Anin VitroModel of Oligodendrocyte Destruction by Nitric Oxide and Its Relevance to Multiple Sclerosis

There is mounting evidence that nitric oxide (NO) is produced in the brains of patients with multiple sclerosis (MS) and in the experimental model of MS, experimental autoimmune encephalomyelitis, after the induction of Type II nitric oxide synthase (iNOS). Because NO can cause a variety of biological insults that compromise or even kill normal cells, we studied the effects of NO on oligodendrocytes since they are a target in MS tissue. In anin vitromodel, we have been able to demonstrate that NO causes damage to oligodendrocytes preferentially, sparing microglia almost completely and affecting some but not all astrocytic functions. This article describes the types of assays used to measure morphological changes, mitochondrial dysfunction, DNA strand breaks, and cell death brought on by NO or peroxynitrite (ONOO-) as well as a comprehensive review of the various techniques and sensitivities of NO and iNOS assays that would be applicable to similarin vitromodels.     

Shifting sources of functional limitation following extensive (70%) lung resection.

We previously found that, following surgical resection of approximately 58% of lung units by right pneumonectomy (PNX) in adult canines, oxygen-diffusing capacity (Dl(O(2))) fell sufficiently to become a major factor limiting exercise capacity, although the decline was mitigated by recruitment, remodeling, and growth of the remaining lung units. To determine whether an upper limit of compensation is reached following the loss of even more lung units, we measured pulmonary gas exchange, hemodynamics, and ventilatory power requirements in adult canines during treadmill exercise following two-stage resection of approximately 70% of lung units in the presence or absence of mediastinal distortion. Results were compared with that in control animals following right PNX or thoracotomy without resection (Sham). Following 70% lung resection, peak O(2) uptake was 45% below normal. Ventilation-perfusion mismatch developed, and pulmonary arterial pressure and ventilatory power requirements became markedly elevated. In contrast, the relationship of Dl(O(2)) to cardiac output remained normal, indicating preservation of Dl(O(2))-to-cardiac output ratio and alveolar-capillary recruitment up to peak exercise. The impairment in airway and vascular function exceeded the impairment in gas exchange and imposed the major limitation to exercise following 70% resection. Mediastinal distortion further reduced air and blood flow conductance, resulting in CO(2) retention. Results suggest that adaptation of extra-acinar airways and blood vessels lagged behind that of acinar tissue. As more lung units were lost, functional compensation became limited by the disproportionately reduced convective conductance rather than by alveolar diffusion disequilibrium.     

An enzyme-linked immunosorbent assay for the association of the catalytic domain of diphthamide-specific ribosyltransferases to eukaryotic elongation factor-2.

The X-ray structure of the catalytic domain of Pseudomonas aeruginosa exotoxin A (PE24) has recently been solved to high resolution, facilitating studies on the interaction of PE24 with its target substrate, eukaryotic elongation factor-2 (eEF-2). PE24 exhibits mono-ADP-ribosyltransferase (ADPRT) activity in a mechanism that has been proposed to feature a nucleophilic attack by the diphthamide residue (nucleophile) of eEF-2 on the C-1 of the nicotinamide ribose of NAD(+). The interaction of wheat germ eEF-2 with PE24 was studied by employing an enzyme-linked immunosorbent assay (ELISA), devised to assess protein-protein interactions. It was shown that the proteins associate with each other only in the presence of the enzyme's nucleotide substrate, NAD(+), and exhibit a dose-dependent association that is saturable. The apparent dissociation constant (K(d)) for this protein-protein interaction is 50 nM and is salt-dependent. The association is maximal at low ionic strength and is progressively weaker at higher salt concentrations, which corroborates previous findings on the salt dependence of ADPRT activity for this toxin. This finding suggests that the sensitivity of ADPRT activity toward high salt resides in the interaction between the catalytic domain of the toxin and eEF-2. A major product of the glycohydrolase activity of PE24, nicotinamide, inhibits the binding between PE24 and eEF-2 with an ID(50) of 20 microM. The naturally occurring, noncatalytic mutant of PE24, H426Y, did not bind eEF-2 in the ELISA, verifying that His 426 is located at the center of the eEF-2 binding site within ETA.