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Biomedical subjects

M Manthorpe

Publications and source records attributed to M Manthorpe.

At least 109 records · Page 6Linked to original sources
PubMed1982

Purified mouse Schwann cells: mitogenic effects of fetal calf serum and fibroblast growth factor.

We present an in vitro bioassay protocol for the detailed examination of mitogenic influences on purified mouse Schwann cells. This involves measurements of (i) increases in Schwann cell number per culture, (ii) incorporation of radiolabeled thymidine per culture, and (iii) the proportion of total Schwann cells that exhibit a labeled nucleus by autoradiography. Using this standard protocol we show that the mitogenic efforts of fetal calf serum must be distinguished from its effects on cell retention to the culture substratum, and preparations of mouse brain or pituitary fibroblast growth factor but not mouse epidermal growth factor are very potent mouse Schwann cell mitogens. These mitogenic effects of serum and fibroblast growth factor have not been reported for rat Schwann cells.

Animals↗
PubMed1981

Neuronotrophic factors and their antibodies: in vitro microassays for titration and screening.

Using nerve growth factor (NGF), anti-NGF sera and dissociated neonatal mouse dorsal root ganglionic neurons we present a microculture assay methodology for (1) the titration of neurotrophic factor (NTF) activity in monolayer culture, (2) the titration of NTF antibodies which 'block' NTF biological activity, (3) the titration of NTF antibodies that bind and remove (sequester) NTF from culture medium and (4) a large-scale, convenient, and rapid screening for NTF biological activity as well as for NTF 'blocking' or 'sequestering' antibodies. These quantitative and qualitative in vitro microimmunoassays should be applicable to any neuronotrophic factor or its antibody, even when the agent is only available in crude, unpurified form. Since the microculture systems permit the simultaneous screening of one thousand samples per day they should be useful for the detection and quantitation of monoclonal antibodies present in hybridoma-conditioned media.

Animals↗
PubMed1981

Polyornithine-attached neurite-promoting factors (PNPFs). Culture sources and responsive neurons.

We have recently reported the existence within chick embryo heart cell conditioned medium (HCM) of two distinct and independently assayable factors. One agent, ciliary neuronotrophic factor (CNTF), supports the in vitro survival of 8-day chick embryo ciliary ganglionic (CG) neurons. The other factor, polyornithine-attachable neurite promoting factor (PNPF) is required for extensive neuritic growth from these same CNTF-supported CG neurons. In the present study we have examined the occurrence of PNPF activity within nearly 100 different conditioned media using our previously described chick CG bioassay system. From this screening we conclude that: (1) PNPF production is a rather widespread property of cultured neural as well as non-neural cells; and (2) the chick bioassay is sensitive to PNPF activity from all the species examined, including mouse, rat, human and chick cells. We next examined the effects of 3 representative PNPF-containing conditioned media (from chick heart, mouse Schwann and rat Schwannoma) on neurite production from 3 other peripheral ganglionic neuronal cultures (8-day chick dorsal root, 11-day chick sympathetic, and neonatal mouse dorsal root ganglia) as well as 4 central neuronal cultures (8-day chick embryo telencephalon, optic lobe and spinal cord and neonatal mouse cerebellum). The results of these studies indicate: (1) that the peripheral neurons exhibit a dramatic increase in neurite production in response to PNPF which can be easily recognized both qualitatively and quantitatively; whereas (2) the CNS neurons showed essentially no PNPF-induced increase in neurite production. The sole exception to the latter was the appearance within the chick spinal cord cultures of a neuronal population which extended very long neurites in response to PNPF.

Animals↗
PubMed1981

Trophic activities for dorsal root and sympathetic ganglionic neurons in media conditioned by Schwann and other peripheral cells.

We describe here the use of 3 established nerve growth factor (NGF) targets (cultured neurons from dissociated chick embryo sympathetic ganglia, and chick embryo or neonatal mouse dorsal root ganglia) to investigate neuronotrophic activities in conditioned media (CMs) from: (i) chick embryo heart; (ii) purified mouse Schwann cells; and (iii) clonal rat Schwannoma RN22 cells. In chick sympathetic and mouse dorsal root ganglionic cultures, all 3 CMs supported survival of the same number of neurons as did mouse submaxillary NGF, and in most cases no increased survival resulted from concurrent administration of NGF and any one CM. NGF and CM activities were quantitated in each of the responsive cell systems. No differences were seen when either test population was used for the same agent, or when different CMs were examined on the same test cells. The CM activity, unlike that of NGF, was not blocked by even excess amounts of antiserum against mouse submaxillary NGF. The neuronotrophic activity of CMs appears to reside with macromolecular constituents. None of the CMs displayed trophic activity on chick embryo dorsal root ganglionic neurons. However, at least one of them (RN22 medium) had drastic effects on these ganglionic cells even in the presence of NGF, leaving open the possibility of a 'toxic' factor overriding putative trophic agents.

