PubMed Health⌕ Search

Biomedical subjects

M Manthorpe

Publications and source records attributed to M Manthorpe.

At least 37 records · Page 2Linked to original sources
PubMed1992

Immunolocalization of ciliary neuronotrophic factor in adult rat sciatic nerve.

Two rabbit polyclonal antibodies were raised against synthetic peptides corresponding to residue numbers 45-59 and 181-200 of rat ciliary neuronotrophic factor (CNTF). The resulting antibodies were purified by affinity chromatography and both purified antibodies reacted by enzyme-linked immunoassay (ELISA) and immunoblotting with rat sciatic nerve CNTF. The anti-CNTF peptide antibodies were used to immunostain sections of adult rat sciatic nerve, previously known as the richest tissue source of CNTF. By light microscopy both antibodies appeared to stain exclusively Schwann cells and axons and both did so with the same pattern of specific staining. Immunostaining was eliminated by absorption of the anti-peptide antibodies with either their corresponding peptide or with purified rat nerve CNTF or by using purified nonspecific IgG. Schwann cells were stained and in semi-thin sections this staining appeared to be in the Schwann cell cytoplasm. Axons could be stained in addition to Schwann cells providing higher concentrations of antibodies were used. Epineurial, endoneurial and endothelial cells appeared unstained. Since all Schwann cells and axons appear to contain CNTF and since CNTF is known to act in vitro to support sensory and sympathetic ganglionic and motor neurons, we suggest that Schwann cells may normally provide CNTF to those neurons contributing axons to the peripheral nerve.

Amino Acid Sequence↗
PubMed1992

CG-4, a new bipotential glial cell line from rat brain, is capable of differentiating in vitro into either mature oligodendrocytes or type-2 astrocytes.

We have established a permanent cell line (CG-4) of rat central nervous system glial precursors from primary cultures of bipotential oligodendrocyte-type 2-astrocyte (O-2A) progenitor cells, which were kept proliferating with the mitogen(s) secreted by the neuronal B104 cell line. The CG-4 cells have a normal karyotype and display the properties of normal O-2A cells. CG-4 cells can be propagated in serum-free culture medium supplemented with medium conditioned by B104 cells for unrestricted periods of time as O-2A cells, characterized by the presence of the A2B5 surface marker and the absence of markers specific for oligodendrocytes (galactocerebroside, myelin basic protein) or type 2-astrocytes (glial acidic fibrillary protein). bFGF and PDGF are potent mitogens for CG-4 cells and their combination can substitute for the B104-derived mitogen(s). CG-4 cells are capable of differentiating into either oligodendrocytes or type 2-astrocytes. Differentiation into oligodendrocytes occurs after withdrawal of the mitogen. Replacement of the mitogen with fetal calf serum (20%), in contrast, induces 50% of the CG-4 cells to differentiate into type 2-astrocytes. Pure cultures of oligodendrocytes or type 2-astrocytes can be generated in substantial amounts from CG-4 cells and maintained for several weeks in medium containing 5% fetal calf serum.

Animals↗
PubMed1992

Merosin promotes cell attachment and neurite outgrowth and is a component of the neurite-promoting factor of RN22 schwannoma cells.

The laminin-like protein merosin was purified from human placenta in intact form and as pepsin fragments and compared to laminin in heparin affinity chromatography and cell binding assays. Intact merosin and a small fragment of merosin comprising the last two repeats of the heavy chain g domain bind to heparin. Intact merosin and large pepsin fragments of merosin, but not the small C-terminal fragment, mediate the attachment and spreading of several types of cells and promote neurite outgrowth from neuronal cells similar to laminin and its corresponding fragments. Cells with various integrin-type receptors for laminin attached equally well to merosin and laminin, suggesting that several of the known laminin binding receptors also bind to merosin. Antibodies to the beta 1 subunit of integrins inhibited neurite outgrowth on merosin as well as on laminin, confirming the involvement of integrin-mediated interaction of cells with both merosin and laminin. Schwannoma cells, which have previously been shown to produce a laminin-like, neurite-promoting factor, synthesize merosin in vivo and in vitro as shown by protein and mRNA analysis. The results suggest that merosin, which is the more abundant basement membrane protein in the laminin family, has properties very similar to laminin despite differences in the structure of the heavy chain. Furthermore, merosin may be identical to or a component of the neurite-promoting factors previously reported from heart, muscle, and Schwann cells.

Base Sequence↗
PubMed1992

Stromelysin generates a fibronectin fragment that inhibits Schwann cell proliferation.

