Biomedical subjects

K Wewetzer

Publications and source records attributed to K Wewetzer.

27 records · Page 2Linked to original sources

PubMed1996

Fibroblast growth factor and its implications for developing and regenerating neurons.

FGF is a multifunctional heparin-binding protein which was characterized by its mitogenic and angiogenic action outside the nervous system. Recent data confirm this multifunctionality also with regard to the nervous system. The distribution of FGF and its receptors seems not to be in agreement with the idea of a single function for one population but argues for a more complex action, which might be dependent on the development stage and cell type. FGF and its receptors are widely distributed in the nervous system. In brainstem and spinal cord motoneurons and in sensory ganglia the FGF-2 staining pattern is developmentally regulated suggesting a functional change during embryonic and postnatal development. In addition, after nerve lesion the FGF-2 expression is altered in sensory and motoneurons. Administration of FGF-2 reveals trophic effects on survival and transmitter metabolism in vivo and in vitro. According to a more general neurotrophic factor concept, a physiological role of FGF for distinct neuron populations during development is likely. In the motor system, for example, FGF could act synergistically with certain neurotrophins, CNTF, or other non-identified co-factors. In the sensory system, a possible non-neurotrophic role for at least postnatal and adult sensory neurons has to be further addressed in the future. In order to further define and characterize the actions of the FGFs a mapping of the different family members and their respective receptor molecules during development and in the adult has to be done.

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PubMed1995

Immunocytochemical localization of the heparin-binding growth-associated molecule (HB-GAM) in the developing and adult rat cerebellar cortex.

The heparin-binding, growth-associated molecule (HB-GAM) is a developmentally regulated protein that belongs to a new family of heparin-binding molecules, not related to the fibroblast growth factors (FGFs), with putative functions during cell growth and differentiation. In order to further study the functional role of HB-GAM we have used a polyclonal antiserum, raised against the purified protein to localize HB-GAM in the developing and adult rat cerebellar cortex. During postnatal development HB-GAM-like immunoreactivity (IR) was found to be present in all layers of the cerebellar cortex. IR was mainly associated with processes or extracellular structures but not with cell bodies. Throughout all the stages examined the molecular layer was clearly labeled, whereas staining in the internal granular layer was diffuse. IR in the external granular layer on postnatal day 1 and 8 was found to be associated with radially oriented fibres connecting the internal granular layer with the pial surface of the cerebellum. The intensity of this staining seemed to increase from day 1 to 8. Staining of corresponding areas with an antiserum against the glial fibrillary acidic protein (GFAP) suggested that the HB-GAM antiserum in the developing cerebellar cortex labels Bergmann glia fibres of Golgi epithelial cells. Because of the diffuse staining of the molecular layer in the adult rat it was not possible to distinguish whether radial fibres in the adult contained any HB-GAM IR. Golgi epithelial cells are considered as crucial for the migration of granular cells during the differentiation of the cerebellar cortex. We therefore speculate that the association of HB-GAM-like IR may be of functional relevance. The fact that molecules, such as tenascin, known to be involved in morphogenetic events show a similar spatiotemporal distribution pattern further underscores this hypothesis. HB-GAM, which possesses a classical signal sequence, might be release in the extracellular space and could mediate adhesion phenomena by binding to heparin-like molecules associated with the neuronal membrane. Therefore, it will be important to investigate whether specific antibodies against HB-GAM are able to interfere with normal cerebellar development in vitro and in vivo.

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PubMed1995

Establishment of a single-step hybridoma cloning protocol using an automated cell transfer system: comparison with limiting dilution.

An easy-to-standardize single-step protocol of hybridoma cloning has been established using a recently introduced, commercially available cell transfer system. By controlling the volume of air within a sealed glass micropipette by means of a Peltier device, single cells are gently collected or ejected. The transfer of cells from a source dish to the wells of a target microplate is controlled by a microprocessor. Since collection as well as expulsion of cells is done under microscopic control seeding of single cells can be guaranteed. Monoclonality is therefore reproducibly achieved in a single step, reducing the time required for cloning enormously, and conserving man-power and material. Since the automated transfer of cells is time-saving and easy-to-standardize, it substantially facilitates cloning of hybridoma. The present protocol therefore represents an alternative to limiting-dilution cloning as well as to other previously introduced techniques of single-cell cloning. It is easily adapted to a wide spectrum of other cell types and can therefore be used in many other applications involving single cell manipulation.

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PubMed1993

Cell blotting and isoelectric focusing of neuroblastoma-derived heparin-binding neurotrophic activities: detection of basic fibroblast growth factor protein and mRNA.

Neuroblastoma cells have been shown to contain growth factors, which may be involved in the regulation of cell proliferation and/or differentiation. We have examined whether human IMR-32 neuroblastoma cells store factors with a capacity to promote neuron survival and differentiation. Heparin affinity chromatography, cell blotting, and isoelectric focusing of IMR-32 cell extracts revealed multiple neurotrophic activities at molecular weights of 16.8, 24.1, 39.0, 45.3, 52.3 and isoelectric points of 4.0, 5.0, 8.3, 9.0. Immunocytochemistry, immunoblotting, and radio-immunoassay with specific antibodies suggests that one neurotrophic activity is immunologically identical with basic fibroblast growth factor (bFGF). This assumption is supported by Northern blot analysis, which shows a 6.0 kb bFGF transcript.

