Tricarboxylic acid cycle enzyme activities in a mouse model of methylmalonic aciduria

Methylmalonic acidemia (MMA) is a propionate pathway disorder caused by dysfunction of the mitochondrial enzyme methylmalonyl-CoA mutase (MMUT). MMUT catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA, an anaplerotic reaction which feeds into the tricarboxylic acid (TCA) cycle. As part of the pathological mechanisms of MMA, previous studies have suggested there is decreased TCA activity due to a toxic inhibition of TCA cycle enzymes by MMA related metabolites, in addition to reduced anaplerosis. Here, we have utilized mitochondria isolated from livers of a mouse model of MMA (Mut-ko/ki) and their littermate controls (Ki/wt) to examine the amounts and enzyme functions of most of the TCA cycle enzymes. We have performed mRNA quantification, protein semi-quantitation, and enzyme activity quantification for TCA cycle enzymes in these samples. Expression profiling showed increased mRNA levels of fumarate hydratase in the Mut-ko/ki samples, which by contrast had reduced protein levels as detected by immunoblot, while all other mRNA levels were unaltered. Immunoblotting also revealed decreased protein levels of 2-oxoglutarate dehydrogenase and malate dehydrogenase 2. Interesting, the decreased protein amount of 2-oxoglutarate dehydrogenase was reflected in decreased activity for this enzyme while there is a trend towards decreased activity of fumarate hydratase and malate dehydrogenase 2. Citrate synthase, isocitrate dehydrogenase 2/3, succinyl-CoA synthase, and succinate dehydrogenase are not statistically different in terms of quantity of enzyme or activity. Finally, we found decreased activity when examining the function of methylmalonyl-CoA mutase in series with succinate synthase and succinate dehydrogenase in the Mut-ko/ki mice compared to their littermate controls, as expected. This study demonstrates decreased activity of certain TCA cycle enzymes and by corollary decreased TCA cycle function, but it supports decreased protein quantity rather than toxic inhibition as the underlying mechanism of action. SUMMARY: Methylmalonic acidemia (MMA) is an inborn metabolic disorder of propionate catabolism. In this disorder, toxic metabolites are considered to be the major pathogenic mechanism for acute and long-term complications. However, despite optimized therapies aimed at reducing metabolite levels, patients continue to suffer from late complications, including metabolic stroke and renal insufficiency. Since the propionate pathway feeds into the tricarboxylic acid (TCA) cycle, we investigated TCA cycle function in a constitutive MMA mouse model. We demonstrated decreased amounts of the TCA enzymes, Mdh2 and Ogdh as semi-quantified by immunoblot. Enzymatic activity of Ogdh is also decreased in the MMA mouse model compared to controls. Thus, when the enzyme amounts are decreased, we see the enzymatic activity also decreased to a similar extent for Ogdh. Further studies to elucidate the structural and/or functional links between the TCA cycle and propionate pathways might lead to new treatment approaches for MMA patients

Distinct ligand binding sites in integrin alpha3beta1 regulate matrix adhesion and cell-cell contact.

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition

Microscopic dynamics of thin hard rods

Based on the collision rules for hard needles we derive a hydrodynamic equation that determines the coupled translational and rotational dynamics of a tagged thin rod in an ensemble of identical rods. Specifically, based on a Pseudo-Liouville operator for binary collisions between rods, the Mori-Zwanzig projection formalism is used to derive a continued fraction representation for the correlation function of the tagged particle's density, specifying its position and orientation. Truncation of the continued fraction gives rise to a generalised Enskog equation, which can be compared to the phenomenological Perrin equation for anisotropic diffusion. Only for sufficiently large density do we observe anisotropic diffusion, as indicated by an anisotropic mean square displacement, growing linearly with time. For lower densities, the Perrin equation is shown to be an insufficient hydrodynamic description for hard needles interacting via binary collisions. We compare our results to simulations and find excellent quantitative agreement for low densities and qualtitative agreement for higher densities.Comment: 21 pages, 6 figures, v2: clarifications and improved readabilit

Proposed guidelines for the diagnosis and management of methylmalonic and propionic acidemia.

Methylmalonic and propionic acidemia (MMA/PA) are inborn errors of metabolism characterized by accumulation of propionic acid and/or methylmalonic acid due to deficiency of methylmalonyl-CoA mutase (MUT) or propionyl-CoA carboxylase (PCC). MMA has an estimated incidence of ~ 1: 50,000 and PA of ~ 1:100\u27000 -150,000. Patients present either shortly after birth with acute deterioration, metabolic acidosis and hyperammonemia or later at any age with a more heterogeneous clinical picture, leading to early death or to severe neurological handicap in many survivors. Mental outcome tends to be worse in PA and late complications include chronic kidney disease almost exclusively in MMA and cardiomyopathy mainly in PA. Except for vitamin B12 responsive forms of MMA the outcome remains poor despite the existence of apparently effective therapy with a low protein diet and carnitine. This may be related to under recognition and delayed diagnosis due to nonspecific clinical presentation and insufficient awareness of health care professionals because of disease rarity

Distinct ligand binding sites in integrin α3β1 regulate matrix adhesion and cell–cell contact

The integrin α3β1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with α3β1 via a surface loop within the α3 β-propeller (residues 242–246) but outside the laminin binding region, suggesting that uPAR–integrin interactions could signal differently from matrix engagement. To explore this, α3−/− epithelial cells were reconstituted with wild-type (wt) α3 or α3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt α3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and γ-catenin. Src kinase inhibition or expression of Src 1–251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that α3β1 regulates both cell–cell contact and matrix adhesion, but through distinct protein interaction sites within its β-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition

Regulation of α5β1 integrin conformation and function by urokinase receptor binding

Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with β1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the β-propeller of α3β1 empowers vitronectin adhesion by this integrin. How uPAR modifies other β1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds α5β1 and rather than blocking, renders fibronectin (Fn) binding by α5β1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of α5β1–uPAR to Fn type III repeats 12–15 in addition to type III repeats 9–11 bound by α5β1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall α5β1 conformation. A β1 peptide (res 224NLDSPEGGF232) that models near the known α-chain uPAR-binding region, or a β1-chain Ser227Ala point mutation, abrogated effects of uPAR on α5β1. Direct binding and regulation of α5β1 by uPAR implies a modified “bent” integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands

Parametric Polyhedra with at least kk Lattice Points: Their Semigroup Structure and the k-Frobenius Problem

Given an integral $d \times n$ matrix $A$, the well-studied affine semigroup \mbox{ Sg} (A)=\{ b : Ax=b, \ x \in {\mathbb Z}^n, x \geq 0\} can be stratified by the number of lattice points inside the parametric polyhedra $P_A(b)=\{x: Ax=b, x\geq0\}$. Such families of parametric polyhedra appear in many areas of combinatorics, convex geometry, algebra and number theory. The key themes of this paper are: (1) A structure theory that characterizes precisely the subset \mbox{ Sg}_{\geq k}(A) of all vectors b \in \mbox{ Sg}(A) such that $P_A(b) \cap {\mathbb Z}^n$ has at least $k$ solutions. We demonstrate that this set is finitely generated, it is a union of translated copies of a semigroup which can be computed explicitly via Hilbert bases computations. Related results can be derived for those right-hand-side vectors $b$ for which $P_A(b) \cap {\mathbb Z}^n$ has exactly $k$ solutions or fewer than $k$ solutions. (2) A computational complexity theory. We show that, when $n$, $k$ are fixed natural numbers, one can compute in polynomial time an encoding of \mbox{ Sg}_{\geq k}(A) as a multivariate generating function, using a short sum of rational functions. As a consequence, one can identify all right-hand-side vectors of bounded norm that have at least $k$ solutions. (3) Applications and computation for the $k$-Frobenius numbers. Using Generating functions we prove that for fixed $n,k$ the $k$-Frobenius number can be computed in polynomial time. This generalizes a well-known result for $k=1$ by R. Kannan. Using some adaptation of dynamic programming we show some practical computations of $k$-Frobenius numbers and their relatives

A North Atlantic tephrostratigraphical framework for 130-60 ka b2k:new tephra discoveries, marine-based correlations, and future challenges

Building chronological frameworks for proxy sequences spanning 130–60 ka b2k is plagued by difficulties and uncertainties. Recent developments in the North Atlantic region, however, affirm the potential offered by tephrochronology and specifically the search for cryptotephra. Here we review the potential offered by tephrostratigraphy for sequences spanning 130–60 ka b2k. We combine newly identified cryptotephra deposits from the NGRIP ice-core and a marine core from the Iceland Basin with previously published data from the ice and marine realms to construct the first tephrostratigraphical framework for this time-interval. Forty-three tephra or cryptotephra deposits are incorporated into this framework; twenty three tephra deposits are found in the Greenland ice-cores, including nine new NGRIP tephras, and twenty separate deposits are preserved in various North Atlantic marine sequences. Major, minor and trace element results are presented for the new NGRIP horizons together with age estimates based on their position within the ice-core record. Basaltic tephras of Icelandic origin dominate the framework with only eight tephras of rhyolitic composition found. New results from marine core MD99-2253 also illustrate some of the complexities and challenges of assessing the depositional integrity of marine cryptotephra deposits. Tephra-based correlations in the marine environment provide independent tie-points for this time-interval and highlight the potential of widening the application of tephrochronology. Further investigations, however, are required, that combine robust geochemical fingerprinting and a rigorous assessment of tephra depositional processes, in order to trace coeval events between the two depositional realms

The global build-up to intrinsic edge localized mode bursts seen in divertor full flux loops in JET

A global signature of the build-up to an intrinsic edge localized mode (ELM) is found in the temporal analytic phase of signals measured in full flux azimuthal loops in the divertor region of JET. Toroidally integrating, full flux loop signals provide a global measurement proportional to the voltage induced by changes in poloidal magnetic flux; they are electromagnetically induced by the dynamics of spatially integrated current density. We perform direct time-domain analysis of the high time-resolution full flux loop signals VLD2 and VLD3. We analyze plasmas where a steady H-mode is sustained over several seconds during which all the observed ELMs are intrinsic; there is no deliberate intent to pace the ELMing process by external means. ELM occurrence times are determined from the Be II emission at the divertor. We previously [Chapman et al., Phys. Plasmas 21, 062302 (2014); Chapman et al., in 41st EPS Conference on Plasma Physics, Europhysics Conference Abstracts (European Physical Society, 2014), Vol. 38F, ISBN 2-914771-90-8] found that the occurrence times of intrinsic ELMs correlate with specific temporal analytic phases of the VLD2 and VLD3 signals. Here, we investigate how the VLD2 and VLD3 temporal analytic phases vary with time in advance of the ELM occurrence time. We identify a build-up to the ELM in which the VLD2 and VLD3 signals progressively align to the temporal analytic phase at which ELMs preferentially occur, on a ∼2−5ms timescale. At the same time, the VLD2 and VLD3 signals become temporally phase synchronized with each other, consistent with the emergence of coherent global dynamics in the integrated current density. In a plasma that remains close to a global magnetic equilibrium, this can reflect bulk displacement or motion of the plasma. This build-up signature to an intrinsic ELM can be extracted from a time interval of data that does not extend beyond the ELM occurrence time, so that these full flux loop signals could assist in ELM prediction or mitigation