibited by either teeth brushing or sterilization with povidone iodine. These observations suggest the presence of H₂-generating bacteria in the oral cavity. The screening of commensal bacteria in the oral cavity revealed that a variety of anaerobic bacteria generate H₂. Among them, Klebsiella pneumoniae (K. pneumoniae) generated significantly large amounts of H₂ in the presence of glucose. Biochemical analysis revealed that various proteins in K. pneumoniae are carbonylated under standard culture conditions, and that oxidative stress induced by the presence of Fe⁺⁺ and H₂O₂ increases the number of carbonylated proteins, particularly when their hydrogenase activity is inhibited by KCN. Inhibition of H₂ generation markedly suppresses the growth of K. pneumoniae. These observations suggest that H₂ generation and/or the reduction of oxidative stress is important for the survival and growth of K. pneumoniae in the oral cavity.     

Active and inactive state structures of unliganded Lactobacillus casei allosteric L‐lactate dehydrogenase

Lactobacillus casei L ‐lactate dehydrogenase (LCLDH) is activated through the homotropic and heterotropic activation effects of pyruvate and fructose 1,6‐bisphosphate (FBP), respectively, and exhibits unusually high pH‐dependence in the allosteric effects of these ligands. The active (R) and inactive (T) state structures of unliganded LCLDH were determined at 2.5 and 2.6 Å resolution, respectively. In the catalytic site, the structural rearrangements are concerned mostly in switching of the orientation of Arg171 through the flexible intersubunit contact at the Q‐axis subunit interface. The distorted orientation of Arg171 in the T state is stabilized by a unique intra‐helix salt bridge between Arg171 and Glu178, which is in striking contrast to the multiple intersubunit salt bridges in Lactobacillus pentosus nonallosteric L ‐lactate dehydrogenase. In the backbone structure, major structural rearrangements of LCLDH are focused in two mobile regions of the catalytic domain. The two regions form an intersubunit linkage through contact at the P‐axis subunit interface involving Arg185, replacement of which with Gln severely decreases the homotropic and hetertropic activation effects on the enzyme. These two regions form another intersubunit linkage in the Q‐axis related dimer through the rigid NAD‐binding domain, and thus constitute a pivotal frame of the intersubunit linkage for the allosteric motion, which is coupled with the concerted structural change of the four subunits in a tetramer, and of the binding sites for pyruvate and FBP. The unique intersubunit salt bridges, which are observed only in the R state structure, are likely involved in the pH‐dependent allosteric equilibrium. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Expression,localization, and biological function of the R3 subtype of receptor-type protein tyrosine phosphatases in mammals

The R3 subtype of receptor-type protein tyrosine phosphatases (RPTPs) includes VE-PTP, DEP-1, PTPRO, and SAP-1. All of these enzymes share a similar structure, with a single catalytic domain and putative tyrosine phosphorylation sites in the cytoplasmic region and fibronectin type III–like domains in the extracellular region. The expression of each R3 RPTP is largely restricted to a single or limited number of cell types, with VE-PTP and DEP-1 being expressed in endothelial or hematopoietic cells, PTPRO in neurons and in podocytes of the renal glomerulus, and SAP-1 in gastrointestinal epithelial cells. In addition, these RPTPs are localized specifically at the apical surface of polarized cells. The structure, expression, and localization of the R3 RPTPs suggest that they perform tissue-specific functions and that they might act through a common mechanism that includes activation of Src family kinases. In this review, we describe recent insights into R3-subtype RPTPs, particularly those of mammals.     

