Diverging patterns of mitochondrial and nuclear DNA diversity in subarctic black spruce: imprint of a founder effect associated with postglacial colonization

High-latitude ecotonal populations at the species margins may exhibit altered patterns of genetic diversity, resulting from more or less recent founder events and from bottleneck effects in response to climate oscillations. Patterns of genetic diversity were investigated in nine populations of the conifer black spruce (Picea mariana [Mill.] BSP.) in northwestern Québec, Canada, using seed-dispersed mitochondrial (mt) DNA and nuclear (nc) DNA. mtDNA diversity (mitotypes) was assessed at three loci, and ncDNA diversity was estimated for nine expressed sequence tag polymorphism (ESTP) loci. Sampling included populations from the boreal forest and the southern and northern subzones of the subarctic forest-tundra, a fire-born ecotone. For ncDNA, populations from all three vegetation zones were highly diverse with little population differentiation (thetaN = 0.014); even the northernmost populations showed no loss of rare alleles. Patterns of mitotype diversity were strikingly different: within-population diversity and population differentiation were high for boreal forest populations [expected heterozygosity per locus (HE) = 0.58 and thetaM = 0.529], but all subarctic populations were fixed for a single mitotype (HE = 0). This lack of variation suggests a founder event caused by long-distance seed establishment during postglacial colonization, consistent with palaeoecological data. The estimated movement of seeds alone (effective number of migrants per generation, NmM < 2) was much restricted compared to that estimated from nuclear variants, which including pollen movement (NmN > 17). This could account for the conservation of a founder imprint in the mtDNA of subarctic black spruce. After reduction, presumably in the early Holocene, the diversity in ncDNA would have been replenished rapidly by pollen-mediated gene flow, and maintained subsequently through vegetative layering during the current cooler period covering the last 3000 years.     

What is the nature of the replicative niche of a stealthy bug named Brucella?

Brucella spp. are facultatively intracellular bacteria that persist and multiply in the macrophages of their mammalian hosts. The so-called phagosome to which they have adapted is their natural living niche. Characterization of this niche would facilitate an understanding of the true relationship between the host cell and the intracellular bacteria. This Opinion analyses and discusses the characteristic properties and genesis of this vacuole during phagocytosis as deduced from the virulence factors necessary for intracellular multiplication of the pathogen. We conclude that the replicative niche of Brucella spp.--the 'brucellosome'--differs from all other cellular organelles, and that it isolates the pathogen from certain cytoplasmic nutrients. Adaptation to the stress conditions encountered and the use of anaerobic respiration enable brucellae to replicate in the compartment they create.     

Imidazole derivatives as a novel class of hybrid compounds with inhibitory histamine N-methyltransferase potencies and histamine hH3 receptor affinities

In this study, a novel series of imidazole-containing compounds with dual properties, that is, inhibitory potency at the enzyme histamine N(tau)-methyltransferase (HMT) and antagonist potency at histamine H(3) receptors was designed and synthesized. Pharmacologically, these new hybrid drugs were evaluated in functional assays for their inhibitory potencies at rat kidney HMT and for their antagonist activities on synaptosomes of rat cerebral cortex. For selected compounds, binding affinities at recombinant human histamine H(3) receptors were determined. The first compounds (1-10) of the series proved to be H(3) receptor ligands of high potency at rat synaptosomes or of high binding affinity at human H(3) receptors, respectively, but of only moderate activity as inhibitors of rat kidney HMT. In contrast, aminoquinoline- or tetrahydroacridine-containing derivatives 11-17 also displayed HMT inhibitory potency in the nanomolar concentration range. Preliminary data from molecular modeling investigations showed that the imidazole derivative 15 and the HMT inhibitor quinacrine possess identical binding areas. The most interesting compound (14) is simultaneously a highly potent H(3) receptor ligand (K(i)=4.1nM) and a highly potent HMT inhibitor (IC(50)=24nM), which makes this derivative a valuable pharmacological tool for further development.     

Parallel liquid synthesis of N,N'-Disubstituted 3-amino azepin-2-ones as potent and specific farnesyl transferase inhibitors

A rapid structure-activity study was performed by parallel liquid synthesis on N,N'-disubstitution of 3-amino azepin-2-one to afford potent and specific farnesyl transferase inhibitors with low nM enzymatic and cellular activities. The activities of the selected compounds were validated in vivo, and compounds 41a and 44a presented significant antitumour activity.     

