Contrasting expression of canonical Wnt signaling reporters TOPGAL, BATGAL and Axin2(LacZ) during murine lung development and repair

Canonical WNT signaling plays multiple roles in lung organogenesis and repair by regulating early progenitor cell fates: investigation has been enhanced by canonical Wnt reporter mice, TOPGAL, BATGAL and Axin2(LacZ). Although widely used, it remains unclear whether these reporters convey the same information about canonical Wnt signaling. We therefore compared beta-galactosidase expression patterns in canonical Wnt signaling of these reporter mice in whole embryo versus isolated prenatal lungs. To determine if expression varied further during repair, we analyzed comparative pulmonary expression of beta-galactosidase after naphthalene injury. Our data show important differences between reporter mice. While TOPGAL and BATGAL lines demonstrate Wnt signaling well in early lung epithelium, BATGAL expression is markedly reduced in late embryonic and adult lungs. By contrast, Axin2(LacZ) expression is sustained in embryonic lung mesenchyme as well as epithelium. Three days into repair after naphthalene, BATGAL expression is induced in bronchial epithelium as well as TOPGAL expression (already strongly expressed without injury). Axin2(LacZ) expression is increased in bronchial epithelium of injured lungs. Interestingly, both TOPGAL and Axin2(LacZ) are up regulated in parabronchial smooth muscle cells during repair. Therefore the optimal choice of Wnt reporter line depends on whether up- or down-regulation of canonical Wnt signal reporting in either lung epithelium or mesenchyme is being compared.     

A kinetic model for Xylella fastidiosa adhesion, biofilm formation, and virulence

Xylella fastidiosa is the causal agent of citrus variegated chlorosis and Pierce's disease which are the major threat to the citrus and wine industries. The most accepted hypothesis for Xf diseases affirms that it is a vascular occlusion caused by bacterial biofilm, embedded in an extracellular translucent matrix that was deduced to be the exopolysaccharide fastidian. Fourier transform infrared spectroscopy analysis demonstrated that virulent cells which form biofilm on glass have low fastidian content similar to the weak virulent ones. This indicates that high amounts of fastidian are not necessary for adhesion. In this paper we propose a kinetic model for X. fastidiosa adhesion, biofilm formation, and virulence based on electrostatic attraction between bacterial surface proteins and xylem walls. Fastidian is involved in final biofilm formation and cation sequestration in dilute sap.     

Fragmentation gradients differentially affect the species range distributions of four taxonomic groups in semi‐deciduous Atlantic forest

Human activities often cause habitat fragmentation and how forest fragments affect species range distributions has implications for ecology and conservation. However, few studies have considered communities within the same landscape. Here, we analyzed metacommunity structure to determine the range distributions for species in four taxonomic groups (amphibians, birds, social wasps, and trees) in a patchy landscape of semi‐deciduous Atlantic forest in southwestern Brazil. Although trees are a key component of the environment for animals in forested patches, the ranges of bird, wasp, and amphibian species did not change in concert with the species ranges of trees. The species ranges of amphibians and social wasps were unaffected by fragmentation gradients and exhibited independent distribution patterns (i.e., random structure). In contrast, birds and trees exhibited range turnover along different fragmentation gradients, indicating that species show idiosyncratic responses to abiotic factors (i.e., Gleasonian structure). For birds, some less‐resilient species occurred only in fragments with a large area of native vegetation at a radius of 5 km from the center of the sampled forest fragments, whereas other more stress‐tolerant species occurred only in sites with small areas of native vegetation. For trees, some later succession species (e.g., animal‐dispersed seeds) occurred only in fragments with high connectivity, whereas earlier‐recruiting species (e.g., wind‐dispersed seeds) occurred in fragments with low connectivity. Thus, determining the effects of human‐modified landscapes on species range distributions, even within the same landscape, might not be a trivial task.     

