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Biomedical subjects

G B Grunwald

Publications and source records attributed to G B Grunwald.

34 records · Page 2Linked to original sources
PubMed1990

Immunocytochemical analysis of embryonic compartmentation with a monoclonal antibody against a cytokeratin-related antigen.

Mab 113F4, a monoclonal antibody recognizing an antigen in the outer synaptic layer of the chick neural retina, also recognizes an antigen appearing in all three germ layers of the gastrulating chick embryo. However, as neurulation proceeds, the antigen is down-regulated in three distinct patterns. First, the antigen is lost specifically from those trunk ectodermal cells destined to form the neural plate and, later, the neural tube. It remains absent from any neural derivative until day 13 when it appears in the outer synaptic layer of the neural retina, coincident with synaptogenesis in this region. Second, the entirety of the head ectoderm loses this antigen as the head lifts off the blastoderm. This down-regulation is followed later by a similar loss of antigen expression in the trunk ectoderm. Third, expression in the mesoderm becomes limited to the lateral plate and extraembryonic epithelia. Endodermal derivatives continue to express the antigen throughout development. Antigen 113F4 is localized within the cytoplasm and is organized in a fibrillar pattern. The intracellular localization of this antigen and its characteristic spatio-temporal tissue distribution are consistent with the antigen being a cytokeratin or cytokeratin-related antigen. The changes in tissue distribution suggest a possible role in tissue modelling in response to inductive interactions during development.

Animals↗
PubMed1990

Generation and characterization of monoclonal antibodies specific for the retinal pigment epithelium.

Although both the neural retina and the retinal pigment epithelium (RPE) arise as neighboring portions of the embryonic optic cup, these two tissues follow very different developmental pathways. In order to obtain probes for the analysis of RPE development from its earliest divergence from the neural retina to late stages of differentiation, we have developed a panel of monoclonal antibodies which recognize antigens specific to the RPE. These probes have been applied to an immunohistochemical analysis of RPE development. The results indicate that the RPE is antigenically distinct from the neural retina even before the onset of overt differentiation. In addition, the RPE layer of the retina becomes further subdivided antigenically as its distinct anterior and posterior derivatives develop. These antibodies will be useful markers in the analysis of RPE development.

Animals↗
PubMed1990

Identification of an adhesion-associated protein of the retinal pigment epithelium.

The normal function of the retinal pigment epithelium (RPE) is dependent on the maintenance of tight adhesions between cells. In order to identify cell surface molecules which may be important for maintaining the integrity of the RPE, we have undertaken a combined functional, biochemical, and immunohistochemical analysis of cell surface proteins of the RPE. These studies have led to the identification of a 100-kD cell surface protein whose presence correlates with the maintenance of calcium-dependent adhesions between RPE cells. In intact RPE tissue the protein is concentrated at the junctions between RPE cells. The properties of the protein suggest that it may be a member of the cadherin family of calcium-dependent cell adhesion proteins.

Animals↗
PubMed1989

Expression of calcium-dependent cell adhesion during ocular development: a biochemical, histochemical and functional analysis.

Previous studies of the adhesive properties of embryonic chick neural retina cells indicate a gradual decrease in the expression of calcium-dependent adhesions during retinal histogenesis, a function which has been attributed in part to gp130/4.8, a retinal calcium-dependent adhesion-associated cell surface membrane glycoprotein with a molecular weight of approximately 130 kDa and an isoelectric point of 4.8 (G. B. Grunwald, R. Pratt, and J. Lilien, 1982, J. Cell Sci. 55, 69-83). The experiments described here were done to define the relationship of gp130/4.8 to N-cadherin, another calcium-dependent adhesion molecule found in chick retina, which has a reported molecular weight of 127 kDa and which is recognized by monoclonal antibody NCD-2 (K. Hatta and M. Takeichi, 1986, Nature (London) 320, 447-449). Using two-dimensional gel electrophoresis followed by Western blotting as well as quantitative solid-phase immunoassays, polyspecific antisera recognizing gp130/4.8 were compared with monoclonal antibody NCD-2 for reactivity with proteins of retina and other tissues. The data lead us to conclude that retinal calcium-dependent adhesion proteins gp130/4.8 and N-cadherin are likely to be the same molecule. In order to obtain evidence for a direct correlation of changes in expression of these adhesion proteins with changes in retinal cell adhesivity and related morphogenetic events, parallel studies were carried out with cells from various ocular tissues to examine the functional, biochemical, and immunohistochemical expression of N-cadherin during ocular development. Immunohistochemical mapping of N-cadherin in the developing chick eye reveals three modes of N-cadherin expression which occur simultaneously in different ocular tissues: (1) down-regulation, (2) up-regulation, and (3) steady-state expression. These patterns of expression correlate with changes in the adhesive behavior of cells as well as with discrete stages in the morphogenesis of several ocular tissues. The results suggest that N-cadherin is a versatile cell adhesion protein with a role in both the development of several ocular tissues and the maintenance of specialized structures in the mature eye.