Animals↗
PubMed1981

Neurite-promoting factor in conditioned medium from RN22 Schwannoma cultures: bioassay, fractionation, and properties.

On polyornithine (PORN) substrata dissociated 8-day chick embryo ciliary ganglionic neurons will survive if the culture medium is supplemented with Ciliary neuronotrophic Factor. However, neuritic growth will not occur unless the substratum is derivatized with a PORN-bindable Neurite Promoting Factor (PNPF). In this preliminary study we report that soluble PNPF can be (1) assayed by a convenient in vitro system; (2) obtained in relatively large amounts from serum-free media conditioned over RN22 Schwannoma cultures; (3) concentrated by using Amicon XM100 ultrafiltration; and (4) separated from nearly all of the non-active protein by using ion-exchange chromatography. The partially purified PNPF can be concentrated using XM100 and is heat- and protease-sensitive. In the course of these fractionation studies we observed in some cases a concentration-dependent interference with the expression of PNPF activity in the bioassay; we propose graphical methods to permit the simultaneous determination of PNPF and the extent of such interference. Different treatments that affected the interference property did not always affect PNPF activity in a reciprocal manner, leaving open the possibility that the interference with PNPF activity results from reversible alteration of the PNPF molecule, or that there exists a separate interfering agent.

Animals↗
PubMed1980

Purification of mouse Schwann cells using neurite-induced proliferation in serum-free monolayer culture.

We have recently reported that neonatal mouse dorsal root ganglionic Schwann cells will (i) survive and assume characteristic morphologies in a serum-free, fully defined cultured medium (N1 medium), (ii) proliferate extensively in the same N1 medium if neurons are also present and maintained by nerve growth factor, and (iii) display a strong proliferative response to serum even in the absence of neuronal elements, while also undergoing marked changes in their morphology and their associative behavior toward neurites. In this report, we present a detailed procedure, based upon these earlier observations, which yields purified cultures of either neurons plus associated Schwann cells or Schwann cells in the absence of neurons. The procedure utilizes the neuritic mitogen for selective expansion of Schwann cell numbers in serum-free primary cultures, and a secondary culture step involving neuronal removal and additional Schwann cell expansion using the serum mitogen. The procedure requires 9 days for the generation of 3-4 X 10(6) Schwann cells from 12 newborn mice (with a Schwann cell to neuron ration of 10) and an additional 6-7 days for the generation of a neuron-free secondary population of 40 X 10(6) Schwann cells with less than 3% contamination by identifiable ganglionic fibroblasts.

Animals↗
PubMed1980

Survival, proliferation and morphological specialization of mouse Schwann cells in a serum-free, fully defined medium.

Neonatal mouse dorsal root ganglionic (DRG) cell dissociates were cultured in a synthetic medium with horse serum or the serum-free supplement N1 (insulin, transferrin, progesterone, putrescine, selenium). Serum-supplemented cultures with added nerve growth factor (NGF) yielded neurons, small flat and spindle cells (Schwann) and large flat cells (fibroblastic elements). However, in serum-free, N1-supplemented medium plus exogenous NGF, neurons and Schwann cells predominated, with very few large flat cells. In the N1 medium most Schwann cells assumed a typical spindle shape and were associated with neuritic processes when neurons were present. Upon addition of serum, virtually all of the Schwann cells appeared to abandon physical contact with the neurites and develop a more flattened morphology. In N1 medium without NGF (no neurites), most Schwann cells still assumed a spindle shape and formed characteristic chain-like associations. Autoradiographic techniques, as well as numerical analyses, demonstrated that in N1 medium Schwann cells were able to proliferate when associated with neurites but only slightly so in their absence. These Schwann cells showed a marked increase in proliferation when serum was added regardless of the presence or absence of neurites. The above observations may provide a basis for the preparation of purified Schwann cells, alone or in combination with their neurons.

Animals↗
PubMed1980

Cholinergic neuronotrophic factors: fractionation properties of an extract from selected chick embryonic eye tissues.

An aqueous extract derived from selected intraocular tissues of 15-day chick embryos contains a soluble macromolecular agent which is capable of ensuring the survival of 8-day chick embryonic ciliary ganglionic neurons in monolayer culture. When this ciliary neuronotrophic factor (CNTF) was concentrated using ultrafiltration and subjected to Sephadex G100 and G200 chromatography, activity was detected in most of the eluted fractions. A peak of the most active fractions was eluted in a region corresponding to a molecular weight of 35-40 X 10(3) and contained about 20-30% of the applied protein. CNTF activity bound readily to DE-52 cellulose resin at neutral pH and was eluted with NaCl in a narrow region containing about 20-40% of the applied protein. Gel electrophoretic staining profiles of the active DE52 fraction indicated considerable (but still only partial) simplification in protein composition. While significant CNTF activity losses were incurred in response to each of the above treatments, an active material could be conveniently generated in one working day in milligram amounts having a specific activity of 60,000 trophic units/mg protein. This trophic activity is in the same range as that of the only other known neuronotrophic factor, Nerve Growth Factor.

