Akt Phosphorylates Both Tsc1 and Tsc2 in Drosophila,but Neither Phosphorylation Is Required for Normal Animal Growth

Akt, an essential component of the insulin pathway, is a potent inducer of tissue growth. One of Akt''s phosphorylation targets is Tsc2, an inhibitor of the anabolic kinase TOR. This could account for part of Akt''s growth promoting activity. Although phosphorylation of Tsc2 by Akt does occur in vivo, and under certain circumstances can lead to reduced Tsc2 activity, the functional significance of this event is unclear since flies lacking Akt phosphorylation sites on Tsc2 are viable and normal in size and growth rate. Since Drosophila Tsc1, the obligate partner of Tsc2, has an Akt phosphorylation motif that is not conserved in mammals, we investigate here whether Akt redundantly phosphorylates the Tsc complex on Tsc1 and Tsc2. We provide evidence that Akt phosphorylates Tsc1 at Ser533. We show that flies lacking Akt phosphorylation sites on Tsc1 alone, or on both Tsc1 and Tsc2 concurrently, are viable and normal in size. This shows that phosphorylation of the Tsc1/2 complex by Akt is not required for Akt to activate TORC1 and to promote tissue growth in Drosophila.     

Apoptotic Engulfment Pathway and Schizophrenia

Background

Apoptosis has been speculated to be involved in schizophrenia. In a previously study, we reported the association of the MEGF10 gene with the disease. In this study, we followed the apoptotic engulfment pathway involving the MEGF10, GULP1, ABCA1 and ABCA7 genes and tested their association with the disease.

Methodology/Principal Findings

Ten, eleven and five SNPs were genotyped in the GULP1, ABCA1 and ABCA7 genes respectively for the ISHDSF and ICCSS samples. In all 3 genes, we observed nominally significant associations. Rs2004888 at GULP1 was significant in both ISHDSF and ICCSS samples (p = 0.0083 and 0.0437 respectively). We sought replication in independent samples for this marker and found highly significant association (p = 0.0003) in 3 Caucasian replication samples. But it was not significant in the 2 Chinese replication samples. In addition, we found a significant 2-marker (rs2242436 * rs3858075) interaction between the ABCA1 and ABCA7 genes in the ISHDSF sample (p = 0.0022) and a 3-marker interaction (rs246896 * rs4522565 * rs3858075) amongst the MEGF10, GULP1 and ABCA1 genes in the ICCSS sample (p = 0.0120). Rs3858075 in the ABCA1 gene was involved in both 2- and 3-marker interactions in the two samples.

Conclusions/Significance

From these data, we concluded that the GULP1 gene and the apoptotic engulfment pathway are involved in schizophrenia in subjects of European ancestry and multiple genes in the pathway may interactively increase the risks to the disease.     

Prevalence Study of Yaws in the Democratic Republic of Congo Using the Lot Quality Assurance Sampling Method

Background

Until the 1970s the prevalence of non-venereal trepanomatosis, including yaws, was greatly reduced after worldwide mass treatment. In 2005, cases were again reported in the Democratic Republic of the Congo. We carried out a survey to estimate the village-level prevalence of yaws in the region of Equator in the north of the country in order to define appropriate strategies to effectively treat the affected population.

Methodology/Principal Findings

We designed a community-based survey using the Lot Quality Assurance Sampling method to classify the prevalence of active yaws in 14 groups of villages (lots). The classification into high, moderate, or low yaws prevalence corresponded to World Health Organization prevalence thresholds for identifying appropriate operational treatment strategies. Active yaws cases were defined by suggestive clinical signs and positive rapid plasma reagin and Treponema pallidum hemagglutination serological tests. The overall prevalence in the study area was 4.7% (95% confidence interval: 3.4–6.0). Two of 14 lots had high prevalence (>10%), three moderate prevalence (5–10%) and nine low prevalence (<5%.).

