p53 and p21(Waf1) Are Recruited to Distinct PML‐Containing Nuclear Foci in Irradiated and Nutlin‐3a‐Treated U2OS Cells

Promyelocytic leukemia nuclear bodies (PML‐NBs) are multiprotein complexes that include PML protein and localize in nuclear foci. PML‐NBs are implicated in multiple stress responses, including apoptosis, DNA repair, and p53‐dependent growth inhibition. ALT‐associated PML bodies (APBs) are specialized PML‐NBs that include telomere‐repeat binding‐factor TRF1 and are exclusively in telomerase‐negative tumors where telomere length is maintained through alternative (ALT) recombination mechanisms. We compared cell‐cycle and p53 responses in ALT‐positive cancer cells (U2OS) exposed to ionizing radiation (IR) or the p53 stabilizer Nutlin‐3a. Both IR and Nutlin‐3a caused growth arrest and comparable induction of p53. However, p21, whose gene p53 activates, displayed biphasic induction following IR and monophasic induction following Nutlin‐3a. p53 was recruited to PML‐NBs 3–4 days after IR, approximately coincident with the secondary p21 increase. These p53/PML‐NBs marked sites of apparently unrepaired DNA double‐strand breaks (DSBs), identified by colocalization with phosphorylated histone H2AX. Both Nutlin‐3a and IR caused a large increase in APBs that was dependent on p53 and p21 expression. Moreover, p21, and to a lesser extent p53, was recruited to APBs in a fraction of Nutlin‐3a‐treated cells. These data indicate (1) p53 is recruited to PML‐NBs after IR that likely mark unrepaired DSBs, suggesting p53 may either be further activated at these sites and/or function in their repair; (2) p53–p21 pathway activation increases the percentage of APB‐positive cells, (3) p21 and p53 are recruited to ALT‐associated PML‐NBs after Nutlin‐3a treatment, suggesting that they may play a previously unrecognized role in telomere maintenance. J. Cell. Biochem. 111: 1280–1290, 2010. © 2010 Wiley‐Liss, Inc.     

Applying geographic profiling used in the field of criminology for predicting the nest locations of bumble bees

We tested whether geographic profiling (GP) can predict multiple nest locations of bumble bees. GP was originally developed in the field of criminology for predicting the area where an offender most likely resides on the basis of the actual crime sites and the predefined probability of crime interaction. The predefined probability of crime interaction in the GP model depends on the distance of a site from an offender's residence. We applied GP for predicting nest locations, assuming that foraging and nest sites were the crime sites and the offenders’ residences, respectively. We identified the foraging and nest sites of the invasive species Bombus terrestris in 2004, 2005, and 2006. We fitted GP model coefficients to the field data of the foraging and nest sites, and used GP with the fitting coefficients. GP succeeded in predicting about 10-30% of actual nests. Sensitivity analysis showed that the predictability of the GP model mainly depended on the coefficient value of buffer zone, the distance at the mode of the foraging probability. GP will be able to predict the nest locations of bumble bees in other area by using the fitting coefficient values measured in this study. It will be possible to further improve the predictability of the GP model by considering food site preference and nest density.     

Bottromycin derivatives: Efficient chemical modifications of the ester moiety and evaluation of anti-MRSA and anti-VRE activities

Novel bottromycin derivatives were synthesized from bottromycin A₂ via a hydrazide derivative as a common intermediate. Seventeen derivatives were subjected to in vitro evaluation against drug-resistant gram-positive bacteria. Some compounds showed potent anti-MRSA and anti-VRE activity, as did bottromycin A₂. Notably, a propyl ketone derivative exhibited good antibacterial activity with excellent metabolite stability.     

Acute induction of Eya3 by late-night light stimulation triggers TSHβ expression in photoperiodism

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Highlights? Genome-wide expression data identify “long-day” and “short-day” genes ? Late-night light stimulation acutely induces Eya3 in the mouse pars tuberalis ? Eya3 and Six1 synergistically induce TSHβ expression via the Six consensus sequence ? Activation of the TSHβ promoter is further enhanced by Tef and Hlf via the D box     

3-(2-Aminocarbonylphenyl)propanoic acid analogs as potent and selective EP3 receptor antagonists. Part 2: Optimization of the side chains to improve in vitro and in vivo potencies

A series of 3-[2-{[(3-methyl-1-phenylbutyl)amino]carbonyl}-4-(phenoxymethyl)phenyl]propanoic acid analogs were synthesized and evaluated for their in vitro potency. In most cases, introduction of one or two substituents into the two phenyl moieties resulted in the tendency of an increase or retention of in vitro activities. Several compounds, which showed excellent subtype selectivity, were evaluated for their inhibitory effect against PGE₂-induced uterine contraction in pregnant rats, which is thought to be mediated by the EP3 receptor subtype. The structure–activity relationships (SARs) are also discussed.     

Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis.

C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G(-/-) homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared fibroblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras. Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G-deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.     

Ectopic expression of a rice plasma membrane intrinsic protein (OsPIP1;3) promotes plant growth and water uptake

Plasma membrane intrinsic proteins (PIPs) are known to be major facilitators of the movement of a number of substrates across cell membranes. From a drought‐resistant cultivar of Oryza sativa (rice), we isolated an OsPIP1;3 gene single‐nucleotide polymorphism (SNP) that is mostly expressed in rice roots and is strongly responsive to drought stress. Immunocytochemistry showed that OsPIP1;3 majorly accumulated on the proximal end of the endodermis and the cell surface around the xylem. Expression of GFP‐OsPIP1;3 alone in Xenopus oocytes or rice protoplasts showed OsPIP1;3 mislocalization in the endoplasmic reticulum (ER)‐like neighborhood, whereas co‐expression of OsPIP2;2 recruited OsPIP1;3 to the plasma membrane and led to a significant enhancement of water permeability in oocytes. Moreover, reconstitution of 10×His‐OsPIP1;3 in liposomes demonstrated water channel activity, as revealed by stopped‐flow light scattering. Intriguingly, by patch‐clamp technique, we detected significant NO₃^? conductance of OsPIP1;3 in mammalian cells. To investigate the physiological functions of OsPIP1;3, we ectopically expressed the OsPIP1;3 gene in Nicotiana benthamiana (tobacco). The transgenic tobacco plants exhibited higher photosynthesis rates, root hydraulic conductivity (Lp_r) and water‐use efficiency, resulting in a greater biomass and a higher resistance to water deficit than the wild‐type did. Further experiments suggested that heterologous expression of OsPIP1;3 in cyanobacterium altered bacterial growth under different conditions of CO₂ gas supply. Overall, besides shedding light on the multiple functions played by OsPIP1;3, this work provides insights into the translational value of plant AQPs.     

Genetic consequences of plant edaphic specialization to solfatara fields: Phylogenetic and population genetic analysis of Carex angustisquama (Cyperaceae)

Edaphic specialization is one of the main drivers of plant diversification and has multifaceted effects on population dynamics. Carex angustisquama is a sedge plant growing only on heavily acidified soil in solfatara fields, where only extremophytes can survive. Because of the lack of closely related species in similar habitats and its disjunct distribution, the species offers ideal settings to investigate the effects of adaptation to solfatara fields and of historical biogeography on the genetic consequences of plant edaphic specialization to solfatara fields. Here, genome‐wide single nucleotide polymorphisms were used to reveal the phylogenetic origin of C. angustisquama, and 16 expressed sequence tag–simple sequence repeat markers were employed to infer population demography of C angustisquama. Molecular phylogenetic analysis strongly indicated that C. angustisquama formed a monophyletic clade with Carex doenitzii, a species growing on nonacidified soil in the sympatric subalpine zone. The result of population genetic analysis showed that C. angustisquama has much lower genetic diversity than the sister species, and notably, all 16 loci were completely homozygous in most individuals of C. angustisquama. Approximate Bayesian computation analysis supported the model that assumed hierarchical declines of population size through its evolutionary sequence. We propose that the edaphic specialist in solfatara fields has newly attained the adaptation to solfatara fields in the process of speciation. Furthermore, we found evidence of a drastic reduction in genetic diversity in C. angustisquama, suggesting that the repeated founder effects associated with edaphic specialization and subsequent population demography lead to the loss of genetic diversity of this extremophyte in solfatara fields.     

Planar cell polarity induces local microtubule bundling for coordinated ciliary beating

Multiciliated cells (MCCs) in tracheas generate mucociliary clearance through coordinated ciliary beating. Apical microtubules (MTs) play a crucial role in this process by organizing the planar cell polarity (PCP)–dependent orientation of ciliary basal bodies (BBs), for which the underlying molecular basis remains elusive. Herein, we found that the deficiency of Daple, a dishevelled-associating protein, in tracheal MCCs impaired the planar polarized apical MTs without affecting the core PCP proteins, causing significant defects in the BB orientation at the cell level but not the tissue level. Using live-cell imaging and ultra-high voltage electron microscope tomography, we found that the apical MTs accumulated and were stabilized by side-by-side association with one side of the apical junctional complex, to which Daple was localized. In vitro binding and single-molecule imaging revealed that Daple directly bound to, bundled, and stabilized MTs through its dimerization. These features convey a PCP-related molecular basis for the polarization of apical MTs, which coordinate ciliary beating in tracheal MCCs.