Design, synthesis, modeling, biological evaluation and photoaffinity labeling studies of novel series of photoreactive benzamide probes for histone deacetylase 2

The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are cell permeable and target the nuclear HDACs.     

Scaffold-hopping with zwitterionic CCR3 antagonists: Identification and optimisation of a series with good potency and pharmacokinetics leading to the discovery of AZ12436092

The discovery and optimisation of a series of zwitterionic CCR3 antagonists is described. Optimisation of the structure led to AZ12436092, a compound with excellent selectivity over activity at hERG and outstanding pharmacokinetics in preclinical species.     

Buffering Capacity Explains Signal Variation in Symbiotic Calcium Oscillations

Legumes form symbioses with rhizobial bacteria and arbuscular mycorrhizal fungi that aid plant nutrition. A critical component in the establishment of these symbioses is nuclear-localized calcium (Ca2+) oscillations. Different components on the nuclear envelope have been identified as being required for the generation of the Ca2+ oscillations. Among these an ion channel, Doesn''t Make Infections1, is preferentially localized on the inner nuclear envelope and a Ca2+ ATPase is localized on both the inner and outer nuclear envelopes. Doesn''t Make Infections1 is conserved across plants and has a weak but broad similarity to bacterial potassium channels. A possible role for this cation channel could be hyperpolarization of the nuclear envelope to counterbalance the charge caused by the influx of Ca2+ into the nucleus. Ca2+ channels and Ca2+ pumps are needed for the release and reuptake of Ca2+ from the internal store, which is hypothesized to be the nuclear envelope lumen and endoplasmic reticulum, but the release mechanism of Ca2+ remains to be identified and characterized. Here, we develop a mathematical model based on these components to describe the observed symbiotic Ca2+ oscillations. This model can recapitulate Ca2+ oscillations, and with the inclusion of Ca2+-binding proteins it offers a simple explanation for several previously unexplained phenomena. These include long periods of frequency variation, changes in spike shape, and the initiation and termination of oscillations. The model also predicts that an increase in buffering capacity in the nucleoplasm would cause a period of rapid oscillations. This phenomenon was observed experimentally by adding more of the inducing signal.Plant growth is frequently limited by the essential nutrients nitrogen and phosphorus. Several plant species have established symbiotic relationships with microorganisms to overcome such limitations. In addition to the symbiotic relationship with arbuscular mycorrhizal fungi that many plants establish in order to secure their uptake of water, phosphorus, and other nutrients (Harrison, 2005; Parniske, 2008), legumes have developed interactions with bacteria called rhizobia, resulting in the fixation of atmospheric nitrogen within the plant root (Lhuissier et al., 2001; Gage, 2004; Riely et al., 2006).Root symbioses initiate with signal exchanges in the soil. Plant signals are perceived by the symbionts, triggering the successive release of microbial signals. For rhizobia, the signal molecules are lipochitooligosaccharides termed Nod factors (Dénarié et al., 1996), and the release of lipochitooligosaccharides has also been found in the fungal interaction (Maillet et al., 2011). Upon receiving diffusible signals from the microsymbionts, the plant roots initiate developmental programs that lead to infection by rhizobia or arbuscular mycorrhizal fungi. Both programs employ the same signaling pathway with several components being common to both mycorrhizal and rhizobial interactions (Kistner and Parniske, 2002; Lima et al., 2009). In particular, both the symbioses show characteristic perinuclear and nucleoplasmic localized calcium (Ca2+) oscillations, so-called Ca2+ spiking (Oldroyd and Downie, 2006; Sieberer et al., 2009). It has been suggested that Ca2+ is released from an internal store, most likely the nuclear lumen and associated endoplasmic reticulum (ER; Matzke et al., 2009), with targeted release in the nuclear region (Capoen et al., 2011).Genetic screens in the model legume Medicago truncatula have identified several genes that are required for the plant in the establishment of both symbioses. Two of these, Doesn’t Make Infections1 (DMI1) and DMI2, are essential for the induction of the Ca2+ oscillations, yet the precise roles and mechanisms of these components remain to be determined. DMI2 codes for a plasma membrane receptor-like kinase (Endre et al., 2002; Stracke et al., 2002) that is required to facilitate further signal transduction in the cell (Bersoult et al., 2005). DMI1 is a cation channel located preferentially on the inner nuclear envelope (Ané et al., 2004; Edwards et al., 2007; Riely et al., 2007; Charpentier et al., 2008; Capoen et al., 2011; Venkateshwaran et al., 2012). DMI3 encodes a calcium calmodulin-dependent protein kinase that interacts with downstream components and is thought to be the decoder of the Ca2+ oscillations (Lévy et al., 2004; Mitra et al., 2004; Hayashi et al., 2010). Additional genes coding for three nucleoporins called NUP85, NUP133, and NENA are also required for Ca2+ oscillations (Kanamori et al., 2006; Saito et al., 2007; Groth et al., 2010). The nuclear pore might be involved in trafficking secondary signals and/or ion channels to the inner nuclear membrane. These shared signaling components are collectively referred to as the common Sym pathway.DMI1 plays a key role in the production of Ca2+ oscillations, but its exact mechanism is still unknown. Orthologs of DMI1 have been found; in Lotus japonicus, they are called CASTOR and POLLUX (Charpentier et al., 2008), and in pea (Pisum sativum), SYM8 (Edwards et al., 2007). CASTOR and POLLUX, as well as calcium calmodulin-dependent protein kinase, are highly conserved both in legumes and nonlegumes (Banba et al., 2008; Chen et al., 2009). This highlights the essential role of the Ca2+ oscillations in mycorrhizal signaling.DMI1 is not the channel responsible for the release of Ca2+ (Charpentier et al., 2008; Parniske, 2008; Venkateshwaran et al., 2012) but probably influences the activity of Ca2+ channels. This is similar to how some K⁺ channels act in other plants and yeast (Peiter et al., 2007). Indeed, DMI1 is possibly a K⁺-permeable channel, based on the observation that POLLUX complements K⁺ transport in yeast (Charpentier et al., 2008). In symbiosis, the mode of action of DMI1 could be to allow cations into the nuclear envelope and in that way counterbalance the transmembrane charge that would occur following the release of Ca2+ into the nucleoplasm and cytoplasm. The cation channel could thus change the electrical potential across the nuclear membranes, affecting the opening of the voltage-activated Ca2+ channels (Edwards et al., 2007). This hypothesis is supported by a study reporting a membrane potential over the nuclear envelope in plants (Matzke and Matzke, 1986).Pharmacological evidence and the characteristics of the Ca2+ oscillations supports the involvement of Ca2+ pumps and Ca2+ channels (Engstrom et al., 2002). The pumps are needed to resequester Ca2+ after each release event, actively transporting Ca2+ against the concentration gradient using ATP. A recent study found a SERCA-type Ca2+ ATPase, MCA8, that is located on the inner and outer nuclear envelope of M. truncatula and is required for the symbiotic Ca2+ oscillations (Capoen et al., 2011). Such SERCA pumps are widely distributed on plant membranes, and the variation in their structure points to them being differentially regulated (Sze et al., 2000).Ca2+ channels release Ca2+ from the store, and the mechanism of activating these Ca2+ channels has been hypothesized to be voltage gated (Edwards et al., 2007; Oldroyd and Downie, 2008), but this remains to be verified experimentally. After release of Ca2+ into the cytosol and nucleoplasm, buffers quickly bind to and remove these free ions due to their toxicity to the cell (Sanders et al., 2002). Buffers, i.e. molecules that can bind Ca2+, may play an important role in determining the nonlinear behavior of the oscillatory system for Ca2+ signaling (Falcke, 2004). As numerous Ca2+ buffers are present in cells, it is important to take their contribution into account. Such buffers can also include experimentally introduced dyes and Ca2+ chelators.In Capoen et al. (2011), we investigated the establishment and transmission of spatial waves across the nuclear envelope and demonstrated that the key components for Ca2+ spiking reside on the inner and outer surface of the nuclear membrane. The computational framework we employed for this analysis makes a number of approximations in order to provide the computational efficiency required to perform spatiotemporal simulations. Here, a main focus is to understand the effect of buffers on the Ca2+ oscillations.In this article, we propose a mathematical model based on three key proteins; a Ca2+ ATPase, a voltage-gated Ca2+ channel, and the cation channel DMI1. The model reproduces the symbiotic Ca2+ oscillations, and we further demonstrate that Ca2+-binding proteins can explain initiation, termination, and experimentally observed variation in oscillation patterns. Furthermore, the model predicts that increases in buffering capacity can cause a period of rapid oscillations, and these were observed experimentally.     

