The p53 Tumor Suppressor Causes Congenital Malformations in Rpl24-Deficient Mice and Promotes Their Survival

Hypomorphic mutation in one allele of ribosomal protein l24 gene (Rpl24) is responsible for the Belly Spot and Tail (Bst) mouse, which suffers from defects of the eye, skeleton, and coat pigmentation. It has been hypothesized that these pathological manifestations result exclusively from faulty protein synthesis. We demonstrate here that upregulation of the p53 tumor suppressor during the restricted period of embryonic development significantly contributes to the Bst phenotype. However, in the absence of p53 a large majority of Rpl24^Bst/+ embryos die. We showed that p53 promotes survival of these mice via p21-dependent mechanism. Our results imply that activation of a p53-dependent checkpoint mechanism in response to various ribosomal protein deficiencies might also play a role in the pathogenesis of congenital malformations in humans.Nascent ribosome biogenesis is required during cell growth, proliferation and differentiation (42, 47). It is temporally and spatially organized within the nucleolus, where rRNAs are transcribed, processed, modified, and assembled with ribosomal proteins (RPS) to generate the mature 40S and 60S ribosomal subunits (13). RPS participate in additional steps in ribosome biogenesis in the nucleoplasm and the cytoplasm, such as the transport of ribosomal precursors, stabilization of ribosome structure, and regulation of different steps in protein synthesis (15).The critical role of at least some RPS in mammals is underscored by the pathological or lethal consequences of the deficiency of just one allele. Only a few heterozygous mutations of RP genes have been shown to be viable in mammals, and each of them has shown a relatively specific phenotype. Germ line heterozygous mutation for RPS19, RPS24, RPS17, RPL35a, RPL5, and RPL11 genes have been found in patients with Diamond-Blackfan anemia, which is characterized by absent or decreased erythropoiesis, and less frequently by small stature and various somatic malformations that mostly occur in the cephalic region, as well as an increased incidence of leukemia, osteogenic sarcoma, and myelodisplastic syndrome (7, 9, 12, 17, 18). Recently, a link between heterozygous mutations in Rps19 and Rps20 and dark skin phenotype in mice has been demonstrated (29).Another heterozygous RP mutant is the Belly Spot and Tail (Bst) mouse (34). This is a semidominant, hypomorphic mutation caused by an intronic deletion in the Rpl24 gene, affecting Rpl24 mRNA splicing. Rpl24^Bst/+ mice are characterized by reduced body size, a white ventral middle spot, white hind feet, retinal abnormalities, a kinked tail, and other skeletal abnormalities. Since Rpl24^Bst/+ mouse embryonic fibroblasts from these mice showed a significant reduction in the rate of overall protein synthesis, it has been suggested that their phenotype result exclusively from faulty translation of mRNAs in tissues that depend on rapid and flawless protein synthesis (34).It has been argued that the differential phenotypes of heterozygous mutants of RP genes in mammals might be attributable to the expression levels of the respective RP and the consequent decrease in the amount of ribosomes, impairment of specific steps in protein synthesis, and potential extraribosomal function. However, it should be pointed out that the relative contribution of the impaired protein synthesis or extraribosomal function to these phenotypes remains to be determined (9, 17, 34, 36, 37, 43, 57).Recent evidence indicates that deficiencies in individual RPS could lead to pathological consequences via activation of a p53-dependent checkpoint regulatory mechanism. We demonstrated that inducible deletion of the Rps6 gene in the liver of adult mice inhibits the synthesis of the 40S ribosomal subunit, as well as proliferation of liver cells, after partial hepatectomy, despite seemingly unaffected protein synthesis (54). These observations suggested the existence of a novel checkpoint, downstream of the deficiency in ribosome biogenesis. Likewise, the perigastrulation lethality of Rps6 heterozygote embryos appears to reflect the triggering of a p53-dependent checkpoint response rather than a deficit in protein synthesis (37). We have assumed that activation of a p53-dependent checkpoint is triggered by impaired rRNA processing in the nucleolus in Rps6-deficient cells, since the nucleolar structure and function are compromised by almost all known p53-inducing stresses (37, 41, 50). Based on all of these observations, it could be speculated that the rare occurrence of RP heterozygosity in mammals reflects the fatal consequences of p53-dependent checkpoint activation (36, 37). In addition to function in development, this checkpoint may also play a role in other processes. Since various RP deficiencies in Drosophila melanogaster, zebrafish, and humans pose a great risk for development of malignant tumors, it is possible that induction of a p53-dependent checkpoint response prevents expansion of such potentially hazardous cells (1, 9, 11, 56).Recently, we initiated an RNA interference screen for RP deficiencies that upregulate the p53 tumor suppressor in A549 cells. It has been previously suggested that a defect in ribosome biogenesis in the nucleolus caused by a RP deficiency triggers the p53 response (36, 37, 50). A number of studies in yeast showed that Rpl24 does not participate in ribosome biogenesis in the nucleolus, but it assembles late with the nascent 60S ribosomes in the cytoplasm and regulates the 60S subunit joining step during translation initiation and other steps in protein synthesis (10, 25, 45). Thus, it was surprising to observe that RPL24 deficiency triggered the p53 response in our screen. This observation led us to consider the possibility that p53 is upregulated in Rpl24^Bst/+ mice. In contrast to previous opinion that the phenotype of these mice results exclusively from impaired protein synthesis (34), we demonstrate here that it is largely caused by the aberrant upregulation of p53 protein expression during embryonic development.     