Animals↗
PubMed1979

Cholinergic neuronotrophic factors: I. Survival, neurite outgrowth and choline acetyltransferase activity in monolayer cultures from chick embryo ciliary ganglia.

Two key components of neural development and regeneration, survival of the involved neurons and elongation of neuritic elements, are likely to depend on the availability of an appropriate trophic drive to these neurons. At present, only one trophic factor, Nerve Growth Factor, is known to ensure both survival and neuritic growth for its target neurons. A search for a second such agent, a putative cholinergic neuronotrophic factor (CNTF), has been undertaken using as indicators neuronal survival, neurite outgrowth and choline acetyltransferase (CAT) activity in monolayer cell cultures. Eight-day chick embryo ciliary ganglia yielded two monolayer culture systems which appear to be well suited for a CNTF assay. Ciliary ganglionic dissociates, seeded on a highly adhesive collagen substratum, show no neuronal survival by 24 h if the medium is supplemented only with serum or chick embryo extract. However serum and embryo extract combined support survival of, and extensive neuritic outgrowth from, nearly the theoretical number of ganglionic neurons seeded. Alternatively, ciliary ganglionic neurons can be made to survive and produce a profuse neuritic outgrowth on polyornithine-coated dishes if supplied with medium conditioned over chick embryo heart muscle cultures, as already described by other laboratories. The two trophic sources differ markedly in their effects on the ganglionic neurons when tested on collagen or polyornithine substrata, and in some cases when different serum supplements are used. Neuronal survival, neurite production and, possibly, CAT activity appear to be subject to independent regulation. The culture systems used in this study can be developed into quantitative bioassays for the isolation of the different agents responsible for neuronal survival and neurite promotion, and for the investigation of their activities.

Animals↗
PubMed1979

Cholinergic neuronotrophic factors: intraocular distribution of trophic activity for ciliary neurons.

Chick ciliary ganglionic neurons require an interaction with their peripheral targets for survival during a critical period of their embryonic development in vivo. It has recently been shown that survival of these neurons in dissociated cell cultures is supported by extract from whole chick embryo. In this study, an assay system based on microwell cultures of ciliary ganglionic neurons was used to demonstrate that a very rich source of trophic factor for them is the intraocular target tissues they innervate. Out of 8000 trophic units present in a 12-day embryo, 2500 were contained in the eye. A subdissection of the eye showed its activity to be localized in a fraction containing the ciliary body and choroid coat, with a specific activity almost 20-fold higher than that of the whole embryo. This selective intraocular distribution at a time when survival or death of ciliary ganglionic neurons is decided in vivo suggests that this soluble factor may be involved in the normal development of the ciliary ganglion.

Animals↗
PubMed1979

Development, reactivity and GFA immunofluorescence of astroglia-containing monolayer cultures from rat cerebrum.

This report describes detailed protocols for the dissociation, seeding and growth in vitro of monolayer cultures derived from neonatal rat cerebrum. Primary cultures derived by using different seeding densities and in vitro ages were examined qualitatively and quantitatively for morphological composition in terms of two major cell classes (flat cells and process-bearing cells) and for the presence within these classes of glial fibrillary acidic protein (GFA) as detected by immunofluorescence histochemistry. Also examined was the reaction of the cells to serum withdrawal plus the administration of dibutyryl cyclic AMP in terms of the conversion of flat cells into process-bearing cells. Conditions are defined for the generation of in vitro cell populations, more than 90% of which are GFA-containing flat cells which can all be experimentally converted into cells with processes. These well-defined culture preparations will serve as useful models for future studies of astroglial behaviour.

Animals↗
PubMed1975

Cyclic nucleotide phosphodiesterases associated with bovine retinal outer-segment fragments.

ATP-dependent cyclic GMP phosphodiesterase activity (EC 3.1.4.16) associated with bovine retinal outer-segment fragment preparations was stimulated an order of magnitude by light, confirming the results of Miki et al. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3820-3824 at Yale for the frog system. In contrast to the results of the Yale group, however, light stimulation was not observed for cyclic AMP as substrate. A direct relationship of bovine rhodopsin bleaching to phosphodiesterase activation differs from a previous report by the Yale group that full activation of the frog enzyme was achieved by bleaching of a maximum of 2% rhodopsin. Phosphodiesterase activity could be qualitatively removed from the fresh outer-segment preparations with isotonic sucrose which apparently did not disrupt the plasmalemma or discs. Activity recovered from the washing was not light sensitive. Two Km values were determined for cyclic AMP, 5 and 0.05 mM; for cyclic GMP a Km of 0.22 mM was found. All Km values were determined in the presence of 1 mM ATP in the dark. Sonication of fresh outer segments or storing at -20 degrees C abolished the light response. However, storage at -76 degrees C fully preserved it.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