Conclusions/Significance

Although yaws is no longer a World Health Organization priority disease, the presence of yaws in a region where it was supposed to be eradicated demonstrates the importance of continued surveillance and control efforts. Yaws should remain a public health priority in countries where previously it was known to be endemic. The integration of sensitive surveillance systems together with free access to effective treatment is recommended. As a consequence of our study results, more than 16,000 people received free treatment against yaws.     

Induction of Membrane Ceramides: A Novel Strategy to Interfere with T Lymphocyte Cytoskeletal Reorganisation in Viral Immunosuppression

Silencing of T cell activation and function is a highly efficient strategy of immunosuppression induced by pathogens. By promoting formation of membrane microdomains essential for clustering of receptors and signalling platforms in the plasma membrane, ceramides accumulating as a result of membrane sphingomyelin breakdown are not only essential for assembly of signalling complexes and pathogen entry, but also act as signalling modulators, e. g. by regulating relay of phosphatidyl-inositol-3-kinase (PI3K) signalling. Their role in T lymphocyte functions has not been addressed as yet. We now show that measles virus (MV), which interacts with the surface of T cells and thereby efficiently interferes with stimulated dynamic reorganisation of their actin cytoskeleton, causes ceramide accumulation in human T cells in a neutral (NSM) and acid (ASM) sphingomyelinase–dependent manner. Ceramides induced by MV, but also bacterial sphingomyelinase, efficiently interfered with formation of membrane protrusions and T cell spreading and front/rear polarisation in response to β1 integrin ligation or αCD3/CD28 activation, and this was rescued upon pharmacological or genetic ablation of ASM/NSM activity. Moreover, membrane ceramide accumulation downmodulated chemokine-induced T cell motility on fibronectin. Altogether, these findings highlight an as yet unrecognised concept of pathogens able to cause membrane ceramide accumulation to target essential processes in T cell activation and function by preventing stimulated actin cytoskeletal dynamics.     

Impact of <Emphasis Type="Italic">Quillaja saponaria</Emphasis> saponins on grapevine ecosystem organisms

The control of grapevine pathogens is a rising concern in Vitis vinifera culture. The current international trend is toward banning chemicals that are highly toxic to the environment and human workers, and adopting tighter regulations. We evaluated the impact of saponins on three kinds of organisms found in grapevine culture. The ectoparasitic nematode Xiphinema index, the parasitic fungus Botrytis cinerea and various yeast strains representative of the must fermentation population were incubated on synthetic media supplemented with variable concentrations of Quillaja saponaria saponins. Saponins induced reduction in the growth of B. cinerea and showed nematicide effects on X. index. The control of X. index and Botrytis cinerea is discussed in the context of the potential use of these chemicals as environmentally-friendly grapevine treatments. With Saccharomyces cerevisiae and other yeasts, saponins showed higher toxicity against S. cerevisiae strains isolated from wine or palm wine whereas laboratory strains or strains isolated from oak exhibited better resistance. This indicates that Q. saponaria saponins effects against yeast microflora should be assessed in the field before they can be considered an environmentally-safe new molecule against B. cinerea and X. index.     

Multiple effects of the cellular prion protein on tooth development

The role of the prion protein (PrP) in transmissible spongiform encephalopathies has been the focus of intense investigation. However, less is known about the physiological function of normal cellular PrP (PrP(C)). In adult human teeth, PrP(C) has been identified in odontoblasts, cementoblasts and epithelial remnants of Malassez. In this study, we have localized PrP(C) in developing human and mouse teeth, and investigated the function of PrP using a PrP-knockout (Prnp(0/0) ) mouse model. PrP(C) was detected in developing human and mouse ameloblasts and odontoblasts. In vitro, undifferentiated dental mesenchymal cells from embryonic day 18 (E18) Prnp(0/0) mouse molars proliferated much more rapidly compared to age-matched, wild-type (wt) mouse molar dental mesenchymal cells. Histochemistry and immunohistochemical analyses showed a subtle but measurable phenotype, with the absence of PrP resulting in earlier initiation of both dentin and enamel formation. Consistent with this finding, laser microdissected odontoblasts from newborn Prnp(0/0) mouse incisors had a reduced proliferation rate, as measured by the expression of proliferating cell nuclear antigen (PCNA), and increased type 1 collagen mRNA expression. Dentin microhardness of the fully erupted molars was reduced and incisal enamel mineralization was delayed in Prnp(0/0) compared to age-matched wt mouse teeth. Taken together, these results suggest that PrP(C) affects multiple processes involved in tooth formation, through regulating the differentiation of ameloblasts and odontoblasts.     

