Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

Endoplasmic reticulum–synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins.     

Liquid Serial Dilution Is Inferior to Solid Media for Isolation of Cultures Representative of the Phylum-Level Diversity of Soil Bacteria

Representatives of only four well-characterized bacterial phyla were isolated from a pasture soil by using liquid serial dilution culture. In contrast, members of Acidobacteria, Verrucomicrobia, and Gemmatimonadetes and of other poorly represented bacterial lineages were isolated in earlier experiments with solidified versions of the same media. We conclude that, contrary to expectation, liquid serial dilution culture is inferior to culturing on solid media for isolating representatives of many bacterial phyla from soil.     

Development of an autotrophic culture system for the in vitro mycorrhization of potato plantlets

An autotrophic culture system was developed for the in vitro mycorrhization of potato plantlets. Roots of micropropagated plantlets were associated to an arbuscular mycorrhizal fungus under in vitro conditions, while shoots developed under open air conditions. Several thousand spores, an extensive extraradical mycelium and an abundant root colonization were obtained. Spores were able to colonize new plantlets under the same conditions. These results support the capacity of the autotrophic culture system to continuously culture arbuscular mycorrhizal fungi and may serve as a powerful tool to investigate various aspects of the symbiosis for which a source-sink relationship and photosynthetic active tissues are necessary.     

Caenorhabditis elegans: a versatile platform for drug discovery

Drug discovery and drug target identification are two intimately linked facets of intervention strategies aimed at effectively combating pathological conditions in humans. Simple model organisms provide attractive platforms for devising and streamlining efficient drug discovery and drug target identification methodologies. The nematode worm Caenorhabditis elegans has emerged as a particularly convenient and versatile tool that can be exploited to achieve these goals. Although C. elegans is a relatively modern addition to the arsenal of model organisms, its biology has already been investigated to an exceptional level. This, coupled with effortless handling and a notable low cost of cultivation and maintenance, allows seamless implementation of high-throughput drug screening approaches as well as in-depth genetic and biochemical studies of the molecular pathways targeted by specific drugs. In this review, we introduce C. elegans as a model organism with significant advantages toward the identification of molecular drug targets. In addition, we discuss the value of the worm in the development of drug screening and drug evaluation protocols. The unique features of C. elegans, which greatly facilitate drug studies, hold promise for both deciphering disease pathogenesis and formulating educated and effective therapeutic interventions.     

Axl deficiency does not affect adipogenesis or adipose tissue development

To evaluate a potential role of Axl, the high-affinity receptor of growth arrest-specific protein 6 (GAS6) in adiposity, murine embryonic fibroblasts (MEF) derived from mice with genetic deficiency of Axl (Axl(-/-)) or wild-type littermates (Axl(+/+)) were differentiated into mature adipocytes. In addition, Axl(-/-) and Axl(+/+) mice were kept on standard fat diet (SFD) or on high-fat diet (HFD) for 15 weeks. Deficiency of Axl in MEF did not affect differentiation, as shown by a similar uptake of Oil Red O and expression of the adipogenic markers aP2 and peroxisome proliferator activator receptor γ (PPARγ) at the end of the differentiation. In the first 7 weeks of HFD feeding, Axl(-/-) mice gained less weight than their wild-type littermates. Weight gain for both genotypes on either SFD of HFD over 15 weeks was, however, not significantly different, resulting in comparable body weights, as well as subcutaneous (s.c.) and gonadal (GON) fat mass. Adipocyte size in the fat tissues was not affected by Axl deficiency. Gene expression analysis indicated that the absence of Axl in vivo may be compensated for by the other TAM family members Mer and Tyro3. Glucose and insulin tolerance tests (ITT) in Axl(-/-) and Axl(+/+) mice did not reveal significant differences in glucose homeostasis. Thus, Axl deficiency had no significant effect on adipogenesis in vitro or in vivo.     

Identification of mimotopes with diagnostic potential for Trypanosoma brucei gambiense variant surface glycoproteins using human antibody fractions

Background

At present, screening of the population at risk for gambiense human African trypanosomiasis (HAT) is based on detection of antibodies against native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. Drawbacks of these native VSGs include culture of infective T.b. gambiense trypanosomes in laboratory rodents, necessary for production, and the exposure of non-specific epitopes that may cause cross-reactions. We therefore aimed at identifying peptides that mimic epitopes, hence called “mimotopes,” specific to T.b. gambiense VSGs and that may replace the native proteins in antibody detection tests.