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PubMed1992

Stimulation of neuron survival by basic FGF and CNTF is a direct effect and not mediated by non-neuronal cells: evidence from single cell cultures.

The multifunctional proteins, basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF), share a capacity to promote in vitro and in vivo survival of several, partly overlapping neuron populations. Whether they can affect neurons directly or whether their supportive effects are mediated by non-neuronal cells and their growth factor products has been addressed in this study by establishing single neuron cultures from embryonic chick ciliary ganglia. Cultures with one or two neurons and without any non-neuronal cells were obtained by limiting dilution of ganglionic cell suspensions on 96-well microtiter plates. In the presence of bFGF about 80% of the wells that contained 1 or 2 neurons at the time of seeding, had this (these) neuron(s) maintained after 1 and 5 days. Absence of bFGF resulted in the death of neurons in over 80% of the wells screened. Identical results were obtained with CNTF. These data demonstrate the effectiveness of bFGF and CNTF at the single neuron level, but do not rule out that the factors may act indirectly on neurons, particularly in complex in vitro and in vivo situations.

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PubMed1992

Cytokines in neural regeneration.

Growth factors with already established multiple effects on non-neural cells continue to be of considerable interest to researchers with regard to the nervous system, where regulation of cell maintenance and plasticity in relation to lesion and regeneration is part of their functional repertoire. Fibroblast growth factors, interleukins, and type beta transforming growth factors are prominent representatives of such proteins. Ciliary neurotrophic factor is another multifunctional neurokine. The proposed role of this molecule as a 'lesion factor', however, is still not firmly settled.

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PubMed1991

Effects of basic fibroblast growth factor on survival and choline acetyltransferase development of spinal cord neurons.

To investigate the biological role of basic fibroblast growth factor (bFGF) for the development of the spinal cord we studied the in vitro and in vivo effects of this protein on survival and choline acetyltransferase (ChAT)-activity of embryonic chick and rat spinal cord neurons. In vitro, bFGF (ED50 1-2.8 ng/ml) supported the survival of embryonic neurons from the ventral part of the rat spinal cord (ventral spinal cord, vsc), including motoneurons. Addition of bFGF (100 ng/ml) increased the ChAT-activity in embryonic chick vsc cultures to 150% as compared to untreated cultures (100%). The effect of bFGF was dose-dependent. In vivo-application of bFGF resulted in a similar increase of ChAT-activity in chick spinal cord. Since bFGF stimulates the ChAT-activity of spinal cord neurons in vivo and in vitro we therefore conclude that this protein may have a physiological function for the transmitter development of cholinergic spinal cord neurons.

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PubMed1991

Characterization of a GABAergic population of interstitial amacrine cells in the teleost retina.

We used postembedding immunocytochemistry with an antiserum against BSA-conjugated GABA to study the inner plexiform layer of a cyprinid teleost, the roach. In this part of the retina, we observed a distinct banding pattern of GABA-positive material. There was a narrow unstained region separating the distal sublamina a from the proximal sublamina b; each sublamina was further subdivided into four (a) and two (b) sublayers of heavier staining, respectively. Using three-dimensional reconstruction of series of half-micron tangential sections, we were able to characterize a population of interstitial amacrine cells which contained GABA-like immunoreactive material. These cells had elliptical dendritic fields (area: about 0.04 mm2) and conspicuous, thick processes (dia. 4-5 microns). In tangential sections, the dendrites of individual cells appeared to be in close contact, occasionally resulting in difficulties in defining the boundaries of a single dendritic field. Two sub-populations of these cells were observed, one in each sublamina. By comparison with a catalogue of Golgi impregnated amacrine cells and cells microinjected with Lucifer Yellow or HRP, the identity of this type of interstitial amacrine cell is established and its possible physiological properties discussed. Apart from this GABA positive type, a second population of interstitial amacrine cell was observed which did not show positive reaction to the GABA antiserum used.

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PubMedDate not supplied

CNTF rescues motoneurons from ontogenetic cell death in-vivo, but not in-vitro.

We studied the effect of CNTF (ciliary neurotrophic factor, human recombinant and chick) on the survival of motoneurons in the embryonic chick lumbar spinal cord during the period of ontogenetic cell death. Daily applications of 5 micrograms CNTF to the chorionic-allantoic membrane from embryonic day 6 (E6) to E9 maintained approximately 15,500 motoneurons as opposed to 13,200 in controls. In contrast, CNTF failed to promote the survival of cells in spinal cord cultures enriched for motoneurons. These results suggest that CNTF may regulate motoneuron survival in-vivo, but its mode of action remains to be elucidated.

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