Solubilization and purification of Na, K-ATPase from the outer medulla of rabbit kidney

There is great interest in the histologic localization of the Na, K-pump in various tissues: histochemical methods in this case work poorly, so a specific antibody against a purified soluble Na, K-ATPase is necessary. We approached this problem with a two-step ionic detergent treatment; the separation of the solubilized enzyme was attempted by gel filtration and ion-exchange chromatography. The gel filtration purified form was active and nearly pure, while the ion-exchange on was purer but inactive. PAGE analyses of the various enzyme forms are presented.     

MED and PSACH COMP mutations affect chondrogenesis in chicken limb bud micromass cultures

Mutations in cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). We studied the effects of over‐expression of wild type and mutant COMP on early stages of chondrogenesis in chicken limb bud micromass cultures. Cells were transduced with RCAS virus harboring wild type or mutant (C328R, PSACH; T585R, MED) COMP cDNAs and cultured for 3, 4, and 5 days. The effect of COMP constructs on chondrogenesis was assessed by analyzing mRNA and protein expression of several COMP binding partners. Cell viability was assayed, and evaluation of apoptosis was performed by monitoring caspase 3 processing. Over‐expression of COMP, and especially expression of COMP mutants, had a profound affect on the expression of syndecan 3 and tenascin C, early markers of chondrogenesis. Over‐expression of COMP did not affect levels of type II collagen or matrilin‐3; however, there were increases in type IX collagen expression and sulfated proteoglycan synthesis, particularly at day 5 of harvest. In contrast to cells over‐expressing COMP, cells with mutant COMP showed reduction in type IX collagen expression and increased matrilin 3 expression. Finally, reduction in cell viability, and increased activity of caspase 3, at days 4 and 5, were observed in cultures expressing either wild type or mutant COMP. MED, and PSACH mutations, despite displaying phenotypic differences, demonstrated only subtle differences in their cellular viability and mRNA and protein expression of components of the extracellular matrix, including those that interact with COMP. These results suggest that COMP mutations, by disrupting normal interactions between COMP and its binding partners, significantly affect chondrogenesis. J. Cell. Physiol. 224: 817–826, 2010. © 2010 Wiley‐Liss, Inc.     

Role of environmental factors in autoantibody production - importance of a detailed analysis in a small cohort

In the previous issue of Arthritis Research & Therapy, Muro and colleagues reported a detailed epidemiologic analysis in central Japan on one of the new myositis-specific autoantibodies to MDA-5 (melanoma differentiation-associated gene 5), which is associated with clinically amyopathic dermatomyositis accompanying interstitial lung disease. The increasing prevalence of anti-MDA-5, higher prevalence in small rural towns, and geographical clustering in two areas along the Kiso River suggest a role of environmental factors associated with rural communities or the river/water system or both. A detailed analysis of a small cohort may offer clues, which is ignored in multi-center studies, to the pathogenesis of systemic rheumatic diseases and autoantibody production.     

Is inflammaging an auto[innate]immunity subclinical syndrome?

The low-grade, chronic, systemic inflammatory state that characterizes the aging process (inflammaging) results from late evolutive-based expression of the innate immune system. Inflammaging is characterized by the complex set of five conditions which can be described as 1. low-grade, 2. controlled, 3. asymptomatic, 4. chronic, 5. systemic, inflammatory state, and fits with the antagonistic pleiotropy theory on the evolution of aging postulating that senescence is the late deleterious effect of genes (pro-inflammatory versus anti-inflammatory)that are beneficial in early life. Evolutionary programming of the innate immune system may act via selection on these genetic traits. Here I propose that the already acquired knowledge in this field may pave the way to a new chapter in the pathophysiology of autoimmunity: the auto-innate-immunity syndromes. Indeed, differently from the well known chapter of conventional autoimmune diseases and syndromes where the main actor is the adaptive immunity, inflammaging may constitute the subclinical paradigm of a new chapter of autoimmunity, namely that arising from an autoimmune inflammatory response of the innate-immune-system, an old actor of immunity and yet a new actor of autoimmunity, also acting as a major determinant of elderly frailty and age-associated diseases.     

Fluorocarbons as blood substitutes. Toxicity in the rat]

Liquid fluorocarbons, having high solubility for gases (O2, CO2...) have been used as artificial blood substitutes in animals with variable results. The great diversity of these products and the lack of reproductiveness in their composition have not permitted, up to now, a standardization of their utilisation norms. Our work was to study the toxicity, in the rat, of a new kind of fluorocarbon emulsion (E-66, Ugine-Kuhlmann, France) used as an artificial blood substitute during exchange-perfusion. The short survival of the rats is in opposition to the good in vitro results obtained in other experiments (high solubilities of oxygen and dioxide carbon). The toxicity of this fluorinated emulsion is demonstrated by histologic lesions in lungs, liver and kidneys and the great amount of fluor stored in these organs. The mechanism of this toxicity is still to be demonstrated. Hepatic lesions (hyperhemia with dilatation of central veins and sinusoids) and pulmonary lesions (vascular congestion, alveolar oedema) prove a circulatory disturbance leading to right-sided cardiac failure. However, cellular degenerescence lesions, observed in hepatocytes and renal tubular cells, do not permit to exclude formally a cellular toxicity of the E-66 emulsion.