Paternity assignment and demographic closure in the New Zealand southern right whale

The identification and characterization of reproductively isolated subpopulations or 'stocks' are essential for effective conservation and management decisions. This can be difficult in vagile marine species like marine mammals. We used paternity assignment and 'gametic recapture' to examine the reproductive autonomy of southern right whales (Eubalaena australis) on their New Zealand (NZ) calving grounds. We derived DNA profiles for 34 mother-calf pairs from skin biopsy samples, using sex-specific markers, 13 microsatellite loci and mtDNA haplotypes. We constructed DNA profiles for 314 adult males, representing 30% of the census male abundance of the NZ stock, previously estimated from genotypic mark-recapture modelling to be 1085 (95% CL 855, 1416). Under the hypothesis of demographic closure and the assumption of equal reproductive success among males, we predict: (i) the proportion of paternities assigned will reflect the proportion of the male population sampled and (ii) the gametic mark-recapture (GMR) estimate of male abundance will be equivalent to the census male estimate for the NZ stock. Consistent with these predictions, we found that the proportion of assigned paternities equalled the proportion of the census male population size sampled. Using the sample of males as the initial capture, and paternity assignment as the recapture, the GMR estimate of male abundance was 1001 (95% CL 542, 1469), similar to the male census estimate. These findings suggest that right whales returning to the NZ calving ground are reproductively autonomous on a generational timescale, as well as isolated by maternal fidelity on an evolutionary timescale, from others in the Indo-Pacific region.     

Discovery of a new family of amphibians from northeast India with ancient links to Africa

The limbless, primarily soil-dwelling and tropical caecilian amphibians (Gymnophiona) comprise the least known order of tetrapods. On the basis of unprecedented extensive fieldwork, we report the discovery of a previously overlooked, ancient lineage and radiation of caecilians from threatened habitats in the underexplored states of northeast India. Molecular phylogenetic analyses of mitogenomic and nuclear DNA sequences, and comparative cranial anatomy indicate an unexpected sister-group relationship with the exclusively African family Herpelidae. Relaxed molecular clock analyses indicate that these lineages diverged in the Early Cretaceous, about 140 Ma. The discovery adds a major branch to the amphibian tree of life and sheds light on both the evolution and biogeography of caecilians and the biotic history of northeast India-an area generally interpreted as a gateway between biodiversity hotspots rather than a distinct biogeographic unit with its own ancient endemics. Because of its distinctive morphology, inferred age and phylogenetic relationships, we recognize the newly discovered caecilian radiation as a new family of modern amphibians.     

The Ph1 locus suppresses Cdk2-type activity during premeiosis and meiosis in wheat

Despite possessing multiple sets of related (homoeologous) chromosomes, hexaploid wheat (Triticum aestivum) restricts pairing to just true homologs at meiosis. Deletion of a single major locus, Pairing homoeologous1 (Ph1), allows pairing of homoeologs. How can the same chromosomes be processed as homologs instead of being treated as nonhomologs? Ph1 was recently defined to a cluster of defective cyclin-dependent kinase (Cdk)-like genes showing some similarity to mammalian Cdk2. We reasoned that the cluster might suppress Cdk2-type activity and therefore affect replication and histone H1 phosphorylation. Our study does indeed reveal such effects, suggesting that Cdk2-type phosphorylation has a major role in determining chromosome specificity during meiosis.     

