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31.
32.
Maik Hüttemann Zhan Zhang Chadwick Mullins Denise Bessert Icksoo Lee Klaus-Armin Nave Sunita Appikatla Robert P Skoff 《ASN neuro》2009,1(3)
PMD (Pelizaeus–Merzbacher disease), a CNS (central nervous system) disease characterized by shortened lifespan and severe neural dysfunction, is caused by mutations of the PLP1 (X-linked myelin proteolipid protein) gene. The majority of human PLP1 mutations are caused by duplications; almost all others are caused by missense mutations. The cellular events leading to the phenotype are unknown. The same mutations in non-humans make them ideal models to study the mechanisms that cause neurological sequelae. In the present study we show that mice with Plp1 duplications (Plp1tg) have major mitochondrial deficits with a 50% reduction in ATP, a drastically reduced mitochondrial membrane potential and increased numbers of mitochondria. In contrast, the jp (jimpy) mouse with a Plp1 missense mutation exhibits normal mitochondrial function. We show that PLP in the Plp1tg mice and in Plp1-transfected cells is targeted to mitochondria. PLP has motifs permissive for insertion into mitochondria and deletions near its N-terminus prevent its co-localization to mitochondria. These novel data show that Plp1 missense mutations and duplications of the native Plp1 gene initiate uniquely different cellular responses. 相似文献
33.
Nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) regulate substance P release in adult spinal sensory neurons 总被引:8,自引:0,他引:8
NGF increases expression and content of substance P in developing and mature spinal sensory neurons. The role this neurotrophin plays in peptide release, however, is less clear. Accordingly, we examined substance P release from cultures of mature rat sensory neurons, which do not require NGF for survival. Neurons grown without NGF have a low but detectable basal release, which increases with depolarization by KCl (50 mM) but never achieves statistical significance. In contrast, basal release is 3 times higher from neurons that have been cultured in the presence of NGF, and KCl depolarization triples the amount of SP released. Stimulation with capsaicin (10–7 M) yields similar results. Residual peptide remaining after capsaicin stimulation is refractory to release for up to 24 h. Bradykinin does not induce SP secretion from mature neurons nor does it potentiate the action of capsaicin. GDNF, which also increases SP content, mimics NGF. Addition of NGF to the bath during release does not directly induce SP secretion, nor does it alter the effects of KCl, capsaicin, or bradykinin. It appears therefore that NGF increases SP release indirectly by increasing intracellular stores. 相似文献
34.
Cultured oligodendrocytes mimic in vivo phenotypic characteristics: Cell shape, expression of myelin-specific antigens, and membrane production 总被引:4,自引:0,他引:4
Primary cultures of neonatal mouse cerebra were maintained for up to 4 weeks in the absence of neurons. Oligodendrocytes in these cultures pass through a sequence of cytoarchitectural change and antigen expression which mimics the differentiation of oligodendrocytes in vivo. The cell bodies and processes of oligodendrocytes first express the myelin-specific antigen galactocerebroside (GC) by 2 days in vitro. Myelin basic protein (MBP) appears several days later. The majority of oligodendrocytes then proceed to elaborate large sheets of membranous material from the tips and lengths of cell processes. These membranous sheets, which contain GC and MBP, are reminiscent of unwrapped myelin profiles in vivo. As with the cell bodies and processes, GC is inserted into the sheets several days before MBP. Our results establish that oligodendrocytes cultured without neurons are able to produce extensive membranes containing myelin-specific antigens. They also suggest that oligodendrocyte shape and membrane production are, in part, regulated from within the oligodendrocyte itself. 相似文献
35.
The epithelium of the mouse lens stains intensely with antisera to glial fibrillary acidic protein (GFAP). A protein co-migrating with GFAP and immunoreactive with antisera to GFAP can be demonstrated in lens epithelium protein extracts by immunoblots. GFAP has previously been considered unique to cells of neural origin, but this study demonstrates that ectodermally derived cells express GFAP or a highly similar protein. 相似文献
36.