Clinical and genetic characterization of Chanarin-Dorfman syndrome

We describe the clinical features, muscle pathology features, and molecular studies of seven patients with Chanarin-Dorfman syndrome (CDS) or neutral lipid storage disease and ichthyosis (NLSDI), a multisystem triglyceride storage disease with massive accumulation of lipid droplets in muscle fibers.All patients presented with congenital ichthyosiform erythroderma, cytoplasmic lipid droplets in blood cells, mild to severe hepatomegaly, and increased serum CK levels and liver enzymes. Three patients showed muscle symptoms and three had steathorrea. Molecular analysis identified five mutations, three of which are novel.These findings expand the clinical and mutational spectrum and underline the genetic heterogeneity of this disease.     

Analysis of Intracellular Substrates and Products of Thimet Oligopeptidase in Human Embryonic Kidney 293 Cells

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9–11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8–10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.Intracellular protein turnover is a crucial step for cell functioning, and if this process is impaired, the elevated levels of aged proteins usually lead to the formation of intracellular insoluble aggregates that can cause severe pathologies (1). In mammalian cells, most proteins destined for degradation are initially tagged with a polyubiquitin chain in an energy-dependent process and then digested to small peptides by the 26 S proteasome, a large proteolytic complex involved in the regulation of cell division, gene expression, and other key processes (2, 3). In eukaryotes, 30–90% of newly synthesized proteins may be degraded by proteasomes within minutes of synthesis (3, 4). In addition to proteasomes, other extralysosomal proteolytic systems have been reported (5, 6). The proteasome cleaves proteins into peptides that are typically 2–20 amino acids in length (7). In most cases, these peptides are thought to be rapidly hydrolyzed into amino acids by aminopeptidases (8–10). However, some intracellular peptides escape complete degradation and are imported into the endoplasmic reticulum where they associate with major histocompatibility complex class I (MHC-I)³ molecules and traffic to the cell surface for presentation to the immune system (10–12). Additionally, based on the fact that free peptides added to the intracellular milieu can regulate cellular functions mediated by protein interactions such as gene regulation, metabolism, cell signaling, and protein targeting (13, 14), intracellular peptides generated by proteasomes that escape degradation have been suggested to play a role in regulating protein interactions (15). Indeed, oligopeptides isolated from rat brain tissue using the catalytically inactive EP24.15 (EC 3.4.24.15) were introduced into Chinese hamster ovarian-S and HEK293 cells and were found capable of altering G protein-coupled receptor signal transduction (16). Moreover, EP24.15 overexpression itself changed both angiotensin II and isoproterenol signal transduction, suggesting a physiological function for its intracellular substrates/products (16).EP24.15 is a zinc-dependent peptidase of the metallopeptidase M3 family that contains the HEXXH motif (17). This enzyme was first described as a neuropeptide-degrading enzyme present in the soluble fraction of brain homogenates (18). Whereas EP24.15 can be secreted (19, 20), its predominant location in the cytosol and nucleus suggests that the primary function of this enzyme is not the extracellular degradation of neuropeptides and hormones (21, 22). EP24.15 was shown in vivo to participate in antigen presentation through MHC-I (23–25) and in vitro to bind (26) or degrade (27) some MHC-I associated peptides. EP24.15 has also been shown in vitro to degrade peptides containing 5–17 amino acids produced after proteasome digestion of β-casein (28). EP24.15 shows substrate size restriction to peptides containing from 5 to 17 amino acids because of its catalytic center that is located in a deep channel (29). Despite the size restriction, EP24.15 has a broad substrate specificity (30), probably because a significant portion of the enzyme-binding site is lined with potentially flexible loops that allow reorganization of the active site following substrate binding (29). Recently, it has also been suggested that certain substrates may be cleaved by an open form of EP24.15 (31). This characteristic is supported by the ability of EP24.15 to accommodate different amino acid residues at subsites S4 to S3′, which even includes the uncommon post-proline cleavage (30). Such biochemical and structural features make EP24.15 a versatile enzyme to degrade structurally unrelated oligopeptides.Previously, brain peptides that bound to catalytically inactive EP24.15 were isolated and identified using mass spectrometry (22). The majority of peptides captured by the inactive enzyme were intracellular protein fragments that efficiently interacted with EP24.15; the smallest peptide isolated in these assays contained 5 and the largest 17 amino acids (15, 16, 22, 32), which is within the size range previously reported for natural and synthetic substrates of EP24.15 (18, 30, 33, 34). Interestingly, the peptides released by the proteasome are in the same size range of EP24.15 competitive inhibitors/substrates (7, 35, 36). Taken altogether, these data suggest that in the intracellular environment EP24.15 could further cleave proteasome-generated peptides unrelated to MHC-I antigen presentation (15).Although the mutated inactive enzyme “capture” assay was successful in identifying several cellular protein fragments that were substrates for EP24.15, it also found some interacting peptides that were not substrates. In this study, we used several approaches to directly screen for cellular peptides that were cleaved by EP24.15. The first approach involved the extraction of cellular peptides from the HEK293 cell line, incubation in vitro with purified EP24.15, labeling with isotopic tags, and analysis by mass spectrometry to obtain quantitative data on the extent of cleavage. The second approach examined the effect of EP24.15 overexpression on the cellular levels of peptides in the HEK293 cell line. The third set of experiments tested synthetic peptides with purified EP24.15 in vitro, and examined cleavage by high pressure liquid chromatography and mass spectrometry. Collectively, these studies have identified a large number of intracellular peptides, including those that likely represent the endogenous substrates and products of EP24.15, and this original information contributes to a better understanding of the function of this enzyme in vivo.     

