Immunocytochemical localization of rat peripheral nervous system myelin proteins: P2 protein is not a component of all peripheral nervous system myelin sheaths.

To identify and characterize specific mRNAs that increase in abundance during differentiation of mouse 3T3-L1 preadipocytes, a cDNA library was constructed from poly(A)+ RNA isolated from differentiated 3T3-L1 adipocytes. Mixed probe isotope ratio selection and RNA vine peripheral nerves, respectively, as well as 23% and 30% homology with fatty-acid binding proteins from rat liver and intestine, respectively. Moreover, the mRNA hybrid selected by pAL422 DNA directs the in vitro translation of an =13 kDa polypeptide, and this protein is specifically immunoprecipitated by antiserum against bovine myelin P2. These observations strongly suggest that the 422 protein is a structural, and possibly functional, analog of myelin P2.The 3T3-L1 cell line, isolated by Green and Kehinde (1, 2), represents a useful model system for investigating the mechanisms of cell differentiation. Under appropriate conditions, 3T3-L1 preadipocytes, which initially resemble fibroblasts, differentiate into adipocytes in cell culture (1-3). The differentiation process is accompanied by a dramatic increase in the activities of the enzymes associated with de novo lipogenesis (3-8) and triglyceride mobilization (9), as well as an increased sensitivity to lipogenic and lipolytic hormones (10-12). The cells accumulate triglyceride (1-3) and acquire the morphological characteristics of adipocytes (13) isolated from normal adipose tissue. There is good evidence that, for some of the lipogenic enzymes (e.g., fatty acid synthetase), the increases in activity during differentiation arise from increased rates of enzyme synthesis (6,14,15 MATERIALS AND METHODS Cell Culturing. 3T3-L1 preadipocytes were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum until differentiation was initiated according to the procedure of Reed and Lane (11). In this protocol, confluent cell monolayers were incubated for 48 hr in medium containing 10% fetal bovine serum/methylisobutylxanthine (115 ug/ml)/insulin (10 gg/ml)/dexamethasone (390 ng/ml). After 48 hr, the cells were washed free of methylisobutylxanthinie and dexamethasone and maintained in medium containing 10% fetal bovine serum and insulin at 10 ,ug/ml, at which time the cells express the adipocyte phenotype within 48 to 72 hr.Nucleic Acid Isolation and in Vitro Translations. Total cellular RNA was isolated by the procedures described by Chirgwin et al. (18) except that chloroform/butanol (4:1, vol/ vol) was used for deproteinization. The RNA was separated from any traces of DNA or protein by pelleting through CsCl (19). Poly(A)+-containing RNA was prepared as described by Aviv and Leder (20 5468The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

show abstract

“…Furthermore, the data in Fig. 5 show that this -13-kDa polypeptide is specifically immunoprecipitated* by anti-bovine myelin P2 antiserum (41) …”

Section: Resultsmentioning

confidence: 92%

Expression of specific mRNAs during adipose differentiation: identification of an mRNA encoding a homologue of myelin P2 protein.

Bernlohr¹,

Angus²,

Lane³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

304

183

View full text Add to dashboard Cite

show abstract

“…Sections were stained with a well-characterized P, antiserum (Trapp et al, 1979) by the peroxidase-antiperoxidase procedure as described previously (Trapp et al, 1981). RNA isolation and analysis.…”

Section: Methodsmentioning

confidence: 99%

Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells

1988

View full text Add to dashboard Cite

Upon transection of a peripheral nerve, axons distal to the transection degenerate. As a consequence of this axonal degeneration, myelin- forming Schwann cells cease biosynthesis of new myelin membrane, contribute to phagocytosis of previously formed myelin, and markedly down-regulate expression of myelin-specific markers. Among the most prominent of these down-regulated markers are the major structural proteins of peripheral myelin, Po and myelin basic protein (MBP). We have used slot blot and in situ hybridization techniques to demonstrate that for actively myelinating Schwann cells, down-regulation of the Po and MBP genes occurs primarily at the level of mRNA expression. Together with other recent data, these findings strongly argue for axonal modulation of Po and MBP gene transcription during active myelination.

show abstract

“…Sections were stained by the peroxidase-antiperoxidase procedure as described (14). The antisera directed against MBP (15) and Po protein (16) …”

mentioning

confidence: 99%

Spatial segregation of mRNA encoding myelin-specific proteins.

Trapp

Moench

Pulley

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

216

149

View full text Add to dashboard Cite

The cellular and subcellular distributions of mRNAs encoding three myelin-specific proteins-myelin basic protein (MBP), proteolipid protein (PLP), and Po proteinwere studied in tissue sections of developing rat nervous systems by in situ hybridization. The developmental appearance of these mRNAs closely paralleled the appearance of the proteins they encode as determined by immunocytochemistry. mRNA encoding the extrinsic membrane protein, MBP, was concentrated around oligodendrocyte and Schwann cell nuclei during initial stages of myelination; as myelination proceeded, MBP mRNA became distributed diffusely over myelinated fibers. In contrast, mRNAs encoding the intrinsic membrane proteins, PLP and Po, remained concentrated around oligodendrocyte (PLP) and Schwann cell (Po) nuclei at all stages of myelination. These results establish that myelinating cells spatially segregate certain myelin-specific mRNAs. The presence of MBP mRNA within the cytoplasmic domains of myelin internodes indicates that protein sorting during myelination involves transportation of mRNA to specific subcellular sites.Myelin, a multilamellar compact membrane, surrounds many axons in the central and peripheral nervous systems (CNS and PNS). CNS myelin is formed by oligodendrocytes, which individually have the potential to form 30-40 different myelin internodes by extending long slender cytoplasmic processes that ensheath and spirally wrap around axons to form compact myelin. In the PNS, Schwann cells form single myelin internodes. The protein composition of CNS and PNS myelin is well characterized (1). Ultrastructural studies have shown that CNS and PNS myelination occurs in a systematic and predictable manner (2, 3), suggesting that myelin-forming cells utilize very efficient mechanisms for synthesis, transport, and integration ofmyelin components to accomplish the massive expansion of membrane.Formation of myelin represents a terminal phenotypic expression of oligodendrocytes and Schwann cells that must involve the expression of a myelin-specific genetic program. DNAs complementary (cDNA) to the mRNAs that encode several myelin proteins have been cloned recently (4-10). Using these clones, the time courses for expression of myelin-specific mRNAs have been analyzed during development (4-10). The present study focuses on the cellular distribution of three myelin-specific mRNAs. Using in situ hybridization, we determined when these mRNAs could first be detected during early stages of myelination and asked whether spatial segregation of certain mRNAs might occur. (Kodak) melted at 43°C and diluted 1:1 with 0.6 M ammonium acetate. After development of the emulsion, the sections were stained with hematoxylin, dehydrated, and overlaid with coverslips. Sections were photographed with a Zeiss photomicroscope using bright-field and dark-field optics.The cDNA clones used have been well characterized and described elsewhere: proteolipid protein (PLP) (6), MBP (4), and Po (8). The specificity of cDNA hybridization was demonstrated by pretrea...

show abstract

Immunocytochemical localization of rat peripheral nervous system myelin proteins: P2 protein is not a component of all peripheral nervous system myelin sheaths.

Cited by 114 publications

References 16 publications

Expression of specific mRNAs during adipose differentiation: identification of an mRNA encoding a homologue of myelin P2 protein.

Expression of specific mRNAs during adipose differentiation: identification of an mRNA encoding a homologue of myelin P2 protein.

Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells

Spatial segregation of mRNA encoding myelin-specific proteins.

Contact Info

Product

Resources

About