Sulfated glycoconjugate determinants recognized by monoclonal antibody, SG-1, correlate with the experimental metastatic ability of KHT fibrosarcoma cells.

We have examined the binding and functional activity of monoclonal antibody (MAb) SG-1 that was raised by immunization against embryonal carcinoma cells and screened using KHT fibrosarcoma cells. Quantitative absorption, binding and in situ immunochemical staining assays indicate that the MAb SG-1-defined epitopes are expressed preferentially by the highly metastatic KHT35-L1 cells relative to the weakly metastatic, parental KHTp cells. Furthermore, there was a significant correlation (p less than 0.05) between the expression of MAb SG-1-defined antigen on the cells, following trypsin treatment, and their metastatic ability. Binding of MAb SG-1 to antigen was inhibited by specific sulfated polysaccharides including cerebroside sulfate (brain sulfatide), fucoidan, and dextran sulfate (500 kD) but not by heparan, chondroitin, keratan or dextran (5 kD) sulfates. Initial characterization of antigen from KHT cells indicates that the epitope of MAb SG-1 is defined by sulfated glycoconjugates containing galactose and sulfate but not N-acetylglucosamine. In the total lipid extracts of KHT35-L1 cells the antigen was detected in the delipidated protein fraction as well as in the chloroform/methanol fraction. These results suggest that the sulfated glycoconjugate determinants identified by MAb SG-1 may be relevant to the metastatic process of KHT fibrosarcoma cells.

Direct histochemical localisation of 5,7-dihydroxytryptamine and the uptake of serotonin by a subpopulation of GABA neurones in the rabbit retina.

The same cells in both the intact retina and retinal cultures of the rabbit retina take up exogenous serotonin or 5,7-dihydroxytryptamine. Both substances can be localised by immunohistochemistry using a monoclonal antibody directed against serotonin. The 5,7-dihydroxytryptamine can also be revealed directly in both fixed and living tissues using appropriate U.V. light. Some of the cells in the intact retina and retinal cultures which take up 5,7-dihydroxytryptamine also stain positively for the immunohistochemical localisation of gamma-aminobutyric acid (GABA). Cell counts in the cultures show that 80% of all 5,7-dihydroxytryptamine-positive cells stain positively for GABA, while 20% of the GABA-immunoreactive cells also accumulate 5,7-dihydroxytryptamine. These results demonstrate unambiguously that a subpopulation of GABA cells has the capacity to take up exogenous 5,7-dihydroxytryptamine and therefore utilise GABA and, probably, serotonin. Findings in favour of the 'co-occurrence' of GABA and serotonin come from studies where tissues were exposed to radioactive GABA and unlabelled serotonin so that the dual localisation of these substances by autoradiography and immunohistochemistry could be followed. It could be shown both in cultures and in intact retinal pieces that certain cells take up GABA and serotonin while other cells take up either GABA or serotonin.

Substance P-like immunoreactivity in the retina and optic lobe of the squid.

The distribution of substance P-like immunoreactivity within the squid retina and brain was studied by immunofluorescence. Positive immunoreactivity was observed as a single layer of fibres in the retina. The retina was devoid of tyrosine-hydroxylase, serotonin, gamma-aminobutyric acid, cholecystokinin, neuropeptide Y, somatostatin, enkephalin and vasoactive intestinal peptide immunoreactivities. Substance P immunoreactivity was particularly abundant in the optic lobe. The optic lobe had a distinct layer of substance P fibres near the periphery. Immunoreactive cell bodies, fibres and varicosities were additionally present in various areas of the optic lobe. Substance P immunoreactivity in the other ganglia of the brain was restricted to a few scattered fibres.

The occurrence of gamma-aminobutyric acid (GABA)-containing cells in cultures of retinas from the human fetus.

gamma-Aminobutyric acid (GABA) immunoreactivity is demonstrated by the indirect immunofluorescence technique in a small population of retinal neurons cultured from human fetuses. Positive staining was restricted to a few cells and could be observed as soon as the cells became attached to the substrate (within 5 hr). It is therefore concluded that the GABA-positive cells are determined prenatally. The GABA-positive cells grow processes during development in culture and remain constant in numbers. These cells have a different morphology from either GFAP-positive cells or serotonin-accumulating cells. It is suggested that the GABA-positive cells in culture are probably amacrine neurones. Cultures of human retinal dissociates may therefore provide an alternative means of studying specific cell types should a constant supply of living human retinas be difficult to obtain.

GABA neurones in retinas of different species and their postnatal development in situ and in culture in the rabbit retina.

