Eugene D. Day

Superoxide-dependent production of hydroxyl radical catalyzed by iron-EDTA complex.

Their proposal was supported by the following observations: (i) ethylene production could be inhibited by superoxide dismutase, (ii) by catalase, or (iii) by scavengers of hydroxyl radical such as ethanol and benzoate; and (iv) a lag in ethylene production was found to be due to the time required for hydrogen peroxide to accumulate to a reactive concentration. Reaction 1 was the simplest explanation accommodating all the observed facts, and the reaction had been proposed many years earlier by Haber and Weiss during their studies of the catalytic decomposition of hydrogen peroxide by iron salts [4] . In the last seven years the reaction has become known in the biological literature as the Haber-Weiss reaction, and has been invoked by many investigators to explain diverse biological phenomena which are characterized by the same inhibitory criteria described by Beauchamp and Fridovich and outlined above. On the other hand, attempts to demonstrate directly the simple biomolecular reaction shown as reaction 1 have met with uniform failure [5-71, leading to the conclusion that, although reaction 1 may indicate the stoichiometry of the overall process, it cannot be construed as the reaction mechanism. Under chemically well-defined conditions, it has in fact been shown that reaction 1 does not proceed at any appreciable rate. Koppenol, however, has shown the reaction to be quite feasible thermodynamically [8], even if the oxygen molecule which appears as a product is in the electronically excited ‘AgOz singlet state, as Kellogg and Fridovich have suggested [9] .

Myelin Basic Protein

Myelin basic protein (MBP) has engaged the attention of molecular and cellular immunologists during the past decade because of its implication in the induction of the autoimmune disease experimental allergic encephalomyelitis (EAE) of the central nervous system (CNS). What impresses this reviewer much more, however, beyond the direct role MBP plays in the induction of an autoimmune experimental disease in certain strains of laboratory animals, is the fact that the major portion of the total MBP autoimmunology uncovered in recent years is clearly not involved in the induction of the autoimmune disease. To some the message given by this nonencephalitogenic portion possesses no greater meaning than didactic teachings from the realm of bovine serum albumin immunology; to others, this seemingly irrelevant “other” MBP immunology—an immunology against “self” that is not self-destructive—may be extremely vital. As an autoimmune process it may play a major role in the normal maintenance of protection against encephalomyelitis in most vertebrates either through direct participation or by indirect regulation. From this point of view the occasional emergence of EAE is seen as a defect in an otherwise protective immunology, a case in which horror autotoxicus is an exception to the rule.

Neuroimmunologic Diseases: Effector Cell Responses and Immunoregulatory Mechanisms

CFA CNS EAE HAE-STA GPMBP GPK GPSC IFA Ig K cells LNC MBP MBP-SFs MS RIA(s) RK RMBP RSC T cells antigen binding capacity acute disseminated encephalomyelitis anti-thymocyte serum bone marrow derived or Bursa of Fabricius-equivalent immunoglobulin-sur face bearing lymphocytes complete Freunds adjuvant central nervous system experimental altergic encephalomyeiitis experimental altergic encephalomyetitis-supernate transfer activity guinea pig myelin basic protein guinea pig kidney guinea pig spinal cord incomplete Freunds adjuvant immunoglobulin non-T and non-B cells with high density of surface Fc receptors lymph node cells myelin basic protein myelin basic protein-serum factors multiple sclerosis radioimmunoassay(s) rat kidney rat myelin basic protein rat spinal cord thymus-derived or influenced lymphocytes

Isolation and characterization of a manganese-containing superoxide dismutase from rat liver☆

Abstract A manganese-containing superoxide dismutase has been purified from rat liver and characterized. The enzyme has a molecular weight of 89,000 and is composed of four subunits. One atom of manganese is contained per subunit. The metal content, molecular weight, and amino acid analyses show that the rat enzyme is similar to the manganosuperoxide dismutase isolated from human liver.

Radioimmunoassay of myelin basic protein in sodium sulfate

Abstract A radioimmunoassay of myelin basic protein (BP) of the rat and guinea pig, based upon precipitation of the 125 I-BP antibody complex in 1·27M Na 2 SO 4 , was developed. Salting-out constants for rat and rabbit immunoglobulins, using 125 I-BP as indicator, ranged from 1·97 to 2·63; the steepness of the slopes and the high correlation coefficients of the salting-out curves indicated that the antibodies involved were not widely spread among the various isotypes nor did they have a wide range of solubilities. The binding-activity of BP for a number of IgG preparations and whole antiserums in the region of far antigen excess was linear with correlation coefficients appraoching 1·00. Quantitative adsorption analysis with rat cerebral myelin removed 94 per cent of the BP-binding activity of one antiserum. Activities in rabbit serums ranging from 4·97 to 48·92 μg BP bound/ml serum were obtained. Normal rabbit serums displayed a range of activities from 0·00 to 4·93. Labeled and unlabeled BP were equally competitive for antibody in regions of antigen excess.

