Elizabeth Roboz Einstein

PROTEOLYTIC ACTIVITY AND BASIC PROTEIN LOSS IN AND AROUND MULTIPLE SCLEROSIS PLAQUES: COMBINED BIOCHEMICAL AND EIISTOCHEMICAL OBSERVATIONS

Abstract— This combined histochemical and biochemical study has shown that acid proteinase activity (PH 3.5) is increased around histologically‐defined active plaques of multiple sclerosis (MS). Biochemical estimation showed that the enzyme is more active in most samples of ‘normal’ white matter in MS than in controls. A gradient of enzyme activity was observed: control white matter‐white matter distant from plaqueclose white matter‐edgsplaque. Both electrophoretic and histochemical techniques revealed a reduction or absence of basic (encephalitogenic) protein in the plaques. Electrophoresis showed a diminution of encephalitogenic protein outside some plaques. Phospholipids that remain on the base‐line of thin‐layer chromatoplates were shown to be predominantly phosphoinositides combined with encephalitogenic protein

Basic proteins from the acidic extract of bovine spinal cord: I. Isolation and characterization

Abstract 1. 1. Six cationic proteins have been isolated and purified from the acidic extract of bovine spinal cord by the use of carboxymethyl-cellulose and gel-fractionation techniques. 2. 2. In comparison with histones, four of the isolated proteins have low isoleucine and valine, and high glycine content. 3. 3. A molecular weight of 37 000 was estimated by gel permeability technique for the principal and largest protein isolated. However, the same protein was estimated to have a molecular weight of 12 500 by ultracentrifugation, and 15 800 on the basis of the number of peptides obtained on tryptic hydrolysis and the percentage of lysine and arginine obtained on complete hydrolysis.

Increased protease activity and changes in basic proteins and lipids in multiple sclerosis plaques

Abstract Various sections of multiple sclerosis (MS) brains, including the plaques and the edge of the plaques, have been studied. The lipids were determined by quantitative thin-layer chromatography and the values for cerebroside, cholesterol, and cholesterol ester were found to be in agreement with previous workers. There was little difference between MS and normal brains in their phospholipid content. The significant finding was the demonstration that with a partial or total destruction of the encephalitogenic protein around the plaques, a 4-fold increase in acid proteinase occurred. Previous reports ( Chao and Einstein 1968; Einstein et al. 1968a; 1970) have shown that the encephalitogenic protein is converted in vivo and in vitro to an active polypeptide of smaller molecular weight. These suggest, although they do not necessarily prove, that the acid proteinase which is a lysosomal enzyme(s) plays a role in MS by releasing the encephalitogen as such a polypeptide into the circulation and in this way initiating an immunological reaction.

Protective action of the encephalitogen and other basic proteins in experimental allergic encephalomyelitis.

Abstract Various basic protein preparations have been investigated for their ability to protect guinea pigs from EAE. These protein preparations include identical active proteins isolated from human and bovine neural tissue. Treatment, both before and after challenge, with encephalitogen in complete Freunds adjuvant, was effective as evidence by the survival of all guinea pigs. Partial protection was provided by a basic protein preparation extracted from immature human brain and with a basic homogeneous protein isolated from bovine spinal cord. Both were tested prior to their use and found to be nonencephalitogenic. Some surviving guinea pigs, reinjected after 35 days, did not display signs of disease, indicating a prolonged state of acquired resistance. It was concluded that both human and bovine basic proteins can be used as effective inhibitory agents in EAE. This effect is equally efficient regardless of the species of origin of the antigen. More important, this study also has provided evidence that nonencephalitogenic neural proteins may also greatly reduce tha incidence of physical signs in EAE.

Biochemical maturation of the central nervous system. I. Lipid changes.

Summary Lipid studies have been carried out on rabbit neural tissue at ages 1, 5, 8, 9, 14, 30, 60, 90, 120, and 170 days, and the findings correlated to development. Comparison has been made: (1) between brain and myelin prepared from it; (2) between brain and cord myelins; and (3) between myelins from various species, using our results together with data from the literature. It was found that the consistency of the myelin prepared from brain and cord in the early stage of development — 1, 5, 8, and 9 days — differs greatly from the myelin prepared from adult neural tissue. This ‘immature’ myelin is characterized by high phospholipids and low cholesterol and cerebroside. The greatest changes take place between the 1st and 14th days. There is a 5-fold increase in cholesterol and 12-fold in cerebroside; and after this date, there is an even, but moderate, increase. The total lipid at maturation in the myelin is 73.9%; in the brain, 53.3%. Since the lipid composition of the myelin from cord has not been investigated before, we analyzed the cord myelin at ages 5, 8, 9, 14, and 170 days. The lipid compositions of the myelin from cord and brain during development are not exactly parallel. The cord myelin had a higher phospholipid content during development, although ethanolamine glycerophosphatide at maturity was higher in the brain (difference = 2 mole%). The same applies to cholesterol; on the other hand, cerebroside was higher in the cord. The lipid compositions of the myelin of various species — rabbit, bovine, rat, guinea-pig and human — were compared and found at maturity to be similar.

