Power-law and logarithmic relaxations of hydrated proteins: a molecular dynamics simulations study.

We use molecular dynamics simulations to study anomalous internal protein dynamics observed for the backbone atoms of hydrated elastin and hydrated myoglobin in the picoseconds and nanoseconds regimes. The anomalous dynamics manifests itself in a sublinear increase of the atomic mean square displacements and in a power-law or logarithmic-like decay of correlation functions. We find that several, but not all, observations can be described in the frameworks of rugged potential-energy landscape and fractional Fokker-Planck approaches, in particular, a fractional Ornstein-Uhlenbeck process. Furthermore, mode-coupling theory allows us to rationalize findings at ambient temperatures, but there are deviations between theoretical predictions and simulation results related to the anomalous dynamics at cryogenic temperatures. We argue that the observations are consistent with a scenario where a broad ß-relaxation peak shifts through the picoseconds and nanoseconds regimes when cooling from 300 to 200 K, say. Inspection of trajectories of consecutive nitrogen atoms along the protein backbone reveals that correlated forward-backward jumps, which exhibit a substantial degree of cooperativity, are a key feature of the anomalous dynamics.

Identification of a new exon in the myelin proteolipid protein gene encoding novel protein isoforms that are restricted to the somata of oligodendrocytes and neurons.

The myelin proteolipid protein (PLP) gene (i.e., the PLP/DM20 gene) has been of some interest because of its role in certain human demyelinating diseases, such as Pelizaeus-Merzbacher disease. A substantial amount of evidence, including neuronal pathology in knock-out and transgenic animals, suggests the gene also has functions unrelated to myelin structure, but the products of the gene responsible for these putative functions have not yet been identified. Here we report the identification of a new exon of the PLP/DM20 gene and at least two new products of the gene that contain this exon. The new exon, located between exons 1 and 2, is spliced into PLP and DM20 mRNAs creating a new translation initiation site that generates PLP and DM20 proteins with a 12 amino acid leader sequence. This leader sequence appears to target these proteins to a different cellular compartment within the cell bodies of oligodendrocytes and away from the myelin membranes. Furthermore, these new products are also expressed in a number of neuronal populations within the postnatal mouse brain, including the cerebellum, hippocampus, and olfactory system. We term these products somal-restricted PLP and DM20 proteins to distinguish them from the classic PLP and DM20 proteolipids. They represent putative candidates for some of the nonmyelin-related functions of the PLP/DM20 gene.

Identification of a novel silencer that regulates the myelin basic protein gene in neural cells.

The myelin basic protein gene produces two families of proteins, the golli proteins and the 'classic' myelin basic proteins from three transcription start sites (tsp). The golli proteins are expressed from the first tsp, and little is known about genetic elements that control its activity. We have examined elements that may regulate the expression of the golli products produced from this promoter in neural cell lines with constructs containing upstream portions of the first tsp by transient transfection assays. Three putative regulatory elements were identified, among them a 345bp novel silencer region, termed the golli silencer region (GSR), which was characterized in detail. This silencer was responsible for a significant (approx. 60%) inhibition of luciferase expression in PC12 cells. It was orientation-dependent and a double dose of this GSR completely abolished expression of the luciferase reporter activity. Transfections with deleted constructs identified three critical sites that bind at least two repressor proteins. We postulate that the silencer activity is the result of synergistic interactions between these repressor proteins and might involve the formation of a high-ordered protein-DNA structure.

An AG----GG transition at a splice site in the myelin proteolipid protein gene in jimpy mice results in the removal of an exon.

The myelin proteolipid protein gene was characterized in jimpy mice to identify the specific mutation that produces dysmyelination, oligodendrocyte cell death, and death of the animal by 30 days of age. Exon 5 and flanking intron segments were isolated from jimpy proteolipid protein genomic clones and sequenced. A single nucleotide difference was noted between the normal and jimpy proteolipid protein genes: the conversion of an AG/GT to a GG/GT in the splice acceptor signal preceding exon 5, which apparently destroys the splice signal. Thus, exon 5 of the mouse myelin proteolipid protein gene is skipped during the processing of mRNA, producing a shortened proteolipid protein mRNA.

Expression of the myelin basic protein gene locus in neurons and oligodendrocytes in the human fetal central nervous system.

The myelin basic protein (MBP) gene locus is composed of two overlapping transcription units that share all of the MBP exons. One of these transcription units expresses the MBPs and the other expresses a family of proteins structurally related to the MBPs. This second transcription unit is called the Golli gene, and the entire complex is called the Golli-mbp gene. In this study, the expression of the Golli gene was examined in the human fetal central nervous system (CNS). By using reverse transcriptase-polymerase chain reaction cloning we have identified eight new members of the Golli gene family of transcripts expressed in the human CNS. Golli gene expression was examined by in situ hybridization and immunohistochemistry, and surprisingly, Golli products were found to be expressed in neurons as well as oligodendrocytes. Furthermore, the subcellular distribution of Golli immunoreactivity in fetal spinal cord interneurons shifted between the various laminae. Golli protein was localized within the nuclei of interneurons in the posterior horn, but was found in the cell bodies and processes of interneurons in the anterior horn. Within oligodendrocytes, Golli protein was detected in the cell bodies and processes, including processes which were wrapping axonal segments. Golli mRNA expression was also observed in neurons within the cerebral cortex between 18 and 20 weeks postconception, prior to myelination of this brain region. During this period, there was a striking developmental increase in the numbers and in the locations of neurons expressing Golli mRNAs within the cortical plate. The diverse distribution of Golli proteins within neurons and oligodendrocytes indicates that their function is quite different from that of the MBPs to which they are closely related.

Strain-related differences in the ability of T lymphocytes to recognize proteins encoded by the golli-myelin basic protein gene.

Protein products of the golli-MBP gene complex, expressed in the nervous and lymphoid systems, contain sequences in common with sequences in 'classic' MBP, expressed exclusively in the nervous system. In this report, it was determined whether T cell lines (TCLs) specific for encephalitogenic epitopes of 'classic' MBP were able to recognize sequences within golli-MBP. TCLs derived from SJL mice specific for the immunodominant 83-102 sequence and the subdominant 19-27 sequence of 'classic' MBP recognized golli-MBP J37 and BG21, respectively. In contrast, TCLs derived from PL and B10.PL mice specific for the immunodominant 1-9 sequence of 'classic' MBP did not recognize this sequence within either J37 or BG21. These strain-related differences in the ability of golli-MBP proteins to stimulate 'classic' MBP-specific TCLs are discussed with respect to a possible influence on whether the course of EAE is relapsing (SJL) or monophasic (PL and B10.PL).

Expression of the myelin proteolipid protein gene in the human fetal thymus.

We have analyzed human fetal thymus and spleen for expression of the proteolipid protein (PLP) gene. We demonstrate that the PLP gene is transcribed in both tissues, and that both the PLP and DM-20 mRNAs are produced. Western blot analyses revealed that both the PLP and DM-20 protein isoforms were present in the fetal thymus and spleen. Immunohistochemical analyses indicated that the PLP/DM-20 proteins were detected in cells which have the distribution and morphology of thymic macrophages. These results provide further evidence that the PLP and DM-20 proteins are expressed in cell types other than myelin forming cells and possess function(s) unrelated to myelin structure. Furthermore, these data demonstrate that the PLP and DM-20 proteins are not shielded from the immune system behind the blood-brain barrier. These observations directly impinge upon the debate concerning acquisition of tolerance and the recognition that the encephalitogenic nature of PLP in diseases, such as Multiple Sclerosis, may not simply be related to its 'sequestration' from a 'naive' immune system.

Isolation and characterization of a zebra finch aromatase cDNA: in situ hybridization reveals high aromatase expression in brain.