Auday Maki Salwa Atwan Janan Al-Kaledar Andrew Beaman Robert Skoff 《Biotechnic & histochemistry》1997,72(1):38-44
Technical limitations are associated with conducting successful in situ hybridization. In this study, three cell types including a tumor neuroblastoma cell line (Neuro-2a), an oligodendrocyte primary culture, and a nonneuronal acute lymphoblastic leukemia cell line (Reh) were used to conduct successful nonradioactive in situ hybridization. Two cDNA probes were used. A 1 kb probe was used to identify the expression of proteolipid protein (PLP) mRNA in a primary culture of oligodendrocytes. A 760 bp cDNA was used to identify the expression of ubiquitin C-terminal hydrolase (UCH-L1) mRNA in Neuro-2a and Reh cells. The probes were labeled with digoxigenin-11-dUTP, denatured, and hybridized with cells fixed on coverslips. The efficiency of the labeling was tested using dot blot analysis by comparing the intensity of our labeled probes with known concentration of the probe labeled by the provider. The nonspecific signals were washed off, followed by detection of a signal specific to the gene. The specificity of the probes was determined by treating the cells with RNase A, hybridizing with bacterial Dig-labeled cDNA (pBR322) and hybridizing the tissues in the absence of labeled probe. During the labeling step, we found that addition of co-precipitants, such as tRNA or glycogen, during precipitation of the labeled probe followed by overnight incubation at -20 C is essential for good recovery of labeled cDNA. Dissolving the labeled probe in a buffer solution containing sodium dodecyl sulfate improves the quantity of the labeling. At the cellular level, prehybridization treatments optimize the permeability of the cell and allow efficient penetration of the labeled probe. Fixing with paraformaldehyde or an ethanol-acetic acid mixture can preserve the structure of cultured cells. To increase the signal to noise ratio, cells were treated with 0.2 N HC1 followed by extensive washes using a solution with a high salt concentration and containing dextran sulfate. This treatment significantly improves the signal and reduces the background in cell cultures, but not in tissue sections. The ability to reuse the labeled probe-hybridization mixture is another advantage for using nonradioactive in situ hybridization. 相似文献
37.
Proteolipid protein (PLP) is a major structural component of central nervous system (CNS) myelin. Evidence exists that PLP or the related splice variant DM-20 protein may also play a role in early development of oligodendrocytes (OLs), the cells that form CNS myelin. There are several naturally occurring mutations of the PLP gene that have been used to study the roles of PLP both in myelination and in OL differentiation. The PLP mutation in the jimpy (jp) mouse has been extensively characterized. These mutants produce no detectable PLP and exhibit an almost total lack of CNS myelin. Additionally, most OLs in affected animals die prematurely, before producing myelin sheaths. We have studied cultures of jp CNS in order to understand whether OL survival and myelin formation require production of normal PLP. When grown in primary cultures, jp OLs mimic the relatively undifferentiated phenotype of jp OLs in vivo. They produce little myelin basic protein (MBP), never immunostain for PLP, and rarely elaborate myelin-like membranes. We report here that jp OLs grown in medium conditioned by normal astrocytes synthesize MBP and incorporate it into membrane expansions. Some jp OLs grown in this way stain with PLP antibodies, including an antibody to a peptide sequence specific for the mutant jp PLP. This study shows that: (1) an absence of PLP does not necessarily lead to dysmyelination or OL death; (2) OLs are capable of translating at least a portion of the predicted jp PLP; (3) the abnormal PLP made in the cultured jp cells is not toxic to OLs. These results also highlight the importance of environmental factors in controlling OL phenotype. © 1996 John Wiley & Sons, Inc. 相似文献
38.
