Modelling thermodynamic feedback on the metabolism of hydrogenotrophic methanogens.

The magnitude of the Gibbs free energy change of the substrate transformation that supports the growth of a microbe is decreased when the concentrations of the substrates are decreased and when the concentrations of the products of metabolism are increased. Microbes require a supply of ATP for cell maintenance and growth, and coupling the transformation of substrates to products with the formation of ATP also decreases the magnitude of the Gibbs free energy change. Here we include these three thermodynamic controllers (substrate and product concentration, and ATP formation) in a model of substrate transformation by hydrogenotrophic methanogens that results in a number of realistic behaviours. First, a threshold for substrate use emerges, below which the methanogen cannot metabolise its substrate. Under this model, microbes that capture more of the Gibbs free energy change from substrate transformation in the form of ATP have greater thresholds for their substrate, in line with observations of actual microbes. Second, an apparent saturation constant emerges that is controlled by the thermodynamics of the reaction. This increases with increasing ATP synthesis per substrate, so that methanogens that conserve more ATP grow faster at higher substrate concentrations, but are less competitive at low substrate concentrations. As a result, simply changing the ATP yield (moles of ATP per mole of substrate) results in methanogens with differing ecological strategies through thermodynamic impacts on their metabolism. Third, end-product inhibition through thermodynamic feedback can limit the growth of microbes, and those that capture more ATP per substrate are limited by smaller product concentrations than those that capture less ATP.

A two-site ELISA can quantify upregulation of SMN protein by drugs for spinal muscular atrophy.

OBJECTIVES: Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons during early or postnatal development. Severity is variable and is inversely related to the levels of survival of motor neurons (SMN) protein. The aim of this study was to produce a two-site ELISA capable of measuring both the low, basal levels of SMN protein in cell cultures from patients with severe SMA and small increases in these levels after treatment of cells with drugs. METHODS: A monoclonal antibody against recombinant SMN, MANSMA1, was selected for capture of SMN onto microtiter plates. A selected rabbit antiserum against refolded recombinant SMN was used for detection of the captured SMN. RESULTS: The ratio of SMN levels in control fibroblasts to levels in SMA fibroblasts was greater than 3.0, consistent with Western blot data. The limit of detection was 0.13 ng/mL and SMN could be measured in human NT-2 neuronal precursor cells grown in 96-well culture plates (3 x 10(4) cells per well). Increases in SMN levels of 50% were demonstrable by ELISA after 24 hours treatment of 10(5) SMA fibroblasts with valproate or phenylbutyrate. CONCLUSION: A rapid and specific two-site, 96-well ELISA assay, available in kit format, can now quantify the effects of drugs on survival of motor neurons protein levels in cell cultures.

Association of stress proteins with autoantigens: a possible mechanism for triggering autoimmunity?

Patterns of autoantibody production are diagnostic of many autoimmune disorders; the recent observation of additional autospecificities towards stress-induced proteins may also provide insight into the mechanisms by which such responses arise. Grp78 (also known as BiP) is a target of autoaggressive B and T cell responses in our murine model of anti-Ro (SS-A) autoimmunity and also in rheumatoid arthritis. In this report we demonstrate reciprocal intermolecular spreading occurs between Ro52 and Grp78 in immunized mice, reflecting physiological association of these molecules in vivo. Moreover, we provide direct biochemical evidence that Grp78 associates with the clinically relevant autoantigen, Ro52 (SS-A). Due to the discrete compartmentalization of Ro52 (nucleocytoplasmic) and Grp78 (endoplasmic reticulum; ER) we propose that association of these molecules occurs either in apoptotic cells, where they have been demonstrated indirectly to co-localize in discrete apoptotic bodies, or in B cells themselves where both Ro52 and Grp78 are known to bind to immunoglobulin heavy chains. Tagging of molecules by association with Grp78 may facilitate receptor mediated phagocytotsis of the complex; we show evidence that exogenous Grp78 can associate with cell surface receptors on a subpopulation of murine splenocytes. Given the likelihood that Grp78 will associate with viral glycoproteins in the ER it is possible that it may become a bystander target of the spreading antiviral immune response. Thus, we propose a model whereby immunity elicited towards Grp78 leads to the selection of responses towards the Ro polypeptides and the subsequent cascade of responses observed in human disease.