Variation of mobile carbon reserves in trees at the alpine treeline ecotone is under environmental control

? In low temperature-adapted plants, including treeline trees, light-saturated photosynthesis is considerably less sensitive to temperature than growth. As a consequence, all plants tested so far show increased nonstructural carbohydrate (NSC) tissue concentrations when exposed to low temperatures. Reduced carbon supply is thus an unlikely cause for low temperature range limits of plants. For altitudinal treeline trees there is, however, a possibility that high NSC genotypes have been selected. ? Here, we explored this possibility using afforestations with single-provenance conifers along elevational gradients in the Southern Chilean Andes and the Swiss Alps. ? Tree growth was measured at each of four approximately equidistant elevations at and below the treeline. Additionally, at the same elevations, needle, branch and stem sapwood tissues were collected to determine NSC concentrations. ? Overall, growth decreased and NSC concentrations increased with elevation. Along with previous empirical and experimental studies, the findings of this study provide no indication of NSC reduction at the treeline; NSC increased in most species (each represented by one common population) towards their upper climatic limit. The disparity between carbon acquisition and structural carbon investment at low temperature (accumulation of NSC) thus does occur even among genotypes not adapted to treeline environments.     

Congenital asplenia in mice and humans with mutations in a Pbx/Nkx2-5/p15 module

The molecular determinants of spleen organogenesis and the etiology of isolated congenital asplenia (ICA), a life-threatening human condition, are unknown. We previously reported that Pbx1 deficiency causes organ growth defects including asplenia. Here, we show that mice with splenic mesenchyme-specific Pbx1 inactivation exhibit hyposplenia. Moreover, the loss of Pbx causes downregulation of Nkx2-5 and derepression of p15Ink4b in spleen mesenchymal progenitors, perturbing the cell cycle. Removal of p15Ink4b in Pbx1 spleen-specific mutants partially rescues spleen growth. By whole-exome sequencing of a multiplex kindred with ICA, we identify a heterozygous missense mutation (P236H) in NKX2-5 showing reduced transactivation in vitro. This study establishes that a Pbx/Nkx2-5/p15 regulatory module is essential for spleen development.     

Centronuclear Myopathy in Labrador Retrievers: A Recent Founder Mutation in the PTPLA Gene Has Rapidly Disseminated Worldwide

Centronuclear myopathies (CNM) are inherited congenital disorders characterized by an excessive number of internalized nuclei. In humans, CNM results from ∼70 mutations in three major genes from the myotubularin, dynamin and amphiphysin families. Analysis of animal models with altered expression of these genes revealed common defects in all forms of CNM, paving the way for unified pathogenic and therapeutic mechanisms. Despite these efforts, some CNM cases remain genetically unresolved. We previously identified an autosomal recessive form of CNM in French Labrador retrievers from an experimental pedigree, and showed that a loss-of-function mutation in the protein tyrosine phosphatase-like A (PTPLA) gene segregated with CNM. Around the world, client-owned Labrador retrievers with a similar clinical presentation and histopathological changes in muscle biopsies have been described. We hypothesized that these Labradors share the same PTPLA^cnm mutation. Genotyping of an international panel of 7,426 Labradors led to the identification of PTPLA^cnm carriers in 13 countries. Haplotype analysis demonstrated that the PTPLA^cnm allele resulted from a single and recent mutational event that may have rapidly disseminated through the extensive use of popular sires. PTPLA-deficient Labradors will help define the integrated role of PTPLA in the existing CNM gene network. They will be valuable complementary large animal models to test innovative therapies in CNM.     

Transition state chirality and role of the vicinal hydroxyl in the ribosomal peptidyl transferase reaction

The ribosomal peptidyl transferase is a biologically essential catalyst responsible for protein synthesis. The reaction is expected to proceed through a transition state approaching tetrahedral geometry with a specific chirality. To establish that stereospecificity, we synthesized two diastereomers of a transition state inhibitor with mimics for each of the four ligands around the reactive chiral center. Preferential binding of the inhibitor that mimics a transition state with S chirality establishes the spatial position of the nascent peptide and the oxyanion and places the amine near the critical A76 2'-OH group on the P-site tRNA. Another inhibitor series with 2'-NH 2 and 2'-SH substitutions at the critical 2'-OH group was used to test the neutrality of the 2'-OH group as predicted if the hydroxyl functions as a proton shuttle in the transition state. The lack of significant pH-dependent binding by these inhibitors argues that the 2'-OH group remains neutral in the transition state. Both of these observations are consistent with a proton shuttle mechanism for the peptidyl transferase reaction.     