Chaperone-Like Antibodies in Neurodegenerative Tauopathies: Implication for Immunotherapy

Alzheimer’s disease (AD) belongs to the category of neurodegenerative tauopathies, which are characterized by intracellular and extracellular accumulation of misfolded tau. Structurally, tau belongs to the family of the intrinsically disordered proteins that are characterized by the absence of well-defined three-dimensional structure of the free protein. In the course of neurodegeneration, intrinsically disordered tau protein gains highly ordered misfolded structure. Currently it is widely accepted that misfolded tau proteins represent viable drug target for prospective therapeutic development. Until now several therapeutic approaches targeting misfolded tau were developed. Monoclonal antibodies with chaperone-like activities that would be able to neutralize the toxic gain of function of misfolded tau represent novel promising immunological concept in the treatment of AD. We suggest that antibodies as specific chaperones targeting misfolded proteins may serve as potent therapeutic drugs of AD as well as others conformational diseases.     

Improved reproducibility in preparing precision-cut liver tissue slices

More than 35 years ago double Nobel laureate Linus Carl Pauling published a powerful model of the molecular mechanism of general anesthesia, generally referred to as the hydrate-microcrystal (aqueous-phase) theory. This hypothesis, based on the molecular behavior of water molecules, did not receive serious attention during Pauling’s life time, when scientific tools for examining complex systems such as the brain were still in their infancy. The situation has since drastically changed, and, now, in the twenty first century, many scientific tools are available for examining different types of complex systems. The discovery of aquaporin-4, a subtype of water channel abundantly expressed in glial systems, further highlighted the concept that the dynamics of water molecules in the cerebral cortex play an important role in important physiological brain functions including consciousness and information processing.     

Distribution and Population Densities of Diurnal Primates in the Karst Forests of Phong Nha – Ke Bang National Park,Quang Binh Province,Central Vietnam

We studied the status and distribution of the diurnal primates in the Phong Nha – Ke Bang National Park (PNKB NP) from April to August 2007. In the past, researchers reported 9 primate species and subspecies for the karst forests of PNKB NP, which constitutes the most important protected area for the endangered Hatinh langur (Trachypithecus hatinhensis) in Vietnam. All 9 primate taxa are threatened due to hunting pressure and habitat loss. We applied line transect sampling in 4 areas. During a total of 117 transect inspections along 12 different transect routes, we confirmed 5 primate taxa and the analysis of population densities resulted in 2143 (±467) Hatinh langurs, 1316 (±871) red-shanked douc langurs (Pygathrix nemaeus), 930 (±489) stump-tailed macaques (Macaca arctoides), 986 (±883) eastern Assamese macaques (M. a. assamensis), and 18 (±18) southern white-cheeked crested gibbons (Nomascus siki) in the whole PNKB NP, which covers an area of ca. 85,000 ha. We could not detect the 2 nocturnal lorises, Bengal slow lorises (Nycticebus bengalensis) and pygmy slow lorises (N. pygmaeus), as well as rhesus macaques (Macaca mulatta) and northern pig-tail macaques (M. leonina). The distribution of the primates predominantly depended on human impact. We could not recognize a correlation between habitat constitution and abundance of primates. The population density estimates showed a much higher density of the Hatinh langur than previously assumed. Thus the importance of the PNKB NP for the conservation of this endangered langur increased significantly.     