Evolution of mating within the Candida parapsilosis species group

Candida orthopsilosis and Candida metapsilosis are closely related to Candida parapsilosis, a major cause of infection in premature neonates. Mating has not been observed in these species. We show that ～190 isolates of C. parapsilosis contain only an MTLa idiomorph at the mating-type-like locus. Here, we describe the isolation and characterization of the MTL loci from C. orthopsilosis and C. metapsilosis. Among 16 C. orthopsilosis isolates, 9 were homozygous for MTLa, 5 were homozygous for MTLα, and 2 were MTLa/α heterozygotes. The C. orthopsilosis isolates belonged to two divergent groups, as characterized by restriction patterns at MTL, which probably represent subspecies. We sequenced both idiomorphs from each group and showed that they are 95% identical and that the regulatory genes are intact. In contrast, 18 isolates of C. metapsilosis contain only MTLα idiomorphs. Our results suggest that the role of MTL in determining cell type is being eroded in the C. parapsilosis species complex. The population structure of C. orthopsilosis indicates that mating may occur. However, expression of genes in the mating signal transduction pathway does not respond to exposure to alpha factor. C. parapsilosis is also nonresponsive, even when the GTPase-activating protein gene SST2 is deleted. In addition, splicing of introns in MTLa1 and MTLa2 is defective in C. orthopsilosis. Mating is not detected. The alpha factor peptide, which is the same sequence in C. parapsilosis, C. orthopsilosis, and C. metapsilosis, can induce a mating response in Candida albicans. It is therefore likely either that mating of C. orthopsilosis takes place under certain unidentified conditions or that the mating pathway has been adapted for other functions, such as cross-species communication.     

Photosynthetic pigments content, <Emphasis Type="Italic">δ</Emphasis>-aminolevulinic acid dehydratase and acid phosphatase activities and mineral nutrients concentration in cadmium-exposed <Emphasis Type="Italic">Cucumis sativus</Emphasis> L.

In this study, the effects of cadmium chloride (CdCl₂) on plant growth, histology of roots, photosynthetic pigments content, δ-aminolevulinic acid dehydratase (ALA-D; E.C. 4.2.1.24) and acid phosphatase activities (AP; E.C. 3.1.3.2), soluble phosphorus (Pi) measurement and mineral nutrients content in cucumber seedlings (Cucumis sativus L.) were investigated. Cucumber seedlings were grown in vitro in an agar-solidified substrate containing four CdCl₂ treatments (0, 100, 400, and 1000 μM) for ten days. Cd was readily absorbed by seedlings and its content was greater in the roots than in the shoot. Cd reduced shoot and root length, and fresh and dry biomass of seedlings. Inhibition of root cell elongation in Cd-treated seedlings was observed by the increase of the mean radial size of cells belonging to three zones of the root tip. The highest level of Cd reduced in a similar manner chlorophyll a, chlorophyll b and total chlorophyll contents. Increasing concentrations of Cd resulted in a linear decrease in carotenoids levels of cotyledons. Interestingly, the ALA-D activity in cotyledons was inhibited only at the highest level of Cd. Root and shoot AP activities were, respectively, activated and inhibited at all CdCl₂ concentrations. Root Pi concentration was increased in all Cd treatments and it was not altered in the shoot tissues. Moreover, in general, the nutrient contents were increased in the root and decreased in the shoot. Therefore, we suggest that Cd affects negatively growth, photosynthetic pigments, ALA-D and AP activities and partition of mineral nutrients in cucumber seedlings.     

