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G I Tennekoon

Publications and source records attributed to G I Tennekoon.

35 records · Page 2Linked to original sources
PubMed1991

The role of complex carbohydrates in adhesion of the myelin protein, P0.

The most abundant protein of peripheral nerve myelin, a glycoprotein termed P0, is believed to be involved in the compaction of the myelin sheath and is postulated to be the closest relative to the ancestral gene for the immunoglobulin superfamily. Recently, P0 has indeed been shown to behave like a homophilic adhesion molecule via interactions of its extracellular domains. Here we demonstrate the importance of the oligosaccharide moieties of P0 in its functioning as a homophilic adhesion molecule. Expression of the complex form of P0 glycoprotein in transfected Chinese hamster ovary cells greatly increased the adhesiveness of those cells, whereas expression of the high-mannose form of P0 glycoprotein did not. This is the first step in the dissection of P0-P0 interaction at the molecular level.

Animals↗
PubMed1991

Structure of the mouse myelin P2 protein gene.

Myelin P2 protein is a small (14,800 Da) protein found in peripheral and central nervous system myelin. To investigate the regulation of expression of this myelin protein, a mouse genomic library was screened with a rabbit P2 cDNA (pSN2.2-2), and a single positive phage clone containing a 20-kb insert was obtained. This insert contained a single internal SalI restriction site and several EcoRI sites. The EcoRI fragments from this insert were subcloned into Bluescript. The rabbit P2 cDNA (pSN2.2-2) hybridized with a 4-kb EcoRI fragment, and this 4-kb fragment was then sequenced after the creation of nested deletions. The mouse gene contained four exons: exon 1 coded for amino acids 1-24, exon 2 for amino acids 24-81, exon 3 for amino acids 82-115, and exon 4 for amino acids 116-131. The three introns were 1.2 kb, 150 bp, and 1.3 kb in length. Primer extension analysis revealed two transcription start sites at +1 and +47, consistent with the presence of two P2 mRNAs, with the larger transcript appearing more abundant. The amino acid sequences predicted from the mouse DNA indicate that the mouse protein differs from the rabbit protein at 16 different positions, with most of the differences located in exon 3. When the gene structure of fatty acid binding protein (FABP) genes were compared, the P2 gene had the same overall structure as others in the FABP family, suggesting a common ancestral gene for members of this family. The 5'-flanking region contains candidate TATA and CAAT sequences, as well as two AP-1 binding sites that may be important in modulation of the expression of this gene.

Animals↗
PubMed1990

Role of myelin P0 protein as a homophilic adhesion molecule.

Peripheral nervous system myelin is an extension of the Schwann cell's plasma membrane that tightly enwraps axons in many layers and permits nerve impulses to be rapidly conducted. It is not known how these multiple membrane layers are held together in this compact form. Here we present evidence supporting the hypothesis that the extracellular leaflets of myelin are held together by the most abundant protein of myelin of the peripheral nervous system, P0, by homophilic interaction of its extracellular domains. Transfected Chinese hamster ovary cells expressing P0 protein adhere to each other in suspension, to form large aggregates, whereas cells that are identical but which do not express P0 do not. We also show that this aggregation is mediated by homophilic binding between P0-expressing cells and that the apposing plasma membranes of these cells specifically form desmosomes, whereas control transfected cells do not. As the only difference between the two cell populations is the expression of P0, this protein is apparently responsible for the changes in morphology and adhesion in the cells that express it. The idea that P0 is a homophilic adhesion molecule is supported by its inclusion in the immunoglobulin supergene family, all members of which are involved in recognition and/or adhesion.

Animals↗
PubMed1990

Proliferation and differentiation of a transfected Schwann cell line is altered by an artificial basement membrane.

Rapidly dividing transfected Schwann cells were grown on Matrigel, a reconstituted basement membrane gel. Matrigel decreased the proliferation of the cells by 75% when compared to sister cultures that were grown on an untreated plastic substrate. Some transfected cells plated onto a Matrigel substrate formed colonies similar to that observed when the cells were plated on a plastic substrate. Additionally, many cells on Matrigel assembled themselves into fascicles projecting away from the colonies. These fascicles were composed of transfected Schwann cells that had assumed a bipolar appearance reminiscent of quiescent secondary Schwann cells in culture. Transfected cells grown on Matrigel contained approximately 10-fold less glial fibrillary acidic protein when compared to sister cultures grown on an untreated plastic substrate. By indirect immunofluorescence laminin immunoreactivity appeared as globules within the cytoplasm of the cells which were cultured on a plastic substrate. However, cells that were grown on the Matrigel substrate appear to organize laminin in a linear array around themselves. These results demonstrate that the presence of an artificial basement membrane alters the morphology, rate of proliferation, and state of differentiation of a transfected Schwann cell line.