Methodology/Principal Findings

A Ph.D.-12 peptide phage display library was screened with polyclonal antibodies from patient sera, previously affinity purified on VSG LiTat 1.3 or LiTat 1.5. The peptide sequences were derived from the DNA sequence of the selected phages and synthesised as biotinylated peptides. Respectively, eighteen and twenty different mimotopes were identified for VSG LiTat 1.3 and LiTat 1.5, of which six and five were retained for assessment of their diagnostic performance. Based on alignment of the peptide sequences on the original protein sequence of VSG LiTat 1.3 and 1.5, three additional peptides were synthesised. We evaluated the diagnostic performance of the synthetic peptides in indirect ELISA with 102 sera from HAT patients and 102 endemic negative controls. All mimotopes had areas under the curve (AUCs) of ≥0.85, indicating their diagnostic potential. One peptide corresponding to the VSG LiTat 1.3 protein sequence also had an AUC of ≥0.85, while the peptide based on the sequence of VSG LiTat 1.5 had an AUC of only 0.79.

Conclusions/Significance

We delivered the proof of principle that mimotopes for T.b. gambiense VSGs, with diagnostic potential, can be selected by phage display using polyclonal human antibodies.     

Plasminogen activator inhibitor type I may contribute to transient, non-specific changes in immunity in the subacute phase of murine tuberculosis

Tuberculosis, caused by Mycobacterium (M.) tuberculosis, is a devastating infectious disease causing many deaths worldwide. Non-specific host defense mechanisms such as the coagulation and fibrinolytic system may give insight in possible new therapeutic targets. Plasminogen activator inhibitor type-1 (PAI-1), an important regulator of inflammation and fibrinolysis, might be of interest as tuberculosis patients have elevated plasma levels of PAI-1. In this study we set out to investigate the role of PAI-1 during tuberculosis in vivo. Wildtype (WT) and PAI-1 deficient (PAI-1^?/?) mice were intranasally infected with M. tuberculosis H37rv and sacrificed after 2, 5 and 29 weeks. Five weeks post-infection, bacterial loads in lungs of PAI-1^?/? mice were significantly higher compared to WT mice, while no differences were seen 2 and 29 weeks post-infection. At two weeks post-infection increased influx of macrophages and lymphocytes was observed. PAI-1 deficiency was associated with a reduced cytokine response in the lungs; however, upon stimulation with tuberculin purified protein derivative (PPD), PAI-1^?/? splenocytes released increased levels of IFN-γ compared to WT. No clear differences were found between PAI-1^?/? and WT mice at 29 weeks after infection. In conclusion, these data suggest that PAI-1 contributes to transient, non-specific changes in immunity during the early phase of murine tuberculosis.     

COG5-CDG: expanding the clinical spectrum

Background

The Conserved Oligomeric Golgi (COG) complex is involved in the retrograde trafficking of Golgi components, thereby affecting the localization of Golgi glycosyltransferases. Deficiency of a COG-subunit leads to defective protein glycosylation, and thus Congenital Disorders of Glycosylation (CDG). Mutations in subunits 1, 4, 5, 6, 7 and 8 have been associated with CDG-II. The first patient with COG5-CDG was recently described (Paesold-Burda et al. Hum Mol Genet 2009; 18:4350–6). Contrary to most other COG-CDG cases, the patient presented a mild/moderate phenotype, i.e. moderate psychomotor retardation with language delay, truncal ataxia and slight hypotonia.

Methods

CDG-IIx patients from our database were screened for mutations in COG5. Clinical data were compared. Brefeldin A treatment of fibroblasts and immunoblotting experiments were performed to support the diagnosis.

Results and conclusion

We identified five new patients with proven COG5 deficiency. We conclude that the clinical picture is not always as mild as previously described. It rather comprises a broad spectrum with phenotypes ranging from mild to very severe. Interestingly, on a clinical basis some of the patients present a significant overlap with COG7-CDG, a finding which can probably be explained by subunit interactions at the protein level.

     

Absence of carbon transfer between Medicago truncatula plants linked by a mycorrhizal network, demonstrated in an experimental microcosm

Carbon transfer between plants via a common extraradical network of arbuscular mycorrhizal (AM) fungal hyphae has been investigated abundantly, but the results remain equivocal. We studied the transfer of carbon through this fungal network, from a Medicago truncatula donor plant to a receiver (1) M. truncatula plant growing under decreased light conditions and (2) M. truncatula seedling. Autotrophic plants were grown in bicompartmented Petri plates, with their root systems physically separated, but linked by the extraradical network of Glomus intraradices. A control Myc-/Nod- M. truncatula plant was inserted in the same compartment as the receiver plant. Following labeling of the donor plant with 13CO2, 13C was recovered in the donor plant shoots and roots, in the extraradical mycelium and in the receiver plant roots. Fatty acid analysis of the receiver's roots further demonstrated 13C enrichment in the fungal-specific lipids, while almost no label was detected in the plant-specific compounds. We conclude that carbon was transferred from the donor to the receiver plant via the AM fungal network, but that the transferred carbon remained within the intraradical AM fungal structures of the receiver's root and was not transferred to the receiver's plant tissues.