The brain regions controlling song are much larger in male than in female zebra finches. This sex difference is thought to arise developmentally when the male's brain is exposed to higher levels of estrogen. The synthesis of estrogen from androgen is catalyzed by aromatase, a key enzyme implicated in song development in the zebra finch. To study the role of estrogen synthesis in the organization of brain regions responsible for song, a cDNA encoding aromatase was isolated from a zebra finch ovarian cDNA library. The 3188 bp cDNA contains a 1527 bp open reading frame with 5' and 3' untranslated regions of 116 bp and 1545 bp, respectively. The deduced polypeptide is 509 amino acids in length, and is highly homologous to aromatases reported for chicken (92%), human (72%), mouse (70%), rat (69%) and trout (53%). Northern blot analysis revealed 5.4 kb, 4.8 kb and 3.2 kb aromatase mRNAs in brain and ovary. In situ hybridization histochemistry revealed the expression of aromatase mRNA in ovarian thecal cells of some, but not all, follicles, suggesting that aromatase gene expression is regulated during follicular maturation. In the adult brain, the distribution of aromatase mRNA was surprisingly widespread, and included the preoptic area, hypothalamus, hippocampus and neostriatum. By contrast, little aromatase mRNA expression was noted in the song nuclei (HVC, RA, area X). This study, the first description of aromatase mRNA expression by in situ hybridization in the brain of any species, identifies a surprisingly large number of cells that express aromatase mRNA in the zebra finch telencephalon. This pattern may be a unique feature of all songbirds.

Translational regulation of myelin protein synthesis by steroids.

Various steroids, including glucocorticoids, were observed to exert a direct effect on the rates of translation of several myelin-protein specific transcripts in a cell-free, reticulocyte lysate system. Hydrocortisone caused a twofold stimulation in the translation of mRNAs of myelin basic protein and proteolipid protein. It inhibited the translation of 2',3'-cyclic nucleotide 3'-phosphodiesterase mRNA by 50%, and had no effect on the translation of a number of other mRNAs. The data suggest that steroid-mediated translational regulation may serve as a novel mechanism to modulate the expression of myelin protein genes at the translational level.

Subunit composition of skeletal muscle transverse tubule calcium channels evaluated with the 1,4-dihydropyridine photoaffinity probe, [3H]azidopine.

The arylazide 1,4-dihydropyridine, [3H]azidopine, binds with high affinity to calcium channels in partially purified guinea-pig skeletal muscle transverse tubule membranes. Upon brief exposure to u.v. light, [3H]azidopine incorporates covalently into transverse tubule membrane proteins, as judged by SDS-PAGE. After alkylation of sulfhydryl groups with N-ethylmaleimide three specifically labelled bands of mol wts. 240 kd, 158 kd and 99 kd are always observed with fluorography after one-dimensional SDS-PAGE. Two other specific bands with mol. wts. of 52 kd and 55 kd, respectively, were sometimes observed. Two-dimensional SDS-PAGE (non-reduced but alkylated in the first dimension and reduced in the second dimension) revealed that the 240-kd band after reduction migrates with a mol. wt. of 99 kd. The 158-kd and 99-kd bands do not change in mobility. It is suggested that [3H]azidopine binds in such a way that the arylazide moiety of the ligand comes into contact with at least three calcium channel components: the A component of mol. wt. 240 kd, the B component of mol. wt. 158 kd and a C component of mol. wt. 99 kd. B and C are non-covalently bonded subunits of the channel, whereas A could be a heterodimer consisting of B and C, linked by disulfide bonds. Subunits of smaller mol. wt. may be also part of the ionic pore. Photolabelling of transverse tubule membranes after high energy irradiation with 10 MeV electrons supports this interpretation.

Isolation and characterization of a cDNA encoding the zebra finch myelin proteolipid protein.