Basement Membrane↗
PubMed1990

High level of expression of the myelin protein P0 in Chinese hamster ovary cells.

The major PNS myelin protein, P0, has been expressed in Chinese hamster ovary cells by transfection with a plasmid containing the P0-cDNA. The expression of P0 at both the RNA and the protein level was greatly increased, without detriment to the cell, by the dihydrofolate reductase-methotrexate strategy of gene amplification. The P0 expressed by these cells was glycosylated (containing approximately equal amounts of the complex and high-mannose type glycoproteins) and reached the plasma membrane. This system is suitable not only for addressing the function of P0 directly, but it also applicable to any protein of which an abundance is needed.

Animals↗
PubMed1990

Evidence that secondary rat Schwann cells in culture maintain their differentiated phenotype.

Schwann cells, on receiving the correct signal, will encircle an axon and wrap it with a myelin sheath. To begin examining some of the mechanisms underlying the process of myelination in vitro, we isolated Schwann cells from the sciatic nerves of neonatal rats and generated large cell populations with cholera toxin. The immunological and biochemical properties of these secondary Schwann cells were characterized after five to seven passages in the absence of axonal contact. These cells continued to express antigens found in both myelinating (P0 and 2',3'-cyclic nucleotide phosphohydrolase) and nonmyelinating cells in vivo (A5E3 and glial fibrillary acidic protein) in addition to the markers common to both types of cells (Ran-1, 217c, S-100, and laminin). Biochemical analyses showed that these cells synthesize the very-long-chain fatty acids (22-26 carbon atoms) found in myelin membranes. Moreover, the enzymes required for the synthesis of myelin glycolipids (including sphingosine acyltransferase, UDP-galactose:ceramide galactosyltransferase, and cerebroside sulfotransferase) were still active, and metabolic labeling studies showed that galactocerebroside and sulfatide were synthesized even though the galactocerebroside pool was insufficient to be detected by immunostaining. Secondary Schwann cells also synthesized four species of myelin basic protein and the major structural glycoprotein in myelin, P0. The pathway necessary for glycosylation of P0 protein remained active, and an analysis of the oligosaccharide chain revealed that approximately 70% was processed to a complex form. In summary, we found that secondary Schwann cells still express most of the immunological markers of differentiated cells and continue to synthesize low levels of myelin components. Therefore, Schwann cells do not dedifferentiate in culture, as previously believed.

Animals↗
PubMed1990

Production of Schwann cell lines using a regulated oncogene.

The process of myelination in the central and peripheral nervous systems has been well characterized morphologically by a variety of techniques. It is evident from these studies that, in the peripheral nervous system myelin formation is a multistep process. Clearly, a 1:1 relationship must be established with the axon, which is followed by formation of the basal lamina and eventually myelin. Because immortalized Schwann cell lines obtained using SV40 T antigen under the control of an inducible promoter have many properties of untransfected Schwann cells in culture, including their ability to form myelin in vitro, these cells will enable us to dissect more easily the process of myelination. Having successfully immortalized rat Schwann cells without affecting their ability to differentiate fully, we are applying this approach to generate analogous cell lines from the peripheral nerves of other species such as mouse and human. Unlike rat Schwann cells, there are no known mitogens for human and mouse Schwann cells, making it impossible to expand these cell populations. The ability to produce large numbers of human Schwann cells from nerve biopsy and to analyze their biochemical properties would be of enormous value in identifying the cellular abnormalities that result in demyelinating disease. Likewise, there are several mutant mouse strains with defects in myelin formation, and cell lines from these animals would facilitate our understanding of the process leading to dysmyelination.

Animals↗
PubMed1989

Isolation of rat Schwann cell lines: use of SV40 T antigen gene regulated by synthetic metallothionein promoters.

Synthetic promoter elements from the mouse metallothionein-I promoter controlling the expression of SV40 T antigen have been tested for their efficacy in cloning rat Schwann cell lines that retained the characteristic properties of these cells and could be passed in culture indefinitely. The constructed promoters contained either four (MT4) or five (MT5) copies of a metal regulatory element 5' to the CAAT and TATA elements from the HSV-1 thymidine kinase gene. Characterization of these promoters in transient expression assays and transformation assays showed that both MT5 and MT4 were approximately 10-fold and 15-fold, respectively, weaker than the wild-type MT-I promoter in the presence of heavy metal inducer. However, in the absence of inducer, the basal activity of both MT5 and MT4 was barely detectable and much lower than that of MT-I. Schwann cells were transfected with plasmids containing the SV40 T antigen gene under the control of the different metallothionein promoters and cell lines were established from each. Only with the MT5 and MT4 promoters were lines obtained that resembled secondary Schwann cells in culture in their morphology, generation time, and demonstration of contact inhibition. In the presence of zinc, the expression of T antigen in the lines derived with MT5 and MT4 was about 10-fold lower than that derived with MT-I. On removal of the inducer this level was reduced, and in one cell line T antigen was undetectable. Concomitant with the reduction in T antigen expression there was an increased expression of Po, a protein specific to myelin-forming Schwann cells, and a decreased expression of glial fibrillary acidic protein, a protein expressed only in nonmyelin-forming Schwann cells. These cell lines, therefore, closely resemble untransfected Schwann cells in culture.

