p73 deficiency results in impaired self renewal and premature neuronal differentiation of mouse neural progenitors independently of p53

The question of how neural progenitor cells maintain its self-renewal throughout life is a fundamental problem in cell biology with implications in cancer, aging and neurodegenerative diseases. In this work, we have analyzed the p73 function in embryonic neural progenitor cell biology using the neurosphere (NS)-assay and showed that p73-loss has a significant role in the maintenance of neurosphere-forming cells in the embryonic brain. A comparative study of NS from Trp73−/−, p53KO, p53KO;Trp73−/− and their wild-type counterparts demonstrated that p73 deficiency results in two independent, but related, phenotypes: a smaller NS size (related to the proliferation and survival of the neural-progenitors) and a decreased capacity to form NS (self-renewal). The former seems to be the result of p53 compensatory activity, whereas the latter is p53 independent. We also demonstrate that p73 deficiency increases the population of neuronal progenitors ready to differentiate into neurons at the expense of depleting the pool of undifferentiated neurosphere-forming cells. Analysis of the neurogenic niches demonstrated that p73-loss depletes the number of neural-progenitor cells, rendering deficient niches in the adult mice. Altogether, our study identifies TP73 as a positive regulator of self-renewal with a role in the maintenance of the neurogenic capacity. Thus, proposing p73 as an important player in the development of neurodegenerative diseases and a potential therapeutic target.     

Crystallographic analysis of the intact metal centres [3Fe-4S](1+/0) and [4Fe-4S](2+/1+) in a Zn(2+) -containing ferredoxin

Detailed structural models of di-cluster seven-iron ferredoxins constitute a valuable resource for folding and stability studies relating the metal cofactors' role in protein stability. The here reported, hemihedric twinned crystal structure at 2.0 A resolution from Acidianus ambivalens ferredoxin, shows an integral 103 residues, physiologically relevant native form composed by a N-terminal extension comprising a His/Asp Zn(2+) site and the ferredoxin (betaalphabeta)(2) core, which harbours intact clusters I and II, a [3Fe-4S](1+/0) and a [4Fe-4S](2+/1+) centres. This is in contrast with the previously available ferredoxin structure from Sulfolofus tokodai, which was obtained from an artificial oxidative conversion with two [3Fe-4S](1+/0) centres and poor definition around cluster II.     

Radial Dispersion of Neighbors and the Small-Scale Competitive Impact of Two Annual Grasses on a Native Perennial Grass

In California's Mediterranean type grasslands, native perennial grasses such as Nassella pulchra are surrounded by introduced annual species and these annuals are thought to have displaced natives through much of their range. Amongst other invaders, two grasses Lolium multiflorum and Bromus hordeaceus, commonly dominate portions of the grassland with potential for N. pulchra restoration. We hypothesized that competitor species differences and small‐scale gaps (150 cm²) could be important determinants of N. pulchra survival and performance on these sites. Lolium multiflorum and B. hordeaceus were planted in 20 cm diameter circular plots at a constant rate of 1 seed per cm² surrounding newly transplanted N. pulchra plants. Nassella pulchra showed no significant effect of the species of competitor or from the distribution of the competitors. Both interspersion of patches of bare ground and separation of competitors into patches did not increase N. pulchra pre‐dawn water potential, basal area change, number of seeds produced, or average weight of seeds. The presence of L. multiflorum was associated with a decrease in N. pulchra survival compared with plots with only B. hordeaceus. Plants with increases in basal area of less than 0.75 cm² during the growing season had 74% mortality compared with no mortality in plants with more growth. However, initial N. pulchra plant size was not a good predictor of mortality. Limiting competition from annuals may increase survival of N. pulchra plantings, but 60% of the plants survived for at least 1 year, despite being transplanted into soil containing substantial annual grass seed.     

Tyrosine kinase activity of a Ca2+/calmodulin-dependent protein kinase II catalytic fragment

A 30-kDa fragment of Ca²⁺/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX₄₀-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca²⁺/calmodulin-dependent protein kinase I (CX₄₀) were fused to the N-terminal end of 30K-CaMKII. Although CX₄₀-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX₄₀ region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.     

