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E Barbarese

Publications and source records attributed to E Barbarese.

50 records · Page 3Linked to original sources
PubMed1986

Myelin basic protein-specific protein methylase I activity in shiverer mutant mouse brain.

Myelin basic protein (MBP)-specific protein-arginine N-methyltransferase (protein methylase I) activity in homozygous shiverer (shi/shi) mutant mouse brain is significantly higher than in the normal littermate brain at the onset of myelination. While the enzyme activity (expressed as pmol of S-adenosyl-L-[methyl-14C]methionine used/min/mg enzyme protein) increases coincidently during the period of myelination in the normal brain (15-18 days of age), it decreases significantly in the mutant brain during this period of time. These results are in contrast to those found with another dysmyelinating mutant, jimpy (jp/Y) mice, in which the enzyme activity in the mutant brains is similar to that in the normal animals but remains unchanged during the myelination process. There is no difference in the weight and protein concentration of the normal and shiverer mutant brains with corresponding ages, and the histone-specific protein methylase I activity is also unaffected in the shiverer brain.

Animals↗
PubMed1983

Developmental regulation of myelin basic protein expression in mouse brain.

Developmental regulation of myelin basic protein expression in mouse brain has been examined by comparing the myelin basic protein coding potential of mRNA in vitro with the accumulation of myelin basic protein-related polypeptides in vivo. In vitro translation of mRNA isolated from mouse brain generated eight myelin basic protein-related polypeptides with apparent molecular weights of 34K, 30K, 29K, 26K, 21.5K, 18.5K, 17K, and 14K. A similar set of eight myelin basic protein-related polypeptides with corresponding molecular weights was identified in vivo when total brain proteins were analyzed by immunoblotting. Each of the myelin basic protein-related polypeptides shows a characteristic developmental profile in terms of mRNA level and rate of accumulation implying a complex developmental program of myelin basic protein gene expression with regulation and modulation at several different biosynthetic levels.

Aging↗
PubMed1983

Progenitors of oligodendrocytes: limiting dilution analysis in fetal rat brain culture.

In this paper, we describe the use of a combination of cell culture techniques and limiting dilution analysis to determine the number of oligodendrocyte progenitor cells and the oligodendrocyte clone size in primary dispersed cultures of 20- to 21-day-old fetal rat brain. Single-cell suspensions (1, 2, 3 x 10(6) cells/ml) were plated in either microwell or 100 mm dishes. After 22 days in culture the number of differentiated oligodendrocytes was ascertained by determining the amount of myelin basic protein by radioimmunoassay. The total amount of myelin basic protein was the same in the two types of dish, indicating that proliferation and differentiation were unaffected when oligodendrocytes were grown in microwells. The fraction (F0) of microwells containing no oligodendrocytes was determined at each cell dilution. F0 decreased exponentially with increasing total cell concentration. The linearity of the plot of ln F0 versus cell number indicates that the number of oligodendrocyte progenitor cells is limiting. From the equation describing the Poisson distribution of progenitor cells in microwells we calculate that, at the time of plating, primary cultures of fetal rat brain contain one oligodendrocyte progenitor cell per 1.3 x 10(5) brain cells, or a total population of 300-500 progenitor cells per brain. The mean oligodendrocyte clone size was determined to be approximately 825 at 22 days and close to 2000 by 35 days in culture. Therefore, each progenitor cell must undergo approximately 11 divisions, on the average, during postnatal development.

Animals↗
PubMed1983

The effect of the shiverer mutation on myelin basic protein expression in homozygous and heterozygous mouse brain.

We report (a) that the shiverer mutation has pleiotropic phenotypic effects on myelin basic protein expression in the CNS of homozygous (shi/shi) mice and (b) that each of the effects of the shiverer allele is expressed co-dominantly with the wild-type allele in heterozygous (+/shi) animals. First, the total amount of myelin basic protein, as determined by radioimmunoassay, that accumulates in the CNS is approximately 0.1% of the wild-type amount in shi/shi animals and approximately 50% in +/shi animals. Second, the four major forms of myelin basic protein, with molecular weights of 21,500, 18,500, 17,000, and 14,000, that are present in wild-type mouse CNS are undetectable in either whole brain or purified myelin of shi/shi animals, and each of the four proteins is reduced commensurately in brain and myelin of +/shi animals. Third, the small amount of myelin basic protein-related material that does accumulate in the shi/shi brain consists of several polypeptides, with molecular weights ranging from 25,000 to 100,000, the pattern of which is different from that found in wild-type brain. The pattern of myelin basic protein-related polypeptides in +/shi brain is a composite of the wild type and the shiverer mutant. Fourth, messenger RNA from shi/shi brain, when translated in vitro, encodes a set of myelin basic protein-related polypeptides qualitatively similar to that encoded by wild-type messenger RNA, except that the 18,500 and 14,000 translation products are greatly reduced, while other myelin basic protein-related translation products are spared. The pattern of myelin basic protein-related translation products for +/shi messenger RNA is intermediate between the patterns for +/+ and shi/shi messenger RNAs. The results suggest that the genetic lesion in the shiverer mutation impinges on the structural gene (or genes) encoding myelin basic protein or on a cis-acting regulatory element controlling that gene (or genes).