Our previous report (Muir, D., S. Varon, and M. Manthorpe. 1990. J. Cell Biol. 109:2663-2672) described the isolation and partial characterization of a 55-kD antiproliferative protein found in Schwann cell (SC) and schwannoma cell line-conditioned media and we concluded that SC proliferation is under negative autocrine control. In the present study the 55-kD protein was found to possess metalloprotease activity and stromelysin immunoreactivity. The SC-derived metalloprotease shares many properties with stromelysin isolated from other sources including the ability to cleave fibronectin (FN). Furthermore, limited proteolysis of FN by the SC-derived protease generated a FN fragment which itself expresses a potent antiproliferative activity for SCs. The active FN fragment corresponds to the 29-kD amino-terminal region of the FN molecule which was also identified as an active component in SC CM. Additional evidence that a proteolytic fragment of FN can possess antiproliferative activity for SCs was provided by the finding that plasmin can generate an amino-terminal FN fragment which mimicked the activity of the SC metalloprotease-generated antiproliferative FN fragment. Both the 55-kD SC metalloprotease and the 29-kD FN fragment could completely and reversibly inhibit proliferation of SCs treated with various mitogens and both were largely ineffective at inhibiting proliferation by immortalized or transformed SC lines. Normal and transformed SC types do secrete the proform of stromelysin, however, transformed cultures do not produce activated stromelysin and thus cannot generate the antiproliferative fragment of FN. These results suggest that, once activated, a SC-derived protease similar to stromelysin cleaves FN and generates an antiproliferative activity which can maintain normal SC quiescence in vitro.

Animals↗
PubMed1991

Sequence and structural organization of the human gene encoding ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a potent polypeptide hormone whose actions appear to be restricted to the nervous system where it promotes survival, neurotransmitter synthesis and neurite outgrowth in certain neuronal populations. We have cloned the gene encoding human CNTF (hCNTF) and have characterized its structure and organization. The hCNTF gene appears to be a unique-copy gene with a simple genetic organization, since only a single intron interrupts the coding domain. The hCNTF gene is located on chromosome 11, as determined using human-hamster somatic cell hybrids. The CNTF protein is highly conserved in evolution. The amino acid (aa) sequences of rat and rabbit CNTF translated from cDNAs display approx. 85% homology with the deduced aa sequence encoding hCNTF.

Amino Acid Sequence↗
PubMed1991

Dose-dependent responses to nerve growth factor by adult rat cholinergic medial septum and neostriatum neurons.

This study describes the relationship between the concentration of intraventricularly infused nerve growth factor (NGF) and several responses by axotomized cholinergic medial septum neurons and normal cholinergic neostriatal neurons of the adult rat. NGF infused for 14 days starting either immediately after a unilateral fimbria-fornix transection or after a 2-week delay period elicited similar dose-response relationships for the maintenance or restoration of ChAT and NGF receptor positivity and cell body size and for intraseptal 'sprouting' of the axotomized medial septum neurons. Thus, in the medial septum it appears that the expression of 'marker' molecules, cell body size and the induction of 'sprouting' are regulated by virtually the same concentrations of NGF in the two treatment strategies. This suggests that NGF has a general regulatory role and injured but untreated neurons remain fully susceptible to NGF at least up to 2 weeks after the lesion. A 14-day infusion with NGF also induced an above-normal cell body size (hypertrophy) both in axotomized medial septum and in intact striatal cholinergic neurons. The hypertrophic response of normal striatal neurons required less NGF than did that of medial septum neurons. Since the striatal response began to be detectable at a similar concentration as that required for the full maintenance or restoration of ChAT and NGF receptor positivity it could be seen as an unwanted side-effect. The definition of a sub-optimal dose with which a significant, but not maximal response can be elicited will allow future evaluations of potentially additive or synergistic actions by other agents.

Animals↗
PubMed1991

Nerve growth factor promotes CNS cholinergic axonal regeneration into acellular peripheral nerve grafts.

Peripheral nerve grafts promote vigorous regeneration of adult mammalian CNS axons. Elimination of nerve-associated cells by freeze-thawing abolishes this promoting quality, possibly by creating inhibitory cellular debris and/or destroying the production of stimulatory factors by living Schwann or other cells. Here, debris-free acellular peripheral nerve segments placed between the disconnected septum and the hippocampal formation acquired almost no cholinergic axons after 1 month. However, such acellular nerve grafts treated before implantation with purified beta-nerve growth factor (NGF) contained nearly as many longitudinally oriented cholinergic axons as did fresh cellular nerve grafts. These results suggest that (i) NGF is required for the regeneration of adult CNS cholinergic axons into nerve grafts and (ii) an important function of living cells within peripheral nerve may be the production of neuronotrophic factors such as NGF.

