A global double-fluorescent Cre reporter mouse

The Cre/loxP system has been used extensively for conditional mutagenesis in mice. Reporters of Cre activity are important for defining the spatial and temporal extent of Cre-mediated recombination. Here we describe mT/mG, a double-fluorescent Cre reporter mouse that expresses membrane-targeted tandem dimer Tomato (mT) prior to Cre-mediated excision and membrane-targeted green fluorescent protein (mG) after excision. We show that reporter expression is nearly ubiquitous, allowing visualization of fluorescent markers in live and fixed samples of all tissues examined. We further demonstrate that mG labeling is Cre-dependent, complementary to mT at single cell resolution, and distinguishable by fluorescence-activated cell sorting. Both membrane-targeted markers outline cell morphology, highlight membrane structures, and permit visualization of fine cellular processes. In addition to serving as a global Cre reporter, the mT/mG mouse may also be used as a tool for lineage tracing, transplantation studies, and analysis of cell morphology in vivo.     

Tumor suppressor protein Lgl mediates G1 cell cycle arrest at high cell density by forming an Lgl-VprBP-DDB1 complex

Lethal giant larvae (Lgl) is an evolutionarily conserved tumor suppressor whose loss of function causes disrupted epithelial architecture with enhanced cell proliferation and defects in cell polarity. A role for Lgl in the establishment and maintenance of cell polarity via suppression of the PAR-aPKC polarity complex is established; however, the mechanism by which Lgl regulates cell proliferation is not fully understood. Here we show that depletion of Lgl1 and Lgl2 in MDCK epithelial cells results in overproliferation and overproduction of Lgl2 causes G1 arrest. We also show that Lgl associates with the VprBP-DDB1 complex independently of the PAR-aPKC complex and prevents the VprBP-DDB1 subunits from binding to Cul4A, a central component of the CRL4 [VprBP] ubiquitin E3 ligase complex implicated in G1- to S-phase progression. Consistently, depletion of VprBP or Cul4 rescues the overproliferation of Lgl-depleted cells. In addition, the affinity between Lgl2 and the VprBP-DDB1 complex increases at high cell density. Further, aPKC-mediated phosphorylation of Lgl2 negatively regulates the interaction between Lgl2 and VprBP-DDB1 complex. These results suggest a mechanism protecting overproliferation of epithelial cells in which Lgl plays a critical role by inhibiting formation of the CRL4 [VprBP] complex, resulting in G1 arrest.     

Antiangiogenic Tumor Therapy by DNA Vaccine Inducing Aquaporin-1-Specific CTL Based on Ubiquitin-Proteasome System in Mice

Aquaporin-1 (AQP-1) is a water channel protein highly expressed in the vascular endothelial cells of proliferating tissues including malignant cancers. Given that in APC ubiquitinated peptides are effectively introduced into proteasomes from which CD8 epitopes are excised, we fused ubiquitin with AQP-1 (pUB-AQP-1) to produce a DNA vaccine. In C57BL/6J mice immunized with pUB-AQP-1, the growth of B16F10 melanoma was profoundly inhibited. The antitumor effect of the pUB-AQP-1 DNA vaccine was largely mediated by CD8 T cells, which secrete IFN-γ, perforin, and granzyme-B in the presence of APCs transfected with pUB-AQP-1. AQP-1-specific CD8 T cells possessed cytotoxic activity both in vivo and in vitro. After tumor challenge, the microvessel density decreased and the ratio of total blood vessel area to tumor area was significantly reduced as compared with control mice, resulting in a dramatic suppression of tumor growth. The immunization effect was completely abrogated in immunoproteasome-deficient mice. Strikingly this pUB-AQP-1 DNA vaccine was also effective against Colon 26 colon tumors (BALB/c) and MBT/2 bladder tumors (C3H/HeN). Thus, this ubiquitin-conjugated DNA immunization-targeting tumor vasculature is a valid and promising antitumor therapy. This vaccine works across the barriers of tumor species and MHC class I differences in host mice.     

Crystal Structure of Cucumisin,a Subtilisin-Like Endoprotease from Cucumis melo L.