Coordinate expression of fibulin-5/DANCE and elastin during lung injury repair

Fibulin-5, previously known as DANCE and EVEC, is a secreted extracellular matrix protein that functions as a scaffold for elastin fiber assembly and as a ligand for integrins alphavbeta3, alphavbeta5, and alpha9beta1. Fibulin-5 is developmentally regulated in the lung, and lung air space enlargement develops in mice deficient in fibulin-5. Fibulin-5 is also induced in adult lung following lung injury by hyperoxia. To further examine the role of fibulin-5 during repair of lung injury, we assessed fibulin-5 expression during elastase-induced emphysema in C57/b mice. Mice were treated with either saline or elastase via the trachea, and the lung was examined 20 days after treatment. Fibulin-5 mRNA was induced almost fourfold, whereas elastin mRNA was minimally elevated. Immunohistochemistry studies showed that fibulin-5 was induced in cells within the alveolar wall following elastase treatment. Western analysis demonstrates that fibulin-5 was strongly expressed in isolated primary lung interstitial fibroblasts. Fibulin-5 protein was localized to the fibroblast cell layer in culture, and brief elastase treatment degraded the protein. Intact fibulin-5 did not accumulate in the culture media. Treatment of fibroblasts with the proinflammatory cytokine interleukin-1beta abolished fibulin-5 mRNA expression. Our results indicate that fibulin-5 is coordinately expressed and regulated with elastin in lung fibroblasts and may serve a key role during lung injury and repair.     

T.T pair intercalation and duplex interconversion within i-motif tetramers

The i-motif is a four-stranded structure built by intercalation in head-to-tail orientation of two parallel duplexes associated by hemi-protonated C.C(+) pairs. Using NMR methods, we investigated the structure, the base-pair opening kinetics and the internal motions of three i-motif tetramers: [d(5mCCTCnTCC)](4) (n=1, 2, 3). These tetramers cannot accommodate the intercalation of two T.T pairs in face-to-face orientation. They are built by intercalation of two symmetrical duplexes whose contacting T3/TM thymidine bases (M=5, 6, 7) are either base-paired or unstacked. The arrangement of the unstacked/paired thymidine bases of the two T/T groups results in the formation of two different conformations. One, fully symmetric, whose thymidine bases T3 and TM are unstacked and base-paired respectively. The other is the asymmetric assembly of two duplexes: one where both thymidine bases are unstacked and the other with two T.T pairs. The proportion of the symmetric conformer increases from a value beyond the detection threshold for n=1, to 19% for n=2 and up to more than 95% for n=3. The exchange cross-peaks connecting together the intercalated duplexes of [d(5mCCTCTCC)](4) and [d(5mCCTCCTCC)](4) reveal a structural interconversion induced by the simultaneous opening/closing of the contacting T3/TM thymidine bases. In [d(5mCCTCCTCC)](4) the motion of the T3/T6 groups triggers the interconversion of the symmetric and asymmetric conformations. In [d(5mCCTCTCC)](4) the intercalated duplexes exchange their structures in an apparently concerted motion, suggesting the simultaneous opening/closing of two distant T3/T5* and T5/T3* switching groups. The spectrum of [d(5mCCTCCCTCC)](4) is fully symmetric and, for this reason, its spectrum gives no indication for duplex interconversion. Nevertheless, the imino proton exchange kinetics argues for a switching motion of the T3/T7 group. Duplex interconversion is not detectable in that case, due to the tetramer symmetry. The origin of the structural conflict hindering the intercalation of two T.T pairs into the i-motif is discussed.     

Mg2+-dependent gating and strong inward rectification of the cation channel TRPV6

TRPV6 (CaT1/ECaC2), a highly Ca(2+)-selective member of the TRP superfamily of cation channels, becomes permeable to monovalent cations in the absence of extracellular divalent cations. The monovalent currents display characteristic voltage-dependent gating and almost absolute inward rectification. Here, we show that these two features are dependent on the voltage-dependent block/unblock of the channel by intracellular Mg(2+). Mg(2+) blocks the channel by binding to a site within the transmembrane electrical field where it interacts with permeant cations. The block is relieved at positive potentials, indicating that under these conditions Mg(2+) is able to permeate the selectivity filter of the channel. Although sizeable outward monovalent currents were recorded in the absence of intracellular Mg(2+), outward conductance is still approximately 10 times lower than inward conductance under symmetric, divalent-free ionic conditions. This Mg(2+)-independent rectification was preserved in inside-out patches and not altered by high intracellular concentrations of spermine, indicating that TRPV6 displays intrinsic rectification. Neutralization of a single aspartate residue within the putative pore loop abolished the Mg(2+) sensitivity of the channel, yielding voltage-independent, moderately inwardly rectifying monovalent currents in the presence of intracellular Mg(2+). The effects of intracellular Mg(2+) on TRPV6 are partially reminiscent of the gating mechanism of inwardly rectifying K(+) channels and may represent a novel regulatory mechanism for TRPV6 function in vivo.     

Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function

Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21(waf/CIP1). Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway.