Maternal effects on anogenital distance in a wild marmot population

Peer reviewedPublisher PD

Dystropathology increases energy expenditure and protein turnover in the mdx mouse model of Duchenne muscular dystrophy

The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the dietary requirements for these macronutrients at different stages of the disease, are not well-understood. This study used juvenile (4- to 5- wk-old) and adult (12- to 14-wk-old) male dystrophic C57BL/10ScSn-mdx/J and age-matched C57BL/10ScSn/J control male mice to measure total and resting energy expenditure, food intake, spontaneous activity, body composition, whole body protein turnover, and muscle protein synthesis rates. In juvenile mdx mice that have extensive muscle damage, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were higher than in age-matched controls. Adaptations in food intake and decreased activity were insufficient to meet the increased energy and protein needs of juvenile mdx mice and resulted in stunted growth. In (non-growing) adult mdx mice with less severe dystropathology, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were also higher than in age-matched controls. Food intake was sufficient to meet their protein and energy needs, but insufficient to result in fat deposition. These data show that dystropathology impacts the protein and energy needs of mdx mice and that tailored dietary interventions are necessary to redress this imbalance. If not met, the resultant imbalance blunts growth, and may limit the benefits of therapies designed to protect and repair dystrophic muscles

Phosphorylation of p130Cas initiates Rac activation and membrane ruffling

<p>Abstract</p> <p>Background</p> <p>Non-receptor tyrosine kinases (NTKs) regulate physiological processes such as cell migration, differentiation, proliferation, and survival by interacting with and phosphorylating a large number of substrates simultaneously. This makes it difficult to attribute a particular biological effect to the phosphorylation of a particular substrate. We developed the Functional Interaction Trap (FIT) method to phosphorylate specifically a single substrate of choice in living cells, thereby allowing the biological effect(s) of that phosphorylation to be assessed. In this study we have used FIT to investigate the effects of specific phosphorylation of p130Cas, a protein implicated in cell migration. We have also used this approach to address a controversy regarding whether it is Src family kinases or focal adhesion kinase (FAK) that phosphorylates p130Cas in the trimolecular Src-FAK-p130Cas complex.</p> <p>Results</p> <p>We show here that SYF cells (mouse fibroblasts lacking the NTKs Src, Yes and Fyn) exhibit a low level of basal tyrosine phosphorylation at focal adhesions. FIT-mediated tyrosine phosphorylation of NTK substrates p130Cas, paxillin and FAK and cortactin was observed at focal adhesions, while FIT-mediated phosphorylation of cortactin was also seen at the cell periphery. Phosphorylation of p130Cas in SYF cells led to activation of Rac1 and increased membrane ruffling and lamellipodium formation, events associated with cell migration. We also found that the kinase activity of Src and not FAK is essential for phosphorylation of p130Cas when the three proteins exist as a complex in focal adhesions.</p> <p>Conclusion</p> <p>These results demonstrate that tyrosine phosphorylation of p130Cas is sufficient for its localization to focal adhesions and for activation of downstream signaling events associated with cell migration. FIT provides a valuable tool to evaluate the contribution of individual components of the response to signals with multiple outputs, such as activation of NTKs.</p

Implantation Serine Proteinase 1 Exhibits Mixed Substrate Specificity that Silences Signaling via Proteinase-Activated Receptors

Implantation S1 family serine proteinases (ISPs) are tryptases involved in embryo hatching and uterine implantation in the mouse. The two different ISP proteins (ISP1 and ISP2) have been detected in both pre- and post-implantation embryo tissue. To date, native ISP obtained from uterus and blastocyst tissues has been isolated only as an active hetero-dimer that exhibits trypsin-like substrate specificity. We hypothesised that in isolation, ISP1 might have a unique substrate specificity that could relate to its role when expressed alone in individual tissues. Thus, we isolated recombinant ISP1 expressed in Pichia pastoris and evaluated its substrate specificity. Using several chromogenic substrates and serine proteinase inhibitors, we demonstrate that ISP1 exhibits trypsin-like substrate specificity, having a preference for lysine over arginine at the P1 position. Phage display peptide mimetics revealed an expanded but mixed substrate specificity of ISP1, including chymotryptic and elastase activity. Based upon targets observed using phage display, we hypothesised that ISP1 might signal to cells by cleaving and activating proteinase-activated receptors (PARs) and therefore assessed PARs 1, 2 and 4 as potential ISP1 targets. We observed that ISP1 silenced enzyme-triggered PAR signaling by receptor-disarming. This PAR-disarming action of ISP1 may be important for embryo development and implantation

The male fetal biomarker INSL3 reveals substantial hormone exchange between fetuses in early pig gestation

The peptide hormone INSL3 is uniquely produced by the fetal testis to promote the transabdominal phase of testicular descent. Because it is fetal sex specific, and is present in only very low amounts in the maternal circulation, INSL3 acts as an ideal biomarker with which to monitor the movement of fetal hormones within the pregnant uterus of a polytocous species, the pig. INSL3 production by the fetal testis begins at around GD30. At GD45 of the ca.114 day gestation, a time at which testicular descent is promoted, INSL3 evidently moves from male to female allantoic compartments, presumably impacting also on the female fetal circulation. At later time-points (GD63, GD92) there is less inter-fetal transfer, although there still appears to be significant INSL3, presumably of male origin, in the plasma of female fetuses. This study thus provides evidence for substantial transfer of a peptide hormone between fetuses, and probably also across the placenta, emphasizing the vulnerability of the fetus to extrinsic hormonal influences within the uterus

Localized Fetomaternal Hyperglycemia: Spatial and Kinetic Definition by Positron Emission Tomography

to isolated hyperglycemia in the pregnant rat. mg/dL) localized to the left uterine artery was sustained for at least 48 hours while maternal euglycemia was maintained. fetal effects of isolated hyperglycemia. Broadly, this approach can be extended to study a variety of maternal-sided perturbations suspected to directly affect fetal health

Sequestration of Highly Expressed mRNAs in Cytoplasmic Granules, P-Bodies, and Stress Granules Enhances Cell Viability

Transcriptome analyses indicate that a core 10%–15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress

Metabolic Network for the Biosynthesis of Intra- and Extracellular alpha-Glucans Required for Virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis synthesizes intra- and extracellular alpha-glucans that were believed to originate from separate pathways. The extracellular glucose polymer is the main constituent of the mycobacterial capsule that is thought to be involved in immune evasion and virulence. However, the role of the alpha-glucan capsule in pathogenesis has remained enigmatic due to an incomplete understanding of alpha-glucan biosynthetic pathways preventing the generation of capsule-deficient mutants. Three separate and potentially redundant pathways had been implicated in alpha-glucan biosynthesis in mycobacteria: the GlgC-GlgA, the Rv3032 and the TreS-Pep2-GlgE pathways. We now show that alpha-glucan in mycobacteria is exclusively assembled intracellularly utilizing the building block alpha-maltose-1-phosphate as the substrate for the maltosyltransferase GlgE, with subsequent branching of the polymer by the branching enzyme GlgB. Some alpha-glucan is exported to form the alpha-glucan capsule. There is an unexpected convergence of the TreS-Pep2 and GlgC-GlgA pathways that both generate alpha-maltose-1-phosphate. While the TreS-Pep2 route from trehalose was already known, we have now established that GlgA forms this phosphosugar from ADP-glucose and glucose 1-phosphate 1000-fold more efficiently than its hitherto described glycogen synthase activity. The two routes are connected by the common precursor ADPglucose, allowing compensatory flux from one route to the other. Having elucidated this unexpected configuration of the metabolic pathways underlying alpha-glucan biosynthesis in mycobacteria, an M. tuberculosis double mutant devoid of alpha-glucan could be constructed, showing a direct link between the GlgE pathway, alpha-glucan biosynthesis and virulence in a mouse infection model

Lower glycolysis carries a higher flux than any biochemically possible alternative

The universality of many pathways of core metabolism suggests a strong role for evolutionary selection, but it remains unclear whether existing pathways have been selected from a large or small set of biochemical possibilities. To address this question, we construct "in silico" all possible biochemically feasible alternatives to the trunk pathway of glycolysis and gluconeogenesis, one of the most highly conserved pathways in metabolism. We show that, even though a large number of alternative pathways exist, the alternatives carry lower flux than the real pathway under typical physiological conditions. Alternative pathways that could potentially carry higher flux often lead to infeasible intermediate metabolite concentrations. We also find that if physiological conditions were different, different pathways could outperform those found in nature. Our results demonstrate how the rules of biochemistry restrict the alternatives that are open to evolution, and suggest that the existing trunk pathway of glycolysis and gluconeogenesis represents a maximal flux solution.Comment: 9 pages, 4 figure