Cucumisin is a plant serine protease, isolated as an extracellular glycoprotein from the melon fruit Cucumis melo L. var. Prince. Cucumisin is composed of multiple domain modules, including catalytic, protease-associated, and fibronectin‐III-like domains. The crystal structure of cucumisin was determined by the multiwavelength anomalous dispersion method and refined at 2.75 Å resolution. A structural homology search indicated that the catalytic domain of cucumisin shares structural similarity with subtilisin and subtilisin-like fold enzymes. According to the Z-score, the highest structural similarity is with tomato subtilase 3 (SBT3), with an rmsd of 3.5 Å for the entire region. The dimer formation mediated by the protease-associated domain in SBT3 is a distinctive structural characteristic of cucumisin. On the other hand, analytical ultracentrifugation indicated that cucumisin is mainly monomeric in solution. Although the locations of the amino acid residues composing the catalytic triad are well conserved between cucumisin and SBT3, a disulfide bond is uniquely located near the active site of cucumisin. The steric circumstances of the active site with this disulfide bond are distinct from those of SBT3, and it contributes to the substrate preference of cucumisin, especially at the P2 position. Among the plant serine proteases, the thermostability of cucumisin is higher than that of its structural homologue SBT3, as determined by their melting points. A structural comparison between cucumisin and SBT3 revealed that cucumisin possesses less surface area and shortened loop regions. Consequently, the higher thermostability of cucumisin is achieved by its more compact structure.     

Initial step of B12-dependent enzymatic catalysis: energetic implications regarding involvement of the one-electron-reduced form of adenosylcobalamin cofactor

Density functional theory analysis was performed to elucidate the impact of one-electron reduction upon the initial step of adenosylcobalamin-dependent enzymatic catalysis. The transition state (TS) corresponding to the Co–C bond cleavage and subsequent hydrogen abstraction from the substrate was located. The intrinsic reaction coordinate calculations predicted that the reaction consisting of Co–C5′ bond cleavage in [Co^III(corrin^•)]–Rib (where Rib is ribosyl) and hydrogen-atom abstraction from the CH₃–CH₂–CHO substrate occurs in a concerted fashion. The computed activation energy barrier of the reaction (15.0 kcal/mol) was lowered by approximately 54.5% in comparison with the reaction involving the positively charged cofactor model (Im–[Co^III(corrin)]–Rib⁺, where Im is imidazole; energy barrier = 33.0 kcal/mol). The Im base was detached during the TS search in the reaction involving the one-electron-reduced analogue. Thus, to compare the energetics of the two reactions, the axial Im ligand detachment energy for the Im–[Co^III(corrin^•)]–Rib model was computed [7.6 kcal/mol (gas phase); 4.6 kcal/mol (water)]. Consequently, the effective activation energy barrier for the reaction mediated by the Im-off [Co^III(corrin^•)]–Rib was estimated to be 22.6 kcal/mol, which implied an overall 31.5% reduction in the energetic demands of the reaction. Considering that the lengthened Co–N_axial bond has been observed in X-ray crystal structure studies of B₁₂-dependent mutases, the catalytic impact induced by one-electron reduction of the cofactor is expected to be higher in the presence of the enzymatic environment.     

Individual Variation in Behavioural Reactions to Unfamiliar Conspecific Vocalisation and Hormonal Underpinnings in Male Chimpanzees

It has been established that various species exhibit personality, defined as intra‐individual consistency and inter‐individual variation in behavioural phenotypes. For example, certain individuals may demonstrate consistently greater behavioural reactions and elevated stress responses. We conducted playback experiments and hormonal analyses on male chimpanzees (Pan troglodytes) in captivity to investigate the patterns and proximate mediators of individual variations in behavioural reactions. We found intra‐individual consistency and inter‐individual variation in behavioural reactions (intensive vigilance towards the direction of speakers) to vocalisations by unfamiliar chimpanzees. This behavioural reaction was positively correlated with changes in salivary cortisol concentration, suggesting that stress is a proximate factor mediating the variation in behavioural reactions. The males who were highly responsive to the conspecific vocalisation also exhibited high behavioural reactions towards the neutral broadcast stimulus (the jungle crow’s Corvus macrorhynchos ‘ka’ vocalisation). This observation is consistent with the notion that male chimpanzees vary in intrinsic behavioural tendency to different stimuli.     

Nutritional significance of the selective ingestion of Albizia zygia gum exudate by wild chimpanzees in Bossou, Guinea

The selective ingestion of plant gum exudates by chimpanzees has been frequently observed at various study sites. At Bossou, Guinea, chimpanzees also frequently ingest Albizia zygia gum exudate. A functional explanation for this behavior is lacking, so we evaluated its possible contribution of energy in the form of short-chain fatty acids (SCFA) as well as minerals. An in vitro fermentation study of A. zygia gum using the fecal bacteria of a Bossou chimpanzee showed that carboxylic acids were produced with a 6-hr lag phase up to 44 mmol/l by 18 hr of incubation. Acetate was the most abundant acid produced, followed by lactate and propionate. The energy supplied from the fermentation of a piece of gum exudate (20-30 g) was negligible in comparison with the estimated daily energy requirements of chimpanzees in the wild. However, A. zygia gum exudate (20-30 g) can supply sufficient amounts of calcium, manganese, magnesium, and potassium to fulfill the daily requirements for these minerals in chimpanzees.