Cerebroside Sulfotransferase in Golgi-Enriched Fractions from Rat Brain 总被引:15,自引:11,他引:4
Abstract: Golgi-enriched fractions were prepared from brainstems of 17-day-old rats by first floating off myelin, then fractionating the remaining pellet by a series of differential and density gradient centrifugations in sucrose. Fractions enriched in Golgi membranes were recovered at 0.46/0.76 m and 0.76/0.87 m interfaces on the final sucrose gradient as indicated by morphology and the biochemical markers thiamine pyrophosphatase and [3 H]fucose-labeled glycoprotein. Morphology of the two fractions indicated very little contamination with myelin lamellae; however, the presence of significant levels of 2', 3'-cyclic nucleotidase in the lighter fraction suggested a contribution from oligodendroglial or myelin-related membranes. Cerebroside sulfotransferase was highly enriched in the lighter Golgi-enriched fraction relative to the denser fraction, the post-34, 880 x g microsomes, and the myelin-like fraction. In contrast, ceramide galactosyl transferase was more evenly distributed among the fractions. Our results show a more highly localized distribution of sulfatide synthesis than of galactocerebroside synthesis, probably in Golgi membranes or oligodendroglia-related membranes with similar properties. 相似文献
39.
The divergent domains of the NEFA and nucleobindin proteins are derived from an EF-hand ancestor 总被引:2,自引:0,他引:2
Karabinos A; Bhattacharya D; Morys-Wortmann C; Kroll K; Hirschfeld G; Kratzin HD; Barnikol-Watanabe S; Hilschmann N 《Molecular biology and evolution》1996,13(7):990-998
The human protein NEFA (DNA binding, EF-hand, Acidic region) has previously
been isolated from a KM3 cell line and immunolocalized on the plasma
membrane, in the cytoplasma, and in the culture medium. Sequence analysis
of a cDNA clone encoding NEFA identified a hydrophilic domain, two
EF-hands, and a leucine zipper at the C- terminus. These characters are
shared with nucleobindin (Nuc). In this paper we have further characterized
NEFA and probed its evolutionary origins. Circular dichroism (CD) spectra
of recombinant NEFA indicated a helical content of 51% and showed that the
EF-hands are capable of binding Ca2+. Experiments with recombinant NEFA and
synthesized peptides revealed that the leucine zipper cannot form a
homodimer. The leucine zipper may allow heterodimer formation of NEFA and
an unknown protein. Phylogenetic analyses suggest that this protein is
derived from a four-domain EF-hand ancestor with subsequent duplications
and fusions. The leucine zipper and putative DNA-binding domains of NEFA
have evolved secondarily from existing EF-hand sequences. These analyses
provide insights into how complex proteins may originate and trace the
precursor of NEFA to the common ancestor of eukaryotes.
相似文献
40.
We sequenced the entire control region and portions of flanking genes
(tRNA(Phe), tRNA(Glu), and ND6) in the common chaffinch (Fringilla
coelebs), blue chaffinch (F. teydea), brambling (F. montifringilla), and
greenfinch (Carduelis chloris). In these finches the control region is
similar in length (1,223-1,237 bp) and has the same flanking gene order as
in other birds, and contains a putative TAS element and the highly
conserved CSB-1 and F, D, and C boxes recognizable in most vertebrates.
Cloverleaf-like structures associated with the TAS element at the 5' end
and CSB-1 at the 3' end of the control region may be involved with the stop
and start of D-loop synthesis, respectively. The pattern of nucleotide and
substitution bias is similar to that in other vertebrates, and consequently
the finch control region can be subdivided into a central, conserved G-rich
domain (domain II) flanked by hypervariable 5'-C-rich (domain I) and
3'-AT-rich (domain III) segments. In pairwise comparisons among finch
species, the central domain has unusually low transition/transversion
ratios, which suggests that increased G + T content is a functional
constraint, possibly for DNA primase efficiency. In finches the relative
rates of evolution vary among domains according to a ratio of 4.2 (domain
III) to 2.2 (domain I) to 1 (domain II), and extensively among sites within
domains I and II. Domain I and III sequences are extremely useful in
recovering intraspecific phylogeographic splits between populations in
Africa and Europe, Madeira, and a basal lineage in Nefza, Tunisia. Domain
II sequences are highly conserved, and are therefore only useful in
conjunction with sequences from domains I and III in phylogenetic studies
of closely related species.
相似文献