Animals↗
PubMed1988

Reconstitution of adenosine 3'-phosphate 5'-phosphosulfate transporter from rat brain.

Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the "active" sulfate donor for sulfated macromolecules, is synthesized in the cytosolic fraction of rat brains. This molecule is then translocated into the lumen of the Golgi apparatus so that it is available to the sulfotransferase enzymes. The protein responsible for the PAPS translocating activity has been solubilized from vesicles enriched in enzyme markers for the Golgi apparatus and reconstituted into liposomes. In reconstituted liposomes translocating activity has a pH optimum of 7.0 and activity was increased 3-fold by divalent cations, although EDTA produced no inhibition. The affinity of the reconstituted translocator for PAPS showed a Km of 1.2 mM with a Vmax of 14 pmol of PAPS translocated/min/mg of protein. Specificity of the translocator activity was tested with a number of nucleotide analogues and only 3',5'-adenosine diphosphate was a competitive inhibitor. Inhibitors of the mitochondrial ADP/ATP transporter and the red cell anion channel blocked transport of PAPS only at very high concentrations.

Animals↗
PubMed1987

Transfection of neonatal rat Schwann cells with SV-40 large T antigen gene under control of the metallothionein promoter.

Secondary cultures of Schwann cells were transfected with a plasmid containing the SV-40 T antigen gene expressed under the control of the mouse metallothionein-I promoter. We used the calcium phosphate method for transfection and obtained a transfection efficiency of 0.01%. The colonies were cloned by limited dilution, and these cloned cell lines were carried in medium containing zinc chloride (100 microM). One cloned cell line, which has now been carried for 180 doublings, appears to have a transformed phenotype with a doubling time of 20 h. These cells express SV-40 T antigen while maintaining established Schwann cell properties (positive staining for 217c, Ran-2, A5E3, glial fibrillary acidic protein, presence of 2',3'-cyclic nucleotide phosphohydrolase [CNPase] activity, and the ability to synthesize sulfogalactosylceramide and mRNA for the myelin protein, P0). Removal of zinc chloride from the medium resulted in reduced expression of T antigen and a change in the appearance of the cells to a more bipolar shape, although they still did not exhibit contact inhibition and maintained a doubling time of 20 h. These cells now became Ran-2-negative and showed increases in CNPase activity and in their ability to synthesize sulfogalactosylceramide. The amount of P0 mRNA remained unchanged. Transfected Schwann cells, however, stopped dividing when they contacted either basal lamina or neurites and became bipolar in appearance. The Schwann cells in contact with the neurites then extended processes to wrap around bundles of neurites. Transfection with the SV-40 T antigen gene therefore provides a method for obtaining Schwann cell lines that continue to express properties associated with untransfected cells in culture and may be used to study axon-Schwann cell interaction.

Animals↗
PubMed1987

Selective growth of rat Schwann cells in neuron- and serum-free primary culture.

A serum-free medium, designated S4 [consisting of Eagle's Minimum Essential Medium supplemented with the following components (mg/liter): endothelial mitogen, 100; vitamin C, 1; insulin, 5; cholera toxin, 0.01; putrescine, 8; sodium selenite, 0.0043; MgCl2 X 6H2O, 40; and albumin, fatty acids, amino acids, vitamins, and trace elements], has been established to select for the growth of Schwann cells from primary cultures of dissociated neonatal rat sciatic nerve in the absence of neurons and without exposure of the cells to serum or antimitotic agents. By immunological criteria, confluent primary cultures, grown on a substrate of extracellular matrix derived from cultured bovine aorta endothelial cells, consist of approximately 90% Schwann cells after maintenance in this serum-free medium. Secondary cultures of at least 99% purity may be established after treatment of primary cultures maintained in S4 with antiserum against a fibroblast surface antigen, Thy 1.1, in conjunction with complement. Such secondary Schwann cells continue to proliferate in S4, with no decrease in purity, through at least 4 passages.

Animals↗
PubMed1986

Immunoreactive myelin basic protein in cerebrospinal fluid of patients with peripheral neuropathies.