A cDNA was isolated from a zebra finch telencephalon cDNA library that encodes the myelin proteolipid protein. The clone was 2874 nucleotides long containing an open reading frame of 831 nucleotides that encoded a 277 amino acid myelin proteolipid protein. The 5'- and 3' untranslated regions were 112 and 1931 nucleotides, respectively. In Northern blots the clone hybridized to 3 bands of 3.5, 2.4 and 1.5 Kb in mouse brain RNA, but to only a single band of 3.0 kb in zebra finch brain RNA, suggesting the lack of alternative polyadenylation sites within the 3' untranslated region of the zebra finch PLP mRNAs. There was a small degree of homology between the zebra finch and chicken PLP 5' untranslated regions, but relatively little homology of the 5' untranslated regions of the zebra finch PLP cDNA clone with the homologous regions of PLP cDNAs of many mammalian species. Except for a small stretch of considerable homology, there was little overall homology with the 3' untranslated regions of mammalian PLP mRNAs. Approximately 10% (i.e. 28) of the amino acids in the zebra finch PLP differed from mammalian PLP, with most of these changes located within exon 3. There were 16 amino acid changes between zebra finch and chicken, suggesting that greater sequence variation in PLP structure is tolerated among avian species than among mammalian species.

An analysis of the regions of the myelin basic protein that bind to phosphatidylcholine.

The interactions of phosphatidylcholine (PC) to regions of the myelin basic protein (MBP) was examined. In solid phase binding assays the nature of the binding of unilamellar vesicles of 14C-labeled phosphatidylcholine to bovine 18.5 kDa MBP, its N- and C-terminal peptide fragments, photooxidized 18.5 kDa MBP and the mouse 14 kDa protein, with an internal deletion of residues 117-157, was studied. The data were analyzed by computer-generated Scatchard plots in which non-specific binding was eliminated. Non-cooperative, low affinity binding of PC vesicles to MBP was observed, and this binding found to be sensitive to pH and ionic changes. At an ionic strength of 0.1 and pH 7.4, the binding of PC to the 14 kDa mouse MBP exhibited a Kd similar to that obtained with both the N-terminal and photooxidized 18.5 kDa bovine MBP. The studies indicated that the sites of PC interaction with MBP are located in the N-terminal region of the protein. The C-terminal region appeared to modulate the strength of the interaction slightly. Under similar conditions, lysozyme did not bind PC liposomes, and histone bound them nonspecifically.

The major myelin protein genes are expressed in the human thymus.

Demyelinating diseases, such as multiple sclerosis (MS) in man or experimental allergic encephalomyelitis (EAE) in rodents, may include an associated immune response directed against myelin protein antigens such as the proteolipid protein (PLP) and the myelin basic protein (MBP). Development of an immune response has been attributed, in part, to the sequestration of central nervous system antigens behind the blood-brain barrier. Recently, we identified a novel gene, the golli gene, which overlaps the mbp gene. The Golli transcription unit produces a family of mRNAs, and their corresponding proteins possess MBP epitopes known to be encephalitogenic in EAE. Transcription of the golli gene was detected in immune system tissue. Therefore, we wished to determine whether genes that encode the two major myelin protein components, PLP and MBP, were expressed in the human thymus. Our data demonstrate that both the plp and golli genes are transcribed in the fetal human thymus. Moreover, both the PLP and DM-20 transcripts are produced from the plp gene, and the HOG 7 and HOG 5 transcripts are produced from the golli gene. Confocal fluorescent immunohistochemistry using antibodies for the PLP/DM-20 and Golli proteins, co-localized expression of these antigens to thymic macrophages. Thus, the plp and golli genes are expressed, and their corresponding protein produced, in an antigen presenting cell in the human immune system.

DM20 mRNA splice product of the myelin proteolipid protein gene is expressed in the murine heart.

Northern blot analysis of poly A(+) RNA isolated from mouse heart revealed the expression of 3.3 and 2.4 kb mRNAs that hybridized with a cDNA for the mouse proteolipid protein (PLP). In order to examine the relationship of these RNAs to the myelin PLP/DM20 mRNAs, a mouse heart cDNA library was prepared and screened with a mouse PLP cDNA. A cDNA was isolated, sequenced, and found to encode the DM20 variant of PLP. Polymerase chain reaction (PCR) analysis of heart cDNA with three sets of primers confirmed the presence of DM20 mRNA in mouse heart and indicated that it is the major splice product of the PLP gene expressed in that tissue. In situ hybridization localized the expression of the DM20 mRNA to the myocardial cells. Northern blot analysis indicated that expression of the DM20 mRNA is developmentally regulated in the murine heart, increasing significantly in concentration after 12 days postpartum. Northern analysis also revealed the expression of the DM20 mRNA in the hearts of the jimpy and quaking mutants. These results indicate that the PLP gene is expressed in tissues other than brain and support the concept that products of the PLP gene may have some biological role other than as structural components of myelin.