Animals↗
PubMed1982

Antibody-independent complement activation by myelin via the classical complement pathway.

Murine or rabbit whole brain homogenates were shown to activate human complement via the classical pathway by an antibody-independent reaction. This activity required Ca++ ions. Anticomplementary activity in fractionated murine brain was found to reside in the myelin fraction and in purified myelin. It was absent, however, both from highly purified myelin basic protein (MBP) and from the MBP-free residue. Because purified MBP is a monomer and this protein exists in brain tissue largely as a dimer, the ability of the cross-linked form of MBP to activate complement was investigated. MBP, dimerized with difluorodinitrobenzene, was highly anticomplementary. The murine brain, inactive when taken from the newborn mouse, was shown to first acquire the capacity to activate complement at 7 d after birth. This finding is consistent with the report that the synthesis of myelin protein has been shown to be initiated in murine brain 8 d after birth. Complement activation by MBP could play an important role in the pathological changes observed in neurological disorders.

Animals↗
PubMed1981

Noncoordinate regulation of myelinogenic parameters in primary cultures of dissociated fetal rat brain.

The developmental regulation of sulfogalactosylceramide (sulfatide) synthesis, 2', 3'-cyclic nucleotide-3'-phosphohydrolase activity (CNP), and myelin basic protein (MBP) accumulation, three markers characteristic of myelinogenesis, were observed in dispersed cultures of fetal rat brain in spite of the absence of the formation of compact, multilamellar myelin. Sulfatide synthetic rate and CNP activity began to increase by 8 days in vitro (DIV) (approximately comparable to days post-natal) and reached their maxima by 20 DIV. MBP began to accumulate after 14 DEV and reached a maximum by 38 DIV. Thus, the temporal regulation of the onset of expression of these parameters, which is coordinate in vivo, has been dissociated into two sequential periods in vitro. Similarly, the regulatory mechanisms controlling the subsequent decline of the net expression of these three parameters were dissociated. Whereas, the increase in the net CNP activity ceased on schedule, the decline of sulfatide synthetic rate was delayed 20 days, and the accumulation of MBP underwent a net loss. These data suggest that there are multiple parallel but separate mechanisms of temporal regulation controlling myelinogenic gene expression, and that, among them, those factors that control MBP accumulation are more sensitive to disruption by the rigors of dissociated culture.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1981

Requirement for nonoligodendrocyte cell signals for enhanced myelinogenic gene expression in long-term cultures of purified rat oligodendrocytes.

Comparisons have been made of myelinogenic activities in fetal rat brain mixed primary cultures and cultures of isolated oligodendrocytes of comparable age. The specific activities of the sulfatide synthesis, 2',3'-cyclic-nucleotide 3'-phosphohydrolase (2',3'-cyclic-nucleotide 3'-phosphodiesterase, EC 3.1.4.37), and accumulation of myelin basic protein, when expressed per mg of protein, were as high (or generally higher) in isolated oligodendrocyte cultures as in comparable mixed primary cultures at 29 days. However, when these data were analyzed per oligodendrocyte, it became apparent that the isolated oligodendrocytes were substantially less active than their mixed culture counterparts. The results suggest the necessity of nonologodendrocyte positive signals for the optimal expression by oligodendrocytes of myelin-related differentiated functions. The isolation method involves the selection of oligodendrocytes by shaking them from primary cultures of rat brain, followed by the lysis of other contaminating cells in a balanced salt solution at pH 7.2. More than 99% of the isolated cells are viable, at least initially divide, and can be cultured for at least 60 days. The oligodendrocytes selected in this way were characterized by: (i) morphology, (ii) immunofluorescence labeling by antibodies to myelin basic protein and galactosylceramide, and (iii) biochemical analyses for myelin basic protein, activity of 2',3'-cyclic-nucleotide 3'-phosphohydrolase, and sulfogalactosylceramide synthesis.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1981

Developmental regulation of myelin basic protein in dispersed cultures.

