Expression of Ca2+-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells

The second messenger NAADP triggers Ca²⁺ release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2^−/−), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca²⁺ responses as assessed by single-cell Ca²⁺ imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P₂ were only partially dependent on TPCs. In Tpcn1/2^−/− cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca²⁺-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca²⁺ release. High-affinity [³²P]NAADP binding still occurs in Tpcn1/2^−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca²⁺-permeable channels indispensable for NAADP signalling.     

Lithium Polyacrylate (LiPAA) as an Advanced Binder and a Passivating Agent for High‐Voltage Li‐Ion Batteries

Intensive studies of an advanced energy material are reported and lithium polyacrylate (LiPAA) is proven to be a surprisingly unique, multifunctional binder for high‐voltage Li‐ion batteries. The absence of effective passivation at the interface of high‐voltage cathodes in Li‐ion batteries may negatively affect their electrochemical performance, due to detrimental phenomena such as electrolyte solution oxidation and dissolution of transition metal cations. A strategy is introduced to build a stable cathode–electrolyte solution interphase for LiNi_0.5Mn_1.5O₄ (LNMO) spinel high‐voltage cathodes during the electrode fabrication process by simply using LiPAA as the cathode binder. LiPAA is a superb binder due to unique adhesion, cohesion, and wetting properties. It forms a uniform thin passivating film on LNMO and conducting carbon particles in composite cathodes and also compensates Li‐ion loss in full Li‐ion batteries by acting as an extra Li source. It is shown that these positive roles of LiPAA lead to a significant improvement in the electrochemical performance (e.g., cycle life, cell impedance, and rate capability) of LNMO/graphite battery prototypes, compared with that obtained using traditional polyvinylidene fluoride (PVdF) binder for LNMO cathodes. In addition, replacing PVdF with LiPAA binder for LNMO cathodes offers better adhesion, lower cost, and clear environmental advantages.     

The crystal structure of a high affinity RNA stem-loop complexed with the bacteriophage MS2 capsid: further challenges in the modeling of ligand-RNA interactions

We have determined the structure to 2.8 A of an RNA aptamer (F5), containing 2'-deoxy-2-aminopurine (2AP) at the -10 position, complexed with MS2 coat protein by soaking the RNA into precrystallised MS2 capsids. The -10 position of the RNA is an important determinant of binding affinity for coat protein. Adenine at this position in other RNA stem-loops makes three hydrogen bonds to protein functional groups. Substituting 2AP for the -10 adenine in the F5 aptamer yields an RNA with the highest yet reported affinity for coat protein. The refined X-ray structure shows that the 2AP base makes an additional hydrogen bond to the protein compared to adenine that is presumably the principal origin of the increased affinity. There are also slight changes in phosphate backbone positions compared to unmodified F5 that probably also contribute to affinity. Such phosphate movements are common in structures of RNAs bound to the MS2 T = 3 protein shell and highlight problems for de novo design of RNA binding ligands.     

Prostaglandin synthesis by enterocyte microsomes of rabbit small intestine

We studied the prostaglandin (PG) synthetic capacity of microsomes of a relatively pure population of rabbit enterocytes and determined ideal conditions for product synthesis. The epithelial cells were freed from the basement membrane by a combination of calcium chelation and mechanical vibration, and 100,000 X g microsomes were prepared. These microsomes were found to synthesize PG from exogenously added arachidonic acid. The ideal conditions for the reaction were a microsomal protein concentration of 1.0 mg/ml, an arachidonic acid concentration of 33 uM, a reaction mixture pH of 8.0-9.5 and with epinephrine 1.5 mM added as a cofactor. The product yields increased linearly with time up to 30 min. of incubation and were inhibited by 100 uM indomethacin. Under the above ideal conditions enterocyte microsomes yielded the following products expressed as pmole/mg protein/20 min. incubation: PGF2 alpha 98 +/- 7, PGE2 48 +/- 9, PGD2 28 +/- 7, TxB2 40 +/- 5, 6 Keto PGF1 alpha 15 +/- 6.     

Global genetic diversity of Aedes aegypti

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co‐occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub‐Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans‐Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.     

Non-hinge-binding pyrazolo[1,5-a]pyrimidines as potent B-Raf kinase inhibitors

As part of our research effort to discover B-Raf kinase inhibitors, we prepared a series of C-3 substituted N-(3-(pyrazolo[1,5-a]pyrimidin-7-yl)phenyl)-3-(trifluoromethyl)benzamides. X-ray crystallography studies revealed that one of the more potent inhibitors (10n) bound to B-Raf kinase without forming a hinge-binding hydrogen bond. With basic amine residues appended to C-3 aryl residues, cellular activity and solubility were enhanced over previously described compounds of this class.     