The human myelin basic protein gene is included within a 179-kilobase transcription unit: expression in the immune and central nervous systems.

Two human Golli (for gene expressed in the oligodendrocyte lineage)-MBP (for myelin basic protein) cDNAs have been isolated from a human oligodendroglioma cell line. Analysis of these cDNAs has enabled us to determine the entire structure of the human Golli-MBP gene. The Golli-MBP gene, which encompasses the MBP transcription unit, is approximately 179 kb in length and consists of 10 exons, seven of which constitute the MBP gene. The human Golli-MBP gene contains two transcription start sites, each of which gives rise to a family of alternatively spliced transcripts. At least two Golli-MBP transcripts, containing the first three exons of the gene and one or more MBP exons, are produced from the first transcription start site. The second family of transcripts contains only MBP exons and produces the well-known MBPs. In humans, RNA blot analysis revealed that Golli-MBP transcripts were expressed in fetal thymus, spleen, and human B-cell and macrophage cell lines, as well as in fetal spinal cord. These findings clearly link the expression of exons encoding the autoimmunogen/encephalitogen MBP in the central nervous system to cells and tissues of the immune system through normal expression of the Golli-MBP gene. They also establish that this genetic locus, which includes the MBP gene, is conserved among species, providing further evidence that the MBP transcription unit is an integral part of the Golli transcription unit and suggest that this structural arrangement is important for the genetic function and/or regulation of these genes.

Differential sensitivity in the survival of oligodendrocyte cell lines to overexpression of myelin proteolipid protein gene products.

The proteolipid (PLP) gene encodes at least four proteins, including the classic PLP and DM20, which are important components of the myelin sheath, and the recently identified soma-restricted (sr) isoforms, srPLP and srDM20. The classic PLP and DM20 gene products have been implicated in oligodendrocyte survival by overexpression studies in vitro and in vivo. The classic and sr proteolipids are targeted to different cellular compartments in the oligodendrocyte, suggesting different cellular functions. Accordingly, we examined the effects of in vitro overexpression of the sr-PLP/DM20 isoforms on the survival of stably transfected, conditionally immortalized, oligodendroglial cell lines and compared this to overexpression of the classic and the jimpy-mutated proteolipids. The results indicate that overexpression of either normal or jimpy classic PLP/DM20 resulted in a dramatic reduction in the survival of the oligodendrocyte cell lines at the nonpermissive temperature, but not the COS-7 cell line, a cell line expressing the same oncogene constitutively. Survival of the oligodendrocyte cell lines was significantly less affected when either the sr-PLP/DM20 or the dopamine D-2 receptor, another cell membrane protein, was overexpressed in the cell lines. These results suggest that overexpression of the "classic" PLP or DM20 can compromise the survival of oligodendrocytes whether or not they are mutated. Furthermore, they suggest that the internal mechanisms for normal targeting of the PLP/DM20 isoforms of either the "classic" or the "sr" types influence the oligodendrocyte's ability to survive when these proteolipids are overexpressed.

Myelin basic protein gene expression in neurons: developmental and regional changes in protein targeting within neuronal nuclei, cell bodies, and processes.