The presence of myelin basic protein (MBP)-like material in cerebrospinal fluid (CSF) usually reflects breakdown of central nervous system myelin. Immunoreactive MBP levels were measured in 70 CSF specimens from 66 patients with a variety of peripheral neuropathies. Immunoreactive MBP was present in CSF in 70% (16 of 23) of patients with biopsy-proved chronic demyelinating polyneuropathies, whereas in neuropathies producing primarily axonal damage it was present in only 22% (5 of 23). In Guillain-Barré syndrome, 45% (9 of 20) of patients had immunoreactive MBP in the CSF. We conclude that MBP-like material is present in the CSF of most patients with longstanding demyelinating polyradiculoneuropathies and probably reflects detection of peripheral nervous system P1 protein.

Demyelinating Diseases↗
PubMed1986

Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons.

Nerve growth factor (NGF) is a neuronotrophic protein. Its effects on developing peripheral sensory and sympathetic neurons have been extensively characterized, but it is not clear whether NGF plays a role during the development of central nervous system neurons. To address this point, we examined the effect of NGF on the activity of neurotransmitter enzymes in several brain regions. Intracerebroventricular injections of highly purified mouse NGF had a marked effect on the activity of choline acetyltransferase (ChAT), a selective marker of cholinergic neurons. NGF elicited prominent increases in ChAT activity in the basal forebrain of neonatal rats, including the septum and a region which contains neurons of the nucleus basalis and substantia innominata. NGF also increased ChAT activity in the hippocampus and neocortex, terminal regions for the fibers of basal forebrain cholinergic neurons. In analogy with the response of developing peripheral neurons, the NGF effect was shown to be selective for basal forebrain cholinergic cells and to be dose-dependent. Furthermore, septal neurons closely resembled sympathetic neurons in the time course of their response to NGF. These observations suggest that endogenous NGF does play a role in the development of basal forebrain cholinergic neurons.

Animals↗
PubMed1985

Choline acetyltransferase activity in striatum of neonatal rats increased by nerve growth factor.

Some neurodegenerative disorders may be caused by abnormal synthesis or utilization of trophic molecules required to support neuronal survival. A test of this hypothesis requires that trophic agents specific for the affected neurons be identified. Cholinergic neurons in the corpus striatum of neonatal rats were found to respond to intracerebroventricular administration of nerve growth factor with prominent, dose-dependent, selective increases in choline acetyltransferase activity. Cholinergic neurons in the basal forebrain also respond to nerve growth factor in this way. These actions of nerve growth factor may indicate its involvement in the normal function of forebrain cholinergic neurons as well as in neurodegenerative disorders involving such cells.

Acetylcholine↗
PubMed1981

Cerebroside sulfotransferase: preparation of antibody and localization of antigen in kidney.

This immunohistochemical study describes the localization of the enzyme cerebroside sulfotransferase (phosphoadenosine phosphosulfate: galactosylceramide sulfotransferase, EC 2.8.2.11) in rat kidney. The enzyme was purified from kidney and the preparation was used to raise antibodies for immunocytochemical investigations. In the kidney, the antigen was present only on the brush border of the epithelial cells of the proximal tubules, suggesting that sulfation of glycolipids occurs in the cytoplasm and plasma membranes of these specific cells. Moreover, biochemical and immunocytochemical studies of cerebroside sulfotransferase during development indicate that catalytic activity is correlated with the appearance of enzyme protein.

Animals↗
PubMed1977

Myelinogenesis in optic nerve. A morphological, autoradiographic, and biochemical analysis.

Morphological, autoradiographic, and biochemical methods were used to study the time of appearance, distribution, and nature of sulfated constituents in the developing rat optic nerve. Electron microscope studies showed that myelination begins (6 days postnatal) shortly after the appearance of oligodendroglia (5 days postnatal). Over the ensuing 3 wk, myelination increased rapidly. During the 1st postnatal wk, mucopolysaccharides and glycoproteins were labeled with 35S and autoradiographs showed grains over arachnoidal cells, astroglia, and the glia limitans. These results indicated that astroglia synthesize sulfated mucopolysaccharides of the glia limitans. After the onset of myelination, however, the major portion of [35S]sulfate was incorporated into sulfatide. Autoradiographs showed a shift of radioactive grains from astroglia and arachnoidal cells to myelin, indicating that actively myelinating oligodendroglia incorporate [35S]sulfate into myelin sulfatide; there was a concomitant increase in the activity of cerebroside sulfotransferase. In addition, the increasing amounts of proteolipid protein and myelin basic protein corresponded with the morphological appearance of myelin. These results point to a strict correlation between the structural and biochemical changes occurring during myelination. This system provides a useful model for studies designed to evaluate the effects of various perturbations on the process of myelination.

Animals↗