The expression of myelin basic protein, a major component of the myelin membrane, was studied in the absence of myelin formation in a unique situation in which these two processes have been uncoupled. The oligodendrocytes that contained myelin basic protein were identified by immunofluorescence in primary dispersed cultures derived from 20-day-old fetal rat brain. Their number increased 20-fold between 15 and 34 days in culture. Morphologically identifiable myelin was never observed. The oligodendrocytes elaborated a complex network of processes and membranous sheets resembling "unfurled" myelin. Myelin basic protein appeared concomitantly in the perikaryon, processes, and membranous sheets of the oligodendrocytes and remained distributed in these compartments throughout the culture period. The oligodendrocytes synthesized the four forms of myelin basic protein found in rodent brain with molecular weights of 21,500, 18,500, 17,000, and 14,000 and modulated their expression with time in culture. The onset of rapid myelin basic protein accumulation, as measured by radioimmunoassay, took place after 25 days in culture. Myelin basic protein accumulated at the rate of 0.2 fmol per oligodendrocyte per day and reached a level of 300 pmol/mg of protein by 34 days. By 60 days, the amount of myelin basic protein had declined to 100 pmol/mg of protein, a level maintained up until at least 120 days. When the amount of myelin basic protein was correlated with the number of oligodendrocytes, it was estimated that each induced cell contained on average 1 fmol of this protein at the three time points (15, 28, and 34 days in culture) at which cells were counted. Our results indicate that the accumulation, modulation of the molecular forms, and insertion of myelin basic protein into the membrane can occur in the absence of myelin formation, but that continued metabolic stability, subcellular sequestration, and fine control of the relative proportions of the different forms of myelin basic protein may be dependent on myelin morphogenesis.

Animals↗
PubMed1978

Components in multiple sclerosis cerebrospinal fluid that are detected by radioimmunoassay for myelin basic protein.

Components in cerebrospinal fluid that are antigenically related to myelin basic protein have been identified by a technique described recently [Barbarese, E., Braun, P. E. & Carson, J. H. (1977) Proc. Natl. Acad. Sci. USA 74, 3360-3364] involving separating the cerebrospinal fluid proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and measuring the individual components by radioimmunoassay for myelin basic protein. Samples of cerebrospinal fluid from 48 different patients (23 with definite multiple sclerosis, 4 with suspected multiple sclerosis, and 21 with other neurological diseases) were examined by this technique. The results indicate that cerebrospinal fluid can contain at least three separate components that are detected by radioimmunoassay for myelin basic protein. On the basis of their apparent molecular weights, the three components were identified as follows: component I, intact myelin basic protein; component II, proteolytic fragments of myelin basic protein; and component III, a protein of unknown origin with an apparent molecular weight of 50,000. Most samples of cerebrospinal fluid (45 of 48) from patients with multiple sclerosis and from patients with other neurological diseases contained components I and II. Component III was detected in all of the samples from patients with definite multiple sclerosis, in three of four samples from patients with suspected multiple sclerosis, and in none of the samples from patients with other neurological diseases. Some implications of these findings are discussed.

Humans↗
PubMed1977

Identification of prelarge and presmall basic proteins in mouse myelin and their structural relationship to large and small basic proteins.

A new technique is described to identify antigenically related proteins by radioimmunoassay after sodium dodecyl sulfate/polyacrylamide gel fractionation. When adult mouse myelin was examined by this technique, four proteins that are antigenically related to the small myelin basic protein were identified. They were designated: prelarge (molecular weight 21,500), large (18,500), presmall (17,000), and small (14,000). The four proteins were isolated by elution from polyacrylamide gels, and each protein migrated as a single band when analyzed by either sodium dodecyl sulfate or acidic polyacrylamide gel electrophoresis. Serial dilutions of the purified proteins were measured by radioimmunoassay. Both the slope of the inhibition curve and the level of maximal inhibition for each protein were the same as for the small myelin basic protein, indicating that each of the four proteins contains all of the antigenic sites present in the small basic protein. Structural relationships among the four proteins were examined by using two-dimensional analysis of tryptic digests. The results showed that: large was similar in amino acid sequence to the major myelin basic protein from other species; small was identical in sequence to large, except for an internal deletion of approximately 40 amino acid residues: prelarge contained the sequence of large plus an additional sequence of 25-35 amino acid residues; and presmall contained the sequence of small plus the same additional sequence as in prelarge. The four proteins were also treated with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolienine (BNPS-skatole) which cleaves proteins specifically at tryptophan residues. Analysis of the cleavage products indicated that the additional amino acid sequence in both prelarge and presmall extends from the amino terminus of the molecule. Several implications of these results are discussed.

Animals↗
PubMedDate not supplied

RNA on the road to myelin.

In oligodendrocytes some mRNAs are transported from the perikaryon to the distal processes and localized in the myelin compartment where they are translated. This review describes the cis-acting signals and trans-acting factors that mediate intracellular trafficking of myelin basic protein (MBP) RNA, the prototype for such mRNAs in myelinating glia.

Animals↗