Acetylcholinesterase↗
PubMed1991

Biologically active basic fibroblast growth factor migrates at 27 kD in "non-denaturing" SDS-polyacrylamide gel electrophoresis.

Using mild conditions of SDS-PAGE, i.e. no heating of the sample, and the PhastSystem (Pharmacia), we found that bFGF, either natural bovine or recombinant human migrated at a 27 kD position in addition to the classical 18 kD one. By the cell-blot technique, we found that the biological activity toward rat astroblasts and 3T3 mouse fibroblasts was always restricted to the 27 kD band. Partial heat denaturation experiments revealed a close correlation between the remaining biological activity of bFGF in solution and the ratio of the 27 kD band versus the 18 kD band seen on SDS gels. These observations suggest that the bFGF which is biologically active in solution migrates at an apparent Mr of 27 kD in our conditions of electrophoresis, keeping its biological activity after electrophoresis, and the molecules which are inactive (denatured) in solution migrate at 18 kD and remain inactive. These experimental conditions, in which the biological activity appears to be preserved, could be referred to as "non-denaturing SDS-polyacrylamide gel electrophoresis" and could be useful, associated to cell-blot, for the search and characterization of new growth factors active on cells in culture.

3T3 Cells↗
PubMed1990

Reversibility of ganglioside effects on astrocyte morphology.

The B-subunit of cholera toxin (BCT) induces a morphological change in cultured rat cerebral astrocytes from flat (epithelioid) to stellate (process-bearing). This stellation is reversed by the gangliosides GM1 and GD1a at concentrations of 10 microM or higher. Upon changing to a ganglioside-free medium, the flat astrocytes reacquire the stellate morphology within 3 hr, indicating that the antistellation effect of gangliosides is reversible. The possibility that this reversibility was due to a loss of exogenously acquired gangliosides from the cell membrane can be ruled out since pretreatment with GM1, but not GD1a, which does not bind BCT, results in an increased responsiveness to BCT, which was identical whether measured immediately after withdrawal of the ganglioside or 3 hr later. Asialo-GM1, which neither binds BCT nor reverses BCT-induced stellation by itself, prevents the return to stellation after withdrawal of the gangliosides. These data suggest that while gangliosides remain associated with the cell, their effect on astrocytes can change from opposing to permitting the stellate morphology.

Animals↗
PubMed1990

An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures.

We report a quantitative method by which a single microculture can be examined for (i) cell morphology; (ii) cell number; (iii) DNA synthesis; and (iv) expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.

Animals↗
PubMed1990

Septohippocampal cholinergic axonal regeneration through peripheral nerve bridges: quantification and temporal development.

Axons of the adult mammalian CNS have been shown to regrow vigorously into peripheral nerve grafts. Using a cholinergic septohippocampal model for adult CNS regeneration, involving complete denervation of the hippocampal formation from its basal forebrain cholinergic afferents, this study has established quantitative parameters and a temporal baseline of cholinergic fiber regeneration into the dorsal hippocampal tissue through a peripheral sciatic nerve graft. In nerve-implanted animals (i) the nerve grafts are maximally invaded by AChE-positive fibers between 2 weeks and 1 month postlesion, (ii) the fibers entering the hippocampal formation from the graft show a peak numerical increase and rate of elongation around the first month and/or in the proximal hippocampal region, (iii) an apparently normal innervation pattern and fiber density in the most rostral 1.5 mm of the dorsal hippocampal formation is reached by 6 months postlesion. The present study provides a basis for future quantitative comparisons of manipulations of different components of the system, e.g., the contributing neurons, the bridging material, and the receiving central nervous tissue. The temporal/spatial pattern of fiber regeneration suggests that the hippocampal CNS tissue can be a good axonal growth-promoting environment, albeit with temporal and/or spatial limitations, and is therefore not an immutably restrictive environment for axonal regeneration.

Acetylcholinesterase↗
PubMed1990

Differential effects of axotomy on substance P-containing and nicotinic acetylcholine receptor-containing retinal ganglion cells: time course of degeneration and effects of nerve growth factor.