Lack of support for the association between GAD2 polymorphisms and severe human obesity

The demonstration of association between common genetic variants and chronic human diseases such as obesity could have profound implications for the prediction, prevention, and treatment of these conditions. Unequivocal proof of such an association, however, requires independent replication of initial positive findings. Recently, three (-243 A>G, +61450 C>A, and +83897 T>A) single nucleotide polymorphisms (SNPs) within glutamate decarboxylase 2 (GAD2) were found to be associated with class III obesity (body mass index > 40 kg/m2). The association was observed among 188 families (612 individuals) segregating the condition, and a case-control study of 575 cases and 646 lean controls. Functional data supporting a pathophysiological role for one of the SNPs (-243 A>G) were also presented. The gene GAD2 encodes the 65-kDa subunit of glutamic acid decarboxylase-GAD65. In the present study, we attempted to replicate this association in larger groups of individuals, and to extend the functional studies of the -243 A>G SNP. Among 2,359 individuals comprising 693 German nuclear families with severe, early-onset obesity, we found no evidence for a relationship between the three GAD2 SNPs and obesity, whether SNPs were studied individually or as haplotypes. In two independent case-control studies (a total of 680 class III obesity cases and 1,186 lean controls), there was no significant relationship between the -243 A>G SNP and obesity (OR = 0.99, 95% CI 0.83-1.18, p = 0.89) in the pooled sample. These negative findings were recapitulated in a meta-analysis, incorporating all published data for the association between the -243G allele and class III obesity, which yielded an OR of 1.11 (95% CI 0.90-1.36, p = 0.28) in a total sample of 1,252 class III obese cases and 1,800 lean controls. Moreover, analysis of common haplotypes encompassing the GAD2 locus revealed no association with severe obesity in families with the condition. We also obtained functional data for the -243 A>G SNP that does not support a pathophysiological role for this variant in obesity. Potential confounding variables in association studies involving common variants and complex diseases (low power to detect modest genetic effects, overinterpretation of marginal data, population stratification, and biological plausibility) are also discussed in the context of GAD2 and severe obesity.     

Plasmodium falciparum glycogen synthase kinase-3: molecular model, expression, intracellular localisation and selective inhibitors

Worldwide increasing resistance of Plasmodium falciparum to common anti-malaria agents calls for the urgent identification of new drugs. Glycogen synthase kinase-3 (GSK-3) represents a potential screening target for the identification of such new compounds. We have cloned PfGSK-3, the P. falciparum gene homologue of GSK-3 beta. It encodes a 452-amino-acid, 53-kDa protein with an unusual N-terminal extension but a well-conserved catalytic domain. A PfGSK-3 tridimensional homology model was generated on the basis of the recently crystallised human GSK-3 beta. It illustrates how the regions involved in the active site, in substrate binding (P+4 phosphate binding domain) and in activity regulation are highly conserved. Recombinant PfGSK-3 phosphorylates GS-1, a GSK-3-specific peptide substrate, glycogen synthase, recombinant axin and the microtubule-binding protein tau. Neither native nor recombinant PfGSK-3 binds to axin. Expression and intracellular localisation of PfGSK-3 were investigated in the erythrocytic stages. Although PfGSK-3 mRNA is present in similar amounts at all stages, the PfGSK-3 protein is predominantly expressed at the early trophozoite stage. Once synthesized, PfGSK-3 is rapidly transported to the erythrocyte cytoplasm where it associates with vesicle-like structures. The physiological functions of PfGSK-3 for the parasite remain to be elucidated. A series of GSK-3 beta inhibitors were tested on both PfGSK-3 and mammalian GSK-3beta. Remarkably these enzymes show a partially divergent sensitivity to the compounds, suggesting that PfGSK-3 selective compounds might be identified.