The localisation of GABA immunoreactive neurones in retinas of a variety of animals was examined. Immunoreactivity was associated with specific populations of amacrine neurones in all species examined, viz. rat, rabbit, goldfish, frog, pigeon and guinea-pig. All species, with the exception of the frog, possessed immunoreactive perikarya in their retinal ganglion cell layers. These perikarya are probably displaced amacrine cells because GABA immunoreactivity was absent from the optic nerves and destruction of the rat optic nerve did not result in degeneration of these cells. GABA immunoreactivity was also associated with the outer plexiform layers of all the retinas studied; these processes are derived from GABA-positive horizontal cells in rat, rabbit, frog, pigeon and goldfish retinas, from bipolar-like cells in the frog, and probably from interplexiform cells in the guinea-pig retina. The development of GABA-positive neurones in the rabbit retina was also analysed. Immunoreactivity was clearly associated with subpopulations of amacrine and horizontal cells on the second postnatal day. The immunoreactivity at this stage is strong, and fairly well developed processes are apparent. The intensity of the immunoreactivity increases with development in the case of the amacrine cells. The immunoreactive neurones appear fully developed at about the 8th postnatal day, although the immunoreactivity in the inner plexiform layer becomes more dispersed as development proceeds. The immunoreactive horizontal cells become less apparent as development proceeds, but they can still be seen in the adult retina. The GABA immunoreactive cells in rabbit retinas can be maintained in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on serotonin-accumulating cells in rabbit retinal cultures.

The serotonin-accumulating neurones in rabbit retinal cultures were studied, using immunohistochemistry to localize serotonin. Double-labelling experiments showed that serotonin-accumulating cells in culture and intact retinas react positively to antiserum PGP 9.5, which is neurone-specific. The uptake process of serotonin is very specific; known serotonergic blockers, such as chlorimipramine, abolished transport, while benztropine, a dopamine uptake blocker, was ineffectual. Analogues of serotonin such as tryptamine, tryptophan and 5-hydroxytryptophan at concentrations 100-fold those of exogenous serotonin did not appear to compete with the transport of the amine. Newly dissociated retinal cells from 1-5-day postnatal rabbits which lack processes have the capacity to take up exogenous serotonin; these cells when kept in culture grew processes and appeared to reach maximum development after 6-15 days. Dissociated retinal cells subjected to density centrifugation resulted in the production of an enriched (4-fold) population of serotonin-accumulating cells. Since most of the endogenous serotonin was associated with this fraction, it is concluded that the serotonin-accumulating cells contain serotonin.

Occurrence of serotonin-accumulating neurones in cultures of retina from the human foetus.

Cultures were produced from retinas taken from human foetuses. Specific cells in culture have the capacity to take up exogenous serotonin, which was then visualized by immunofluorescence using an antisera to serotonin. The known serotonergic uptake blocker, chlorimipramine, inhibited the uptake of the amine while benztropine, a dopamine-uptake blocker, is ineffectual. These findings show that serotonin-accumulating cells are formed prenatally in man and demonstrate the usefulness of this preparation for future studies.

Localization of tyrosine-hydroxylase immunoreactive cells in rabbit retinal cultures.

Tyrosine-hydroxylase immunoreactivity is demonstrable by the indirect immunofluorescence technique in a small sub-population of retinal neurones cultured from 2-day-old rabbits. Staining was only weak, restricted to a few neurones, and could not be observed in cultures of less than 10 days. Autoradiographical analysis of [3H]dopamine uptake by retinal cultures strongly suggests that neurones containing tyrosine-hydroxylase immunoreactivity have the capacity to take up exogenous dopamine. These findings are in concordance with studies on rabbit retinal preparations.

Enriched populations of rat retinal ganglion cells: Studies using a cell-type specific surface marker.

Cells dissociated from adult and neonatal rat retinas were separated by density gradient centrifugation. Previous work had shown that rat retinal cells labelled by an immunofluorescence assay for the Thy-1 antigen were chiefly or exclusively ganglion cells, and so the proportion of Thy-1 positive cells in the density gradient fractions was used as an index of the enrichment of ganglion cells. The proportion of Thy-1 positive neonatal cells was increased from about 0.4% in the initial dissociate to about 8% in the most enriched fraction of a Percoll step gradient. Amongst adult cells the initial 0.7% Thy-1 positive cells were increased to roughly 2% in the best fraction of a metrizamide step gradient. The presence of relatively large numbers of Thy-1 positive cells in other fractions suggested that it would be difficult to further increase the proportion of rat ganglion cells by methods based on their sedimentation properties. These results demonstrate the importance of cell-type specific markers in attempts to purify cells from the central nervous system.