Polymorphic assemblies and crystalline arrays of lens tetraspanin MP20

Members of the tetraspanin superfamily function as transmembrane scaffold proteins that mediate the assembly of membrane proteins into specific signaling complexes. Tetraspanins also interact with each other and concentrate membrane proteins into tetraspanin-enriched microdomains (TEMs). Here we report that lens-specific tetraspanin MP20 can form multiple types of higher-order assemblies and we present crystalline arrays of MP20. When isolated in the absence of divalent cations, MP20 is solubilized predominantly in tetrameric form, whereas the presence of divalent cations during solubilization promotes the association of MP20 tetramers into higher-order species. This effect only occurs when divalent cations are present during solubilization but not when divalent cations are added to solubilized tetrameric MP20, suggesting that other factors may also be involved. When purified MP20 tetramers are reconstituted with native lens lipids in the presence of magnesium, MP20 forms two-dimensional (2D) crystals. A projection map at 18 Å resolution calculated from negatively stained 2D crystals showed that the building block of the crystal is an octamer consisting of two tetramers related to each other by 2-fold symmetry. In addition to 2D crystals, reconstitution of MP20 with native lipids also produced a variety of large protein-lipid complexes, and we present three-dimensional (3D) reconstructions of the four most abundant of these complexes in negative stain. The various complexes formed by MP20 most likely reflect the many ways in which tetraspanins can interact with each other to allow formation of TEMs.     

Structure and mode of action of a mosquitocidal holotoxin

The crystal structure of the full mosquitocidal toxin from Bacillus sphaericus (MTX_holo) has been determined at 2.5 Å resolution by the molecular replacement method. The resulting structure revealed essentially the complete chain consisting of four ricin B-type domains curling around the catalytic domain in a hedgehog-like assembly. As the structure was virtually identical in three different crystal packings, it is probably not affected by packing contacts. The structure of MTX_holo explains earlier autoinhibition data. An analysis of published complexes comprising ricin B-type lectin domains and sugar molecules shows that the general construction principle applies to all four lectin domains of MTX_holo, indicating 12 putative sugar-binding sites. These sites are sequence-related to those of the cytotoxin pierisin from cabbage butterfly, which are known to bind glycolipids. It seems therefore likely that MTX_holo also binds glycolipids. The seven contact interfaces between the five domains are predominantly polar and not stronger than common crystal contacts so that in an appropriate environment, the multidomain structure would likely uncurl into a string of single domains. The structure of the isolated catalytic domain plus an extended linker was established earlier in three crystal packings, two of which showed a peculiar association around a 7-fold axis. The catalytic domain of the reported MTX_holo closely resembles all three published structures, except one with an appreciable deviation of the 40 N-terminal residues. A comparison of all structures suggests a possible scenario for the translocation of the toxin into the cytosol.     

Histidine kinase regulation by a cyclophilin-like inhibitor

The sensor histidine kinase A (KinA) from Bacillus subtilis triggers a phosphorelay that activates sporulation. The antikinase KipI prevents sporulation by binding KinA and inhibiting the autophosphorylation reaction. Using neutron contrast variation, mutagenesis, and fluorescence data, we show that two KipI monomers bind via their C-domains at a conserved proline in the KinA dimerization and histidine-phosphotransfer (DHp) domain. Our crystal structure of the KipI C-domain reveals the binding motif has a distinctive hydrophobic groove formed by a five-stranded antiparallel β-sheet; a characteristic of the cyclophilin family of proteins that bind prolines and often act as cis-trans peptidyl-prolyl isomerases. We propose that the DHp domain of KinA transmits conformational signals to regulate kinase activity via this proline-mediated interaction. Given that both KinA and KipI homologues are widespread in the bacterial kingdom, this mechanism has broad significance in bacterial signal transduction.     

Analysis of the DDE Motif in the Mutator Superfamily

The eukaryotic Mutator family of transposable elements is widespread in plants. Active or potentially active copies are also found in fungi and protozoans, and sequences related to this family have been detected in metazoans as well. Members of this family are called Mutator-like elements (MULE s). They encode transposases, which contain a region conserved with transposases of the IS256 prokaryotic family, known to harbor a DDE catalytic domain. Different DDE or D34E motifs have been proposed in some groups of eukaryotic MULEs based on primary sequence conservation. On a large number of protein sequences related to, and representative of, all MULE families, we analyzed global conservation, the close environment of different acidic residues and the secondary structure. This allowed us to identify a potential DDE motif that is likely to be homologous to the one in IS256-like transposases. The characteristics of this motif are depicted in each known family of MULEs. Different hypotheses about the evolution of this triad are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Common wheat is one of the most important cereal crops in the world. The improvement of its yield and quality by the introduction of heterologous gene(s) is very significant. Avena sativa L. (2n = 42), belonging to the Avena tribe, possesses resistance to drought, coldness and many dis-eases. Its contents of proteins and fat in seed, especially lysine and unsaturated fatty acid are highest in crops, therefore it is regarded as healthy food. Sexual hybridization between wheat and Avena sativa…