The time course of degeneration of chick retinal ganglion cells was examined with Nissl stains and immunohistochemical methods for detection of substance P-like immunoreactive and nicotinic acetylcholine receptor immunoreactive neurons. Small lesions were made in the retinae, adjacent to the optic nerve head, and were subsequently sectioned parallel to the vitreal surface, permitting direct comparison of normal and axotomized retinal ganglion cells distal to the site of axon damage. At four and six days after surgery, a large number of degenerating cells with clear cytoplasm and pyknotic nuclei were seen. After eight, 10 and 14 days, many retinal ganglion cells displayed a chromatolytic response with dispersed Nissl granules, eccentric nuclei and the cells appeared crenulated. The number of apparently normal neurons in the ganglion cell layer in the axotomized region was reduced by about 50% six days following surgery, by about 70% on the 10th day and by about 75% on the 17th day. The remaining neurons in the ganglion cell layer were identified as displaced amacrine cells. From day 2 onwards, increased numbers of glial cells were present in the optic fibre, ganglion cell and inner plexiform layers. Many glial cells were enlarged and displayed extensive cytoplasmic processes, while others showed mitotic activity. Somata and proximal dendrites of retinal ganglion cells were intensely stained for substance P-like immunoreactivity at two and four days following surgery. At six, eight and 10 days, staining intensity was markedly reduced though still evident and at 14 and 17 days, substance P-like immunoreactivity had virtually disappeared. The persistence of limited substance P-like immunoreactive ganglion cells 10 days after surgery indicates that these cells have a relatively protracted response to axotomy. Nicotinic acetylcholine receptor-like immunoreactivity in the ganglion cells at two and four days following axotomy was substantially reduced. The majority of faintly stained nicotinic acetylcholine receptor-like immunoreactive ganglion cells, as visualized in counterstained sections, did not exhibit pyknosis in the immediate period following axotomy. Double label studies demonstrated that substance P-like immunoreactive ganglion cells were distinct from the nicotinic acetylcholine receptor-like immunoreactive ganglion cells. In a second set of experiments, nerve growth factor was then placed into the vitreous humor following intra-retinal axotomy. The somata, dendrites and proximal axons of lesioned substance P-like immunoreactive ganglion cells in these retinae were more intensely stained for a longer period of time and appeared more robust than cells from untreated retinae.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Schwann cell proliferation in vitro is under negative autocrine control.

In healthy adult peripheral nerve, Schwann cells are believed to be generally quiescent. Similarly, cultures of isolated rat sciatic nerve Schwann cells hardly proliferate in serum-supplemented medium. The possibility that Schwann cells negatively regulate their own proliferation was supported by the demonstration that conditioned media from Schwann cell cultures inhibited the proliferation of mitogen-stimulated test cultures. The inhibition could be complete, was dose dependent, and was exhibited when the test Schwann cells were under the influence of different types of mitogens such as cholera toxin, laminin, and living neurons. The inhibition of proliferation was completely reversible and a rapid doubling of cell number resulted when treatment with conditioned medium was withdrawn from mitogen-stimulated Schwann cells. Conditioned medium from cholera toxin-stimulated and immortalized Schwann cell cultures contained less antiproliferative activity than that found in medium from quiescent Schwann cell cultures. However, media conditioned by two actively proliferating rat Schwannoma cell lines were rich sources of antiproliferative activity for Schwann cells. Unlike the mitogen-stimulated Schwann cells, whose proliferation could be inhibited completely, the immortalized and transformed Schwann cell types were nearly unresponsive to the antiproliferative activity. The antiproliferative activity in Schwann and Schwannoma cell conditioned media was submitted to gel filtration and SDS-PAGE. The activity exists in at least two distinct forms: (a) a high molecular weight complex with an apparent molecular mass greater than 1,000 kD, and (b) a lower molecular weight form having a molecular mass of 55 kD. The active 55-kD form could be derived from the high molecular weight form by gel filtration performed under dissociating conditions. The 55-kD form was further purified to electrophoretic homogeneity. These results suggest that Schwann cells produce an autocrine factor, which we designate as a "neural antiproliferative protein," which completely inhibits the in vitro proliferation of Schwann cells but not that of immortalized Schwann cells or Schwannoma lines.

Animals↗
PubMed1990

Nerve growth factor infusion into the denervated adult rat hippocampal formation promotes its cholinergic reinnervation.