The myelin basic protein (MBP) gene is part of the golli-mbp gene complex. In mouse, the golli-mbp gene produces two families of mRNAs from different transcription start sites that generate either MBPs or golli proteins (which contain MBP sequences in addition to unique peptide sequences). In situ hybridization and immunocytochemical analyses indicate that golli products are expressed in selected neuronal populations in postnatal mouse brain, in addition to oligodendrocytes, as shown earlier. The principal subcellular location of golli proteins in neurons was in axonal and dendritic processes. In a small subset of neurons, golli proteins were located in nuclei. With development and neuronal maturation, golli-mbp expression decreased and/or there was a striking shift in subcellular localization from nuclei and cell soma to the cell processes in specific neuronal populations. Golli protein was localize in neurites of migrating cerebellar granule cells, but it shifted to a nuclear localization when the cells took up residence in the internal granule cell layer. In some regions, (e.g., olfactory bulb and cerebellum) golli proteins were expressed over the entire postnatal period examined (birth to 75 d). The unique patterns of developmental expression within individual populations of neurons, and the unusual shift in subcellular localization of golli proteins with neuronal migration and maturation, suggest a complex regulation of this gene at both the transcriptional and posttranslational levels. The data also suggest that the cellular function(s) of the golli proteins is very different from the structurally related MBPs.

Identification of genes in the oligodendrocyte lineage through the analysis of conditionally immortalized cell lines.

The mouse oligodendrocyte cell lines, N19 and N20.1, were used as sources of potential stage-specific RNA in order to construct a subtraction library enriched in cDNAs expressed early in the oligodendrocyte (OL) lineage. From this library, 23 clones were examined and three were examined in most detail. The mRNAs of the three library clones were preferentially expressed in the N19 (progenitor) compared to the N20.1 (immature) OL line. One of these corresponded to the intermediate filament protein cytokeratin K19, which has not been reported to be expressed in OLs previously. Another was identified as the mouse homolog of T-cadherin, previously reported not to be present in OLs. Antisera raised against a T-cadherin peptide indicated the protein colocalized with the OL lineage markers A(2)B(5), A007, and 01 in mouse primary glial cultures. However, small round cells resembling OL precursors labeled intensely with T-cadherin, but were negative for the other markers, suggesting that this gene might be expressed earlier in the lineage. In early postnatal brain, in addition to the expected neuronal tracts, the T-cadherin antibody labeled small bipolar cells, approximately 8-10 microm in diameter, in white matter tracts. These cells had the morphology of OLs or their precursors and were identified within the cerebellar white matter and the corpus callosum, regions rich in OLs. The third clone, 3g5, was homologous to the P8 clone isolated from rat pancreas. It encoded an 80-amino-acid polypeptide with a protein kinase C domain suggesting a possible role in signal transduction. Antisera to this peptide also colocalized 3g5 with cells expressing A(2)B(5), A007, and 01 in culture and in cells within white matter tracts which had the same morphology as those labeled by T-cadherin in these regions. In addition to these, beta(10) thymosin and mevalonate kinase clones were also isolated from the screen.

Embryonic expression of the myelin basic protein gene: identification of a promoter region that targets transgene expression to pioneer neurons.

The myelin basic protein (MBP) gene produces two families of structurally related proteins from three different promoters-the golli products, generated from the most upstream promoter, and the MBPs, produced from the two downstream promoters. In this report we describe the expression of golli proteins within some of the earliest neuronal populations of the brain, including Cajal-Retzius cells and preplate neurons of the forebrain, representing a new marker for these cells. To identify elements responsible for neuronal expression of the golli products, we generated transgenic animals from constructs containing different portions of the upstream promoter. A construct containing 1.1 kb immediately upstream of the golli transcription start site targeted expression of beta-galactosidase to preplate neurons and a subset of Cajal-Retzius cells in transgenic mice-the first reported genetic element to target expression to these pioneer cortical populations. Although expression in Cajal-Retzius cells declined with embryonic development, preplate cells continued to express the transgene after arriving at their final destination in the subplate. Interestingly, expression persisted in subplate neurons found within a distinct layer between the white matter and cortical layer VI well into postnatal life. Birth dating studies with bromodeoxyuridine indicated that these neurons were born between E10.5 and E12.5. Thus, the transgene marked subplate neurons from their birth, providing a fate marker for these cells. This work suggests a role for the MBP gene in the early developing brain long before myelination and especially in the pioneer cortical neurons important in the formation of the cortical layers.