Biochemical maturation of the central nervous system. II. Protein and proteolytic enzyme changes.

Abstract In an earlier study, the maturation of the central nervous system was followed by analyzing the lipids 5 . Since in the myelin the lipids are complexed with proteins, we have extended our studies to the proteins of the brain, cord, and myelin and have followed the development from embryonic to mature rabbit brains. It was found that the brain weights are quite uniform in the same age group, and that the greatest increase occurs between 14 and 60 days. For assaying protein changes, the technique of acrylamide electrophoresis was found eminently suitable. It was applied in two forms: one, after complete solubilization of the myelin for demonstration of the proteolipids and the encephalitogenic proteins; two, after acid extraction for the demonstration of all basic proteins, including the encephalitogenic protein. This basic, encephalitogenic protein appears later than the proteolipids, although both are constitutents of the myelin. The embryonal brain contains a fast-moving basic protein, most likely a histone, which is either not present or in minimal quantity after birth. Brain and cord myelin proteins were compared and it was found that the sequence of protein synthesis in the early stage differed in that the encephalitogenic protein appeared earlier in the cord than in the brain.

PHYSICAL PROPERTIES OF THE BOVINE ENCEPHALITOGENIC PROTEIN; MOLECULAR WEIGHT AND CONFORMATION

Abstract— The highly basic encephalitogenic protein isolated from bovine spinal cord was studied by various physicochemical methods:

Basic proteins from the acidic extract of bovine spinal cord: II. Encephalitogenic, immunologic and structural interrelationships

Abstract 1. 1. Of 6 cationic proteins isolated form the acidic extract of bovine spinal cord, 3 induce experimental allergic encephalomyelitis when injected into guinea pigs simultaneously with complete Freunds adjuvant. 2. 2. The 3 encephalitogens were shown to be related immunologically, by the passive cutaneous anaphylaxis test, and structurally, by the peptide mapping technique. Quite likely, they are fragments from one molecule produced by enzymatic degradation during the isolation procedures.

Estimation of the molecular weight of flexible disordered proteins by exclusion chromatography

Abstract Estimation of the molecular weight of the encephalitogenic protein by exclusion chromatography, when globular proteins were used for comparison, resulted in a much higher value (39800) than that obtained by sedimentation equilibrium (17800 ± 600). This protein, unlike the reference compounds, is a flexible, disordered molecule. Therefore, globular proteins are not suitable for calibration. In order to adapt the technique and render it suitable for general use, regardless of molecular shape, all proteins (references and unknowns used in the measurements) were denatured in concentrated guanidine hydrochloride and urea, reduced with β-mercaptoethanol to rupture the disulfide bonds, and then chromatographed on Sephadex G-100 columns. Under these conditions, the protein polypeptide chains are true random coils retaining no elements of their original native conformation. The molecular weight of the encephalitogenic protein estimated under these conditions is 19500. The method was also applied for the encephalitogenic active fragment and the lysine-rich histone. The results obtained are in agreement with the data obtained by physical methods.

Suppression of experimental allergic encephalomyelitis by chemically modified encephalitogen

Abstract The results of enzymatic and chemical degradation studies of the basic myelin protein suggested that tryptophan is essential for encephalitogenic activity in the guinea pig. The object of the present investigation was to modify the protein through its tryptophan residue in such a way that it becomes non-encephalitogenic, yet suppresses experimental allergic encephalomyelitis (EAE). Accordingly, the single tryptophan residue of the protein was modified with specific reagents such as 2-hydroxy-5-nitrobenzylbromide (HNB-BR), 2-nitrophenylsulfenyl chloride (NPSCl) and performic acid. Injection of the modified proteins did not result in neurological disease, although some guinea pigs did exhibit histological changes in the brain. Starting 3 days after injection with the active inoculum, the treatment with the modified proteins was carried out for four weeks, using a total of 900 μg og the modified proteins. The guinea pigs treated with HNB and performic acid oxidized protein did not show neurological signs of the disease, but from the NPSCl group, two out of 10 developed EAE, although at a later data compared with those guinea pigs which were injected with the unmodified encephalitogen. The results not only confirm previous findings that intact tryptophan in the protein is a prime requirement for the production of EAE in guinea pigs, but also provide evidence that tryptophan is not essential for suppression of the disease. The conclusion we may draw from these studies is that the encephalitogenic basic myelin protein has two active sites: one which contains tryptophan required for the paralytic effect, the other without tryptophan which provides protection.