The well-documented but little-understood failure of lengthy axonal regeneration after injury of the adult mammalian CNS may be caused by an insufficient availability of local growth-promoting factors. If so, identifying and supplying the missing factors may result in better central axonal regeneration. This hypothesis was tested in an adult rat CNS model in which peripheral nerve grafts were placed into a lesion cavity between the septum and hippocampal formation. Continuous infusion of nerve growth factor (NGF) into the dorsal hippocampal tissue dramatically enhanced and accelerated the regrowth and penetration of cholinergic axons into the hippocampal formation. Thus, NGF can overcome the apparent resistance of the hippocampal CNS tissue to cholinergic reinnervation.

Acetylcholinesterase↗
PubMed1989

Nerve growth factor (NGF) reverses axotomy-induced decreases in choline acetyltransferase, NGF receptor and size of medial septum cholinergic neurons.

Intraventricular nerve growth factor (NGF) infusion in the adult rat can prevent and also, if delayed, reverse the disappearance of most of the axotomized medial septum cholinergic neurons immunostained for choline acetyltransferase (ChAT). We have utilized the delayed NGF treatment protocol to (i) extend to 3 months the delay time between axotomy and NGF treatment, (ii) define the time course of their recovery, (iii) determine that immunostaining for the (lower affinity) NGF receptor (NGFR) parallels loss and reversal of the ChAT marker, and (iv) evaluate changes in cholinergic somal size following axotomy and subsequent NGF treatment. While NGF treatments starting only 7 days after the fimbria-fornix transection (axotomy) almost entirely restored the number of both ChAT- and NGFR-positive medial septum neurons, longer delayed (2-3 weeks) treatment brought about recovery from the baseline of 20-25% to only about 70% of the control numbers. This limited recoverability, however, persisted even after a 95 day delay period. In all cases examined maximal recoveries were achieved within 3-7 days of NGF treatment. Neuronal size analyses provided evidence for an axotomy-induced atrophy. NGF treatments, started with 1 or 2 week delays, not only reversed fully the average somal size loss but also induced an actual hypertrophy of several of those neurons. These results provide additional evidence that at least half of the apparent loss of cholinergic medial septum neurons upon axotomy is due to a loss of markers such as the transmitter-related enzyme ChAT and NGFR rather than to actual neuronal cell death. These results also show that NGF exerts a genuine trophic influence by regulating the size of its target neurons as well as their content of several proteins.

Animals↗
PubMed1989

Rat sciatic nerve Schwann cell microcultures: responses to mitogens and production of trophic and neurite-promoting factors.

During embryonic development and in response to injury, the growing axons of peripheral neurons may influence the migration and proliferation of Schwann cells which, in return, may present neurons with a critical supply of factors required for neuronal survival, growth and differentiation. The identification and characterization of agents influencing the proliferation of Schwann cells as well as Schwann cell production of factors affecting neurons is greatly facilitated by the use of in vitro techniques. We describe here a simplified method of obtaining large numbers of purified neonatal rat sciatic nerve Schwann cells for use in generating large numbers of replicate microcultures. We then illustrate the use of these microcultures to examine Schwann cell: i) morphology and survival; ii) proliferation; and iii) production of neuronotrophic and neurite-promoting activities. We report that rat Schwann cells in microculture proliferate in response to serum, laminin and fibronectin, cholera toxin, and chick embryo parasympathetic ciliary neurons. Also, extracts of Schwann cell microcultures contain independently regulated activities which support the survival and neurite outgrowth of peripheral ganglionic neurons.

Animals↗
PubMed1989

An improved device for continuous intraventricular infusions prevents the introduction of pump-derived toxins and increases the effectiveness of NGF treatments.

The recent demonstrations of the ability of nerve growth factor (NGF) to protect and promote the welfare of certain cholinergic neurons in the adult CNS have increased the need for safe, accurate, and reliable procedures for intracerebral administration of protein and other experimental agents. Osmotic minipumps have been used to infuse NGF into the lateral ventricle of adult rats, but a sustained and harmless performance of such infusions has not been fully evaluated. The study reported here has led to (i) the recognition that cytotoxic substances, released from some minipumps into the infusion fluid, may be responsible for various degrees of periventricular tissue damage, and (ii) the redesigning of an infusion device which, among other modifications, uses the osmotic pump to propel infusion fluid into the ventricle but prevents pump-derived materials from entering the infusate itself. Besides several other advantages, the modified infusion device has permitted the demonstration that NGF can fully protect experimentally axotomized medial septum cholinergic neurons and can do so with less variability than previously observed and without creating tissue damage.

Animals↗