Structure and Function Relationship of the Autotransport and Proteolytic Activity of EspP from Shiga Toxin-Producing Escherichia coli

Background

The serine protease autotransporter EspP is a proposed virulence factor of Shiga toxin-producing Escherichia coli (STEC). We recently distinguished four EspP subtypes (EspPα, EspPβ, EspPγ, and EspPδ), which display large differences in transport and proteolytic activities and differ widely concerning their distribution within the STEC population. The mechanisms underlying these functional variations in EspP subtypes are, however, unknown.

Methodology/Principal Findings

The structural basis of proteolytic and autotransport activity was investigated using transposon-based linker scanning mutagenesis, site-directed mutagenesis and structure-function analysis derived from homology modelling of the EspP passenger domain. Transposon mutagenesis of the passenger domain inactivated autotransport when pentapeptide linker insertions occurred in regions essential for overall correct folding or in a loop protruding from the β-helical core. Loss of proteolytic function was limited to mutations in Domain 1 in the N-terminal third of the EspP passenger. Site-directed mutagenesis demonstrated that His¹²⁷, Asp¹⁵⁶ and Ser²⁶³ in Domain 1 form the catalytic triad of EspP.

Conclusions/Significance

Our data indicate that in EspP i) the correct formation of the tertiary structure of the passenger domain is essential for efficient autotransport, and ii) an elastase-like serine protease domain in the N-terminal Domain 1 is responsible for the proteolytic phenotype. Lack of stabilizing interactions of Domain 1 with the core structure of the passenger domain ablates proteolytic activity in subtypes EspPβ and EspPδ.     

A Novel Tandem Mass Spectrometry Method for Rapid Confirmation of Medium- and Very Long-Chain acyl-CoA Dehydrogenase Deficiency in Newborns

Background

Newborn screening for medium- and very long-chain acyl-CoA dehydrogenase (MCAD and VLCAD, respectively) deficiency, using acylcarnitine profiling with tandem mass spectrometry, has increased the number of patients with fatty acid oxidation disorders due to the identification of additional milder, and so far silent, phenotypes. However, especially for VLCADD, the acylcarnitine profile can not constitute the sole parameter in order to reliably confirm disease. Therefore, we developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method to rapidly determine both MCAD- and/or VLCAD-activity in human lymphocytes in order to confirm diagnosis.

Methodology

LC-MS/MS was used to measure MCAD- or VLCAD-catalyzed production of enoyl-CoA and hydroxyacyl-CoA, in human lymphocytes.

Principal Findings

VLCAD activity in controls was 6.95±0.42 mU/mg (range 1.95 to 11.91 mU/mg). Residual VLCAD activity of 4 patients with confirmed VLCAD-deficiency was between 0.3 and 1.1%. Heterozygous ACADVL mutation carriers showed residual VLCAD activities of 23.7 to 54.2%. MCAD activity in controls was 2.38±0.18 mU/mg. In total, 28 patients with suspected MCAD-deficiency were assayed. Nearly all patients with residual MCAD activities below 2.5% were homozygous 985A>G carriers. MCAD-deficient patients with one other than the 985A>G mutation had higher MCAD residual activities, ranging from 5.7 to 13.9%. All patients with the 199T>C mutation had residual activities above 10%.

Conclusions

Our newly developed LC-MS/MS method is able to provide ample sensitivity to correctly and rapidly determine MCAD and VLCAD residual activity in human lymphocytes. Importantly, based on measured MCAD residual activities in correlation with genotype, new insights were obtained on the expected clinical phenotype.     