Seasonal shift in factors controlling net ecosystem production in a high Arctic terrestrial ecosystem

We examined factors controlling temporal changes in net ecosystem production (NEP) in a high Arctic polar semi-desert ecosystem in the snow-free season. We examined the relationships between NEP and biotic and abiotic factors in a dominant plant community (Salix polaris–moss) in the Norwegian high Arctic. Just after snowmelt in early July, the ecosystem released CO₂ into the atmosphere. A few days after snowmelt, however, the ecosystem became a CO₂ sink as the leaves of S. polaris developed. Diurnal changes in NEP mirrored changes in light incidence (photosynthetic photon flux density, PPFD) in summer. NEP was significantly correlated with PPFD when S. polaris had fully developed leaves, i.e., high photosynthetic activity. In autumn, NEP values decreased as S. polaris underwent senescence. During this time, CO₂ was sometimes released into the atmosphere. In wet conditions, moss made a larger contribution to NEP. In fact, the water content of the moss regulated NEP during autumn. Our results indicate that the main factors controlling NEP in summer are coverage and growth of S. polaris, PPFD, and precipitation. In autumn, the main factor controlling NEP is moss water content.     

Epitope analysis of peanut allergen Ara h1 with human monoclonal IgM antibody 92-2

A human-mouse hybridoma clone 92-2 secreting IgM-class human monoclonal antibody to peanut allergen protein Ara h1 was established. To detect antibody-binding sequences on Ara h1, we synthesized a series of peptides of the Ara h1 protein on a multi-pin apparatus for the pin-peptide ELISA. The 92-2 human monoclonal antibody was found to recognize a sequence of GREGEQEWGTPGSHVREETS. Further analysis with shorter pin-peptides with eight amino acid-long showed that the sequence of QEWGTPGS was an essential linear sequence of this epitope. When the QEW part of the sequence was replaced by alanine, the 92-2 monoclonal antibody did not bind to the substituted peptide, showing that those amino acids play an important role in the binding of the 92-2 monoclonal antibody.     

In vitro selection of a photoresponsive RNA aptamer to hemin

A photoresponsive RNA aptamer to hemin was selected in vitro from a random sequence library of RNAs with azobenzene residues. The aptamer bound to hemin under visible light irradiation and was released by ultraviolet light.     

Construction of simultaneous SPR and QCM sensing platform

To construct a novel simultaneous SPR and QCM sensing system, an AT-cut quartz crystal is fabricated by sputtering 250 nm of ITO on one side of the quartz plate over a 5-nm thick underlay of titanium, while a 50-nm thick layer of gold is sputter-deposited on the other side to induce a total light reflection of an incident laser beam on the thin gold layer. The signals of the sensing system are detected using a Handy-SPR and QCA922 when dropping 200 μL of Milli-Q water into the sensing cell. A decrease in the SPR reflected light intensity is clearly identified. In the same experiment, the changes in the resonant frequency and resistance are about 2 kHz and 200 Ω, respectively. Furthermore, the oscillation stabilities of the resonant frequency and resistance are about 50 Hz and 2 Ω, respectively, which are sufficient to detect a large mass change on the QCM/SPR chip.     

Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer

The hemibiotrophic ascomycete Colletotrichum higginsianum is the casual agent of anthracnose disease of cruciferous plants. High efficiency transformation by Agrobacterium tumefaciens-mediated gene transfer has been established for this fungus. However, targeted gene mutagenesis through homologous recombination rarely occurs in C. higginsianum. We have identified and disrupted the C. higginsianum homologue of the human Ku70 gene, ChKU70, which encodes a protein that plays a role in non-homologous end-joining for repair of DNA breaks. chku70 mutants showed a dramatic increase in the frequency of integration of introduced exogenous DNA fragments by homologous recombination without any detectable phenotypic defects. This result demonstrates that the chku70 mutant is an efficient recipient for targeted gene mutagenesis in C. higginsianum. We have also developed a novel approach [named direct repeat recombination-mediated gene targeting (DRGT)] for targeted gene disruption through Agrobacterium tumefaciens-mediated gene transfer. DRGT utilizes homologous recombination between repeated sequences on the T-DNA flanking a partial fragment of the target gene. Our results suggest that DRGT in the chku70 mutant background could be a useful tool for rapid isolation of targeted gene disruptants in C. higginsianum.