The phycocyanin-associated rod linker proteins of the phycobilisome of Gloeobacter violaceus PCC 7421 contain unusually located rod-capping domains

Gloeobacter violaceus PCC 7421 is a unique cyanobacterium that has no thylakoids and whose genome has been sequenced [Y. Nakamura, T. Kaneko, S. Sato, M. Mimuro, H. Miyashita, T. Tsuchiya, S. Sasamoto, A. Watanabe, K. Kawashima, Y. Kishida, C. Kiyokawa, M. Kohara, M. Matsumoto, A. Matsuno, N. Nakazaki, S. Shimpo, C. Takeuchi, M. Yamada, S. Tabata, Complete Genome Structure of Gloeobacter violaceus PCC 7421, a cyanobacterium that lacks thylakoids. DNA Research 10 (2003) 137-145]. Phycobilisomes of G. violaceus were isolated and analyzed by SDS-PAGE followed by N-terminal sequencing. Three rod-linker subunits (CpeC, CpeD and CpeE) were identified as predicted from the genome sequence. The cpcC1 and cpcC2 genes at order locus named (OLN) glr0950 and gll 3219 encoding phycocyanin-associated linker proteins from G. violaceus are 56 and 55 amino acids longer at the N-terminus than the open reading frame proposed in the genome. The two amino acid extensions showed a 66% identity to one another. Also, the N-terminal extensions of these sequences were similar to domains in both the rod-capping-linker protein CpcD2 and to the C-terminus domain of the phycoerythrin-associated linker protein CpeC. These domains are not only unusual in their N-terminal location, but are unusual in that they are more closely related in sequence similarity to the C-terminus domain of the phycoerythrin-associated linker, CpeC of G. violaceus, than to the C-terminus domain of phycocyanin-associated linker CpcC in other cyanobacteria. These linker proteins with unique special domains are indicators of the unusual structure of the phycobilisomes of G. violaceus.     

The absence of sharks from abyssal regions of the world's oceans

The oceanic abyss (depths greater than 3000 m), one of the largest environments on the planet, is characterized by absence of solar light, high pressures and remoteness from surface food supply necessitating special molecular, physiological, behavioural and ecological adaptations of organisms that live there. Sampling by trawl, baited hooks and cameras we show that the Chondrichthyes (sharks, rays and chimaeras) are absent from, or very rare in this region. Analysis of a global data set shows a trend of rapid disappearance of chondrichthyan species with depth when compared with bony fishes. Sharks, apparently well adapted to life at high pressures are conspicuous on slopes down to 2000 m including scavenging at food falls such as dead whales. We propose that they are excluded from the abyss by high-energy demand, including an oil-rich liver for buoyancy, which cannot be sustained in extreme oligotrophic conditions. Sharks are apparently confined to ca 30% of the total ocean and distribution of many species is fragmented around sea mounts, ocean ridges and ocean margins. All populations are therefore within reach of human fisheries, and there is no hidden reserve of chondrichthyan biomass or biodiversity in the deep sea. Sharks may be more vulnerable to over-exploitation than previously thought.     

Synthesis and biological evaluation of fused thio- and selenopyrans as new indolocarbazole analogues with aryl hydrocarbon receptor affinity

A series of thio- and selenopyrans having two fused indole units, structurally related to indolocarbazoles, have been prepared and evaluated for aryl hydrocarbon receptor (AhR) affinity, leading to the identification of several new significant AhR ligands. In particular, the parent thiopyrano[2,3-b:6,5-b′]diindole and its derivative having a methyl group in the central ring, as well as the two corresponding selenopyrans, displayed the highest potencies of the compounds tested.