Phylogenetic analysis of Fosterella L.B. Sm. (Pitcairnioideae, Bromeliaceae) based on four chloroplast DNA regions

The about 31 species of Fosterella L.B. Sm. (Bromeliaceae) are terrestrial herbs with a centre of diversity in the central South American Andes. To resolve infra- and intergeneric relationships among Fosterella and their putative allies, we conducted a phylogenetic analysis based on sequence data from four chloroplast DNA regions (matK gene, rps16 intron, atpB-rbcL and psbB-psbH intergenic spacers). Sequences were generated for 96 accessions corresponding to 60 species from 18 genera. Among these, 57 accessions represented 22 of the 31 recognized Fosterella species and one undescribed morphospecies. Maximum parsimony and Bayesian inference methods yielded well-resolved phylogenies. The monophyly of Fosterella was strongly supported, as was its sister relationship with a clade comprising Deuterocohnia, Dyckia and Encholirium. Six distinct evolutionary lineages were distinguished within Fosterella. Character mapping indicated that parallel evolution of identical character states is common in the genus. Relationships between species and lineages are discussed in the context of morphological, ecological and biogeographical data as well as the results of a previous amplified fragment length polymorphism (AFLP) study.     

Effects of carrion resources on herbivore spatial distribution are mediated by facultative scavengers

Carcasses of large herbivores are pulsed resources whose impact on animal communities and ecological processes is poorly understood. In temperate forests, long-lasting ungulate carcasses are a prime resource for many species of birds and mammals during winter. Facultative carrion-eaters also consume live prey, thus potentially leading to unexpected secondary effects on populations of species not directly linked to carcass exploitation. By snow-tracking and direct observations we investigated in Bia?owie?a Forest (E. Poland) whether large ungulate carcasses elicit spatial responses in facultative scavengers and their prey. We found that in the vicinity of carcass sites the probability of the presence of common ravens Corvus corax, jays Garrulus glandarius and red foxes Vulpes vulpes increased significantly. Indeed, large groups of the two bird species were exclusively found in those places. Because of these aggregations, the probability of predator–prey encounters (red foxes and brown hares Lepus europaeus) was significantly higher near carcass sites. Accordingly, the abundance of hares and other live prey such as red squirrels Sciurus vulgaris decreased at their vicinities, probably as a consequence of direct killing and/or predator avoidance. This study provides the first evidence of carrion pulses permeating into apparently distant trophic levels, such as herbivores, via facultative scavengers, thus highlighting some unnoticed but relevant effects of carrion resources on community structure.     

Preparation and biological activity of 6-benzylaminopurine derivatives in plants and human cancer cells

To study the structure-activity relationships of aromatic cytokinins, the cytokinin activity at both the receptor and cellular levels, as well as CDK inhibitory and anticancer properties of 38 6-benzylaminopurine (BAP) derivatives were compared in various in vitro assays. The compounds were prepared by the condensation of 6-chloropurine with corresponding substituted benzylamines. The majority of synthesised derivatives exhibited high activity in all three of the cytokinin bioassays employed (tobacco callus, wheat senescence and Amaranthus bioassay). The highest activities were obtained in the senescence bioassay. For some compounds tested, significant differences of activity were found in the bioassays used, indicating that diverse recognition systems may operate and suggesting that it may be possible to modulate particular cytokinin-dependent processes with specific compounds. Position-specific steric and hydrophobic effects of different phenyl ring substituents on the variation of biological activity were confirmed. In contrast to their high activity in bioassays, the BAP derivatives were recognised with much lower sensitivity than trans-zeatin in both Arabidopsis thaliana AHK3 and AHK4 receptor assays. The compounds were also investigated for their effects on cyclin-dependent kinase 2 (CDK2) and for antiproliferative properties on cancer and normal cell lines. Several of the tested compounds showed stronger inhibitory activity and cytotoxicity than BAP. There was also a significant positive correlation of the inhibitory effects on human and plant CDKs with cell proliferation of cancer and cytokinin-dependent tobacco cells, respectively. This suggests that at least a part of the antiproliferative effect of the new cytokinins was due to the inhibition of CDK activity.