Molecular characterisation of inter and intra-specific somatic hybrids of potato using randomly amplified polymorphic DNA (RAPD) markers

Summary Protoplast fusion allows the transfer of both mono- and polygenic traits between species that are sexually incompatible. This approach has particular relevance for potato, and somatic hybridisation has been used to introduce a range of disease resistance genes from sexually incompatible wild species into the cultivated potato gene pool. In addition, protoplast fusion allows the resynthesis of tetraploid genotypes from pre-selected diploid or dihaploid donor parents. A limiting factor for the efficient exploitation of this technology in potato breeding is the difficulty of unequivocally identifying nuclear hybrids (heterokaryons). In order to facilitate the identification of hybrids at an early stage following fusion, Randomly Amplified Polymorphic DNA markers (RAPDs) have been used to characterise molecularly both inter- and intra-specific somatic hybrids of potato. RAPD markers detect naturally occurring polymorphism in the donor genotypes and utilise short oligonucleotide primers of arbitrary nucleotide sequence in combination with the polymerase chain reaction (PCR). The exploitation of RAPDs in the characterisation of both somatic and sexual hybrids is discussed.     

Transepithelial prostacyclin gradient in isolated canine trachea

Cyclooxygenase products of arachidonic acid, potential modulators of airway smooth muscle, have recently been described in bronchoalveolar lavage from canine lungs. To evaluate the possibility that airway epithelium represents a barrier to movement of prostacyclin (PGI2), an important bronchodilator synthesized by isolated airway, we measured the concentrations of 6-oxoprostaglandin F1 alpha (6-oxo-PGF1 alpha), the stable degradation product of PGI2, on the mucosal and serosal sides of isolated canine tracheal segments (CTS) mounted in Ussing chambers. 6-oxo-PGF1 alpha was measured by radioimmunoassay after purification by high-performance liquid chromatography. The concentration of 6-oxo-PGF1 alpha was significantly higher on the serosal than the mucosal side of CTS (1,262 +/- 252 vs. 390 +/- 168 pg.min-1.g-1, n = 8, P less than 0.05). A significant correlation was present between 6-oxo-PGF1 alpha measured on both sides of each CTS (r = 0.778, n = 26, P less than 0.01). 6-oxo-PGF1 alpha production from CTS stripped of mucosa was significantly greater than from isolated mucosa. Radiochromatograms obtained after incubation with [3H]arachidonic acid and calcium ionophore A23187 confirmed PGI2 as the predominant cyclooxygenase product of the submucosa, whereas the mucosa produced only small amounts of PGI2 in proportion to other cyclooxygenase products. PGI2 (10(-8) to 10(-6) M) applied to the mucosal surface of closed tracheal segments precontracted with histamine resulted in no significant relaxation, whereas serosal application showed a concentration-dependent effect. Radiolabeled 6-oxo-PGF1 alpha did not cross the isolated epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aboveground resource allocation in response to root herbivory as affected by the arbuscular mycorrhizal symbiosis

Aims

Arbuscular mycorrhizal (AM) fungi associate with the majority of terrestrial plants, influencing their growth, nutrient uptake and defence chemistry. Consequently, AM fungi can significantly impact plant-herbivore interactions, yet surprisingly few studies have investigated how AM fungi affect plant responses to root herbivores. This study aimed to investigate how AM fungi affect plant tolerance mechanisms to belowground herbivory.

Methods

We examined how AM fungi affect plant (Saccharum spp. hybrid) growth, nutrient dynamics and secondary chemistry (phenolics) in response to attack from a root-feeding insect (Dermolepida albohirtum).

Results

Root herbivory reduced root mass by almost 27%. In response, plants augmented investment in aboveground biomass by 25%, as well as increasing carbon concentrations. The AM fungi increased aboveground biomass, phosphorus and carbon. Meanwhile, root herbivory increased foliar phenolics by 31% in mycorrhizal plants, and increased arbuscular colonisation of roots by 75% overall. AM fungi also decreased herbivore performance, potentially via increasing root silicon concentrations.

Conclusions

Our results suggest that AM fungi may be able to augment plant tolerance to root herbivory via resource allocation aboveground and, at the same time, enhance plant root resistance by increasing root silicon. The ability of AM fungi to facilitate resource allocation aboveground in this way may be a more widespread strategy for plants to cope with belowground herbivory.