Endogenous myelin basic protein-serum factors (MBP-SFs) and anti-MBP antibodies in humans: Occurrence in sera of clinically well subjects and patients with multiple sclerosis

Sera of normal subjects and patients wtih multiple sclerosis (MS) have been frequently found to contain picomolar quantities of endogenous myelin basic protein-serum factors (MBP-SFs). These serum factors, collectively representing a heterogeneous spectrum, were detected and measured by means of a competitive inhibition radioimmunoassay (RIA) designed to distinguish their respective binding affinities with anti-MBP reagent antiserum. Anti-MBP antibodies in these same normal and patient sera were also detected and their differing binding affinities determined. In general, when sera of normal subjects were found to contain free MBP-SFs, the reagent anti-MBP antibodies in the reagent antiserum used to detect them were of relatively high binding affinity (8 X 10(8) M-1). When normal sera were found to contain free anti-MBP antibodies, the affinities of such antibodies were invariably lower (0.06-0.7 X 10(8) M-1). In contrast, sera of patients with active MS and exhibiting clinical fluctuations in their disease, infrequently contained high or medium high affinity MBP-SFs, whereas higher affinity anti-MBP antibodies were commonly detected. These patterns of MBP-SFs and anti-MBP antibodies in normal and MS human sera resemble those previously observed in studies of normal Lewis rats and rats developing experimental allergic encephalomyelitis (EAE). The findings here reported provide additional support for the view that circulating endogenous MBP-SFs may function as neuroautotolerogens that restrict expansion of MBP-reactive lymphoid cell clones having potentially injurious effector activity for central nervous system (CNS) tissue.

The isolation and biochemical characterization of three subfractions of myelin from central nervous tissue of the adult rat.

Abstract— By techniques of isosmotic density gradient ultracentrifugation three subfractions of myelin were isolated from homogenates of whole rat brain at densities of 1.054 g/ml (myelin I), 1.060 g/ml (myelin II) and 1.066 g/ml (myelin III). The stability of these fractions was demonstrated by the zonal centrifuge profile analysis of recycled fractions. Examination of the three myelin subfractions by techniques of electron microscopy and thin layer chromatography detected no obvious morphological or chemical differences. However, analysis for protein, cholesterol, phospholipids and cerebrosides did reveal differences among myelin I, myelin II and myelin III. Myelin I contained relatively more cholesterol than II or III. Myelin III contained relatively more phospholipids than I or II. The cerebroside‐to‐protein ratios were the same in all three fractions. Quantitative differences in fatty acid composition (as detected by gas‐liquid chromatography) were also observed.

Monoclonal and polyclonal antibodies to myelin basic protein determinants

A detailed immunochemical examination of monoclonal and polyclonal antibody responses to myelin basic protein (MBP) and its peptides has revealed the existence of as many as 27 antigenic determinants, many of them conformational. Topological mapping of the potential antigenic determinants onto a model of MBP secondary structure places these determinants within 11 separate regions of the molecule, including those portions that have been found to be encephalitogenic. MBP and its peptides, therefore, fall under the umbrella of the Multideterminant-Regulatory Model of Benjamin et al. (1984). However, in the case of MBP, multideterminant immunogenicity appears to represent mainly an escape from tight regulation through the avenue of conformational change.

Zonal centrifuge profiles of rat brain homogenates: instability in sucrose, stability in iso-osmotic Ficoll-sucrose.

Abstract By substituting iso-osmotic Ficoll-sucrose for hyperosmotic sucrose between the densities of 1.043 and 1.088 in sucrose density gradients in the B-XV rotor of an Anderson-NIH-AEC zonal centrifuge system, it was possible to stabilize the zonal centrifuge absorbancy profiles of adult rat brain homogenates. The reason for the instability in ordinary sucrose gradients was found to be the interaction of myelin with other brain structures in hyperosmotic sucrose. No such interaction occurred in isoosmotic sucrose (0.32 M) with or without Ficoll. In Ficoll-sucrose, myelin was separated at three reproducible densities of 1.054, 1.060, and 1.066 gm/ml. No myelin appeared at a density if 1.094 gm/ml, which represented the main collection point in ordinary sucrose. Synaptosomes were separated at peak densities of 1.072 and 1.152 gm/ml. Mitochondria were obtained at a density of 1.176 gm/ml. Areas under zonal centrifuge absorbancy profiles of rat brain homogenates were found to be constant regardless of the values of ω2t that were reached.

Relative affinity of antisera for myelin basic protein (MBP) and degree of affinity heterogeneity.

Abstract A method based essentially on that of Steward and Petty (1972) was used to evaluate the relative binding affiinities of several syngeneic Lewis-rat antisera to myelin basic protein (MBP)†. The syngeneic immune reactions to this ‘self’ antigen were found to contain only populations of relatively high affinity antibodies, ranging from a K0 of 7 × 107 to 2.6 × 108M−1 when measured at 100 nM MBP; and from 4 × 108 to 9 × 109M−1 at 1 nM. The biphasic nature of the plots of antigen binding capacity (ABC) vs log MBP concentration suggested that MBP binding was usually distributed among two or more major discontinuous antibody populations. Advantage was taken of the dual-dilution phenomenon to obtain constants interpreted as degrees of relative affinity heterogeneity of syngeneic antisera. It was found that the dual- dilution phenomenon (the decrease of ABC with increasing antigen P dilution) could be expressed mathematically in the form of a power curve ABC = bP 1−α with b (the intercept at 1 nMP) related to the total number of antibody molecules in the system and α, the degree of affinity heterogeneity of antibodies against the multideterminant antigen. The more heterogeneous the system the higher the number of antibody molecules required to maintain a constant ABC at a given P and the more rapid the decrease of ABC with decreasing P. The degree of affinity heterogeneity of the syngeneic Lewis rat antisera ranged from pronounced (α = 0.14) to moderately restricted (α = 0.8). The heterogeneity index of a rabbit anti-MBP serum was 0.821. An antiserum refractory to the dual-dilution effect was also found for which α was 0.98.