IFN-γ Mediates the Rejection of Haematopoietic Stem Cells in IFN-γR1-Deficient Hosts

Background

Interferon-γ receptor 1 (IFN-γR1) deficiency is a life-threatening inherited disorder, conferring predisposition to mycobacterial diseases. Haematopoietic stem cell transplantation (HSCT) is the only curative treatment available, but is hampered by a very high rate of graft rejection, even with intra-familial HLA-identical transplants. This high rejection rate is not seen in any other congenital disorders and remains unexplained. We studied the underlying mechanism in a mouse model of HSCT for IFN-γR1 deficiency.

Methods and Findings

We demonstrated that HSCT with cells from a syngenic C57BL/6 Ifngr1 ^+/+ donor engrafted well and restored anti-mycobacterial immunity in naive, non-infected C57BL/6 Ifngr1 ^−/− recipients. However, Ifngr1 ^−/− mice previously infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) rejected HSCT. Like infected IFN-γR1-deficient humans, infected Ifngr1 ^−/− mice displayed very high serum IFN-γ levels before HSCT. The administration of a recombinant IFN-γ-expressing AAV vector to Ifngr1 ^−/− naive recipients also resulted in HSCT graft rejection. Transplantation was successful in Ifngr1 ^−/− × Ifng ^−/− double-mutant mice, even after BCG infection. Finally, efficient antibody-mediated IFN-γ depletion in infected Ifngr1 ^−/− mice in vivo allowed subsequent engraftment.

Conclusions

High serum IFN-γ concentration is both necessary and sufficient for graft rejection in IFN-γR1-deficient mice, inhibiting the development of heterologous, IFN-γR1-expressing, haematopoietic cell lineages. These results confirm that IFN-γ is an anti-haematopoietic cytokine in vivo. They also pave the way for HSCT management in IFN-γR1-deficient patients through IFN-γ depletion from the blood. They further raise the possibility that depleting IFN-γ may improve engraftment in other settings, such as HSCT from a haplo-identical or unrelated donor.     

Divergent functional properties of the ribosome-associated molecular chaperone Ssb compared with other Hsp70s.

Ssbs of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, which can be cross-linked to nascent polypeptide chains. Because Ssbs are members of a divergent subclass of Hsp70s found thus far only in fungi, we asked if the structural requirements for in vivo function were similar to those of "classic" Hsp70s. An intact peptide-binding domain is essential and an alteration of a conserved residue in the peptide-binding cleft (V442) affects function. However, Ssb tolerates a number of alterations in the peptide-binding cleft, revealing a high degree of flexibility in its functional requirements. Because binding of Ssb to peptide substrates in vitro was undetectable, we assessed the importance of substrate binding using the chimera BAB, in which the peptide binding domain of Ssb is exchanged for the analogous domain of the more "classical" Hsp70, Ssa. BAB, which binds peptide substrates in vitro, can substitute for Ssb in vivo. Alteration of a residue in the peptide-binding cleft of BAB creates a protein with a reduced affinity for peptide and altered ribosome binding that is unable to substitute for Ssb in vivo. These results indicate that Ssb's ability to bind unfolded polypeptides is likely critical for its function. This binding accounts, in part, for its stable interaction with translating ribosomes, even although it has a low affinity for peptides that detectably bind to other Hsp70s in vitro. These unusual properties may allow Ssb to function efficiently as a chaperone for ribosome-bound nascent chains.     

Gain-of-glycosylation mutations

Disease-causing missense (and other in-frame) mutations can exert their deleterious effects at the cellular level through multiple mechanisms. A pathogenic mechanism involves the addition of a novel N-linked glycan. Up to 1.4% of known disease-causing missense mutations are predicted to give rise to gains-of-glycosylation. For some of these mutations, the novel glycans have been shown to be both necessary and sufficient to account for the deleterious impact of the mutation. The chemical complementation of cells from patients in vitro with various modifiers of glycosylation has been demonstrated and raises the possibility of specific chemical treatments for patients bearing gain-of-glycosylation mutations.