The BAR domain protein PICK1 regulates cell recognition and morphogenesis by interacting with Neph proteins

Neph proteins are evolutionarily conserved membrane proteins of the immunoglobulin superfamily that control the formation of specific intercellular contacts. Cell recognition through these proteins is essential in diverse cellular contexts such as patterning of the compound eye in Drosophila melanogaster, neuronal connectivity in Caenorhabditis elegans, and the formation of the kidney filtration barrier in mammals. Here we identify the PDZ and BAR domain protein PICK1 (protein interacting with C-kinase 1) as a Neph-interacting protein. Binding required dimerization of PICK1, was dependent on PDZ domain protein interactions, and mediated stabilization of Neph1 at the plasma membrane. Moreover, protein kinase C (PKCα) activity facilitated the interaction through releasing Neph proteins from their binding to the multidomain scaffolding protein zonula occludens 1 (ZO-1), another PDZ domain protein. In Drosophila, the Neph homologue Roughest is essential for sorting of interommatidial precursor cells and patterning of the compound eye. RNA interference-mediated knockdown of PICK1 in the Drosophila eye imaginal disc caused a Roughest destabilization at the plasma membrane and a phenotype that resembled rst mutation. These data indicate that Neph proteins and PICK1 synergistically regulate cell recognition and contact formation.     

Nematotoxicity of Marasmius oreades agglutinin (MOA) depends on glycolipid binding and cysteine protease activity

Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a Galα1,3Gal/GalNAc-specific lectin from the fairy ring mushroom that consists of an N-terminal ricin B-type lectin domain and a C-terminal dimerization domain. The latter domain shows structural similarity to catalytically active proteins, suggesting that, in addition to its carbohydrate-binding activity, MOA has an enzymatic function. Here, we demonstrate toxicity of MOA toward the model nematode Caenorhabditis elegans. This toxicity depends on binding of MOA to glycosphingolipids of the worm via its lectin domain. We show further that MOA has cysteine protease activity and demonstrate a critical role of this catalytic function in MOA-mediated nematotoxicity. The proteolytic activity of MOA was dependent on high Ca(2+) concentrations and favored by slightly alkaline pH, suggesting that these conditions trigger activation of the toxin at the target location. Our results suggest that MOA is a fungal toxin with intriguing similarities to bacterial binary toxins and has a protective function against fungivorous soil nematodes.     

High-resolution SNP scan of chromosome 6p21 in pooled samples from patients with complex diseases

We apply a high-throughput protocol of chip-based mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight; MALDI-TOF) as a method of screening for differences in single-nucleotide polymorphism (SNP) allele frequencies. Using pooled DNA from individuals with asthma, Crohn's disease (CD), schizophrenia, type 1 diabetes (T1D), and controls, we selected 534 SNPs from an initial set of 1435 SNPs spanning a 25-Mb region on chromosome 6p21. The standard deviations of measurements of time of flight at different dots, from different PCRs, and from different pools indicate reliable results on each analysis step. In 90% of the disease-control comparisons we found allelic differences of <10%. Of the T1D samples, which served as a positive control, 10 SNPs with significant differences were observed after taking into account multiple testing. Of these 10 SNPs, 5 are located between DQB1 and DRB1, confirming the known association with the DR3 and DR4 haplotypes whereas two additional SNPs also reproduced known associations of T1D with DOB and LTA. In the CD pool also, two earlier described associations were found with SNPs close to DRB1 and MICA. Additional associations were found in the schizophrenia and asthma pools. They should be confirmed in individual samples or can be used to develop further quality criteria for accepting true differences between pools. The determination of SNP allele frequencies in pooled DNA appears to be of value in assigning further genotyping priorities also in large linkage regions.