Age Factors↗
PubMed1988

Effects of epidermal growth factor and phorbol 12-myristate 13-acetate on protein phosphorylation in mouse embryo palate mesenchyme cells in vitro.

Epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulated mouse embryo palate mesenchyme (MEPM) cells to incorporate [32P]O(3-)4 into a protein with an apparent molecular weight of 80 kDa, in vitro. Agents known to elevate intracellular levels of cyclic AMP did not stimulate phosphorylation of this phosphoprotein. Since there is a significant amount of evidence obtained with other cells indicating that phosphorylation of such an 80-kDa phosphoprotein reflects specifically the activation of protein kinase C in response to PMA and other agents, including mitogens, these findings raise the possibility that EGF may activate protein kinase C in MEPM cells.

Animals↗
PubMed1987

Autoimmune basis for visual paraneoplastic syndrome in patients with small cell lung carcinoma. Retinal immune deposits and ablation of retinal ganglion cells.

Recently, patients with visual paraneoplastic syndrome (VPS) were described, a binocular loss of vision found in patients with small cell carcinoma of the lung (SCCL). The patients have serum antibodies against a small number of discrete antigens which are shared by the retina and small cell carcinoma cells, and which are associated with cells and processes of the ganglion cell layer of the retina. Pathologic findings are presented with regard to the presence of immunoglobulins in, and the nature of the lesions in, the central nervous system of a VPS patient. The patient's blood-brain barrier was shown to be compromised, as demonstrated by the finding of high immunoglobulin levels in the cerebrospinal fluid and immune deposits in the retina. It is further shown that within the central nervous system only the retina and optic nerve show any tissue damage with the specific loss of retinal ganglion cells and their processes. The findings support the hypothesis of an autoimmune cause for this remote effect of cancer.

Aged↗
PubMed1986

Monoclonal antibody 18B8, which detects synapse-associated antigens, binds to ganglioside GT3 (II3 (NeuAc)3LacCer).

By immunofluorescence, mouse monoclonal antibody 18B8 detects developmentally regulated antigens in chick neural retina. In older embryos and in adults these antigens are localized in discrete laminae within the inner and outer synaptic layers. The antibody binds to several gangliosides that undergo both qualitative and quantitative changes during neuronal development (Grunwald, G.B., Fredman, P., Magnani, J.L., Trisler, D., Ginsburg, V., and Nirenberg, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4008-4012). The simplest of these gangliosides was isolated from lipid extracts of 10-day chick embryonic retinas by DEAE-Sepharose and silicic acid column chromatography. About 300 micrograms was obtained from 9.3 g (wet weight) of retina. The isolated ganglioside was identified as GT3 by enzymatic analysis and by a comparison of its properties with the authentic ganglioside. By immunostaining thin-layer chromatograms with antibody 18B8, GT3 was detected in gangliosides from human neural tissue including cerebellum, optic nerve, and spinal cord, but not in gangliosides from human liver, pancreas, small intestine, adrenals, thyroid, or erythrocytes. GT3 was also found in five of seven human melanoma cell lines.

Animals↗
PubMed1986

Detection of alpha-transducin in retinal rods but not cones.

The distribution in chicken retina of the alpha subunit of transducin, the guanine nucleotide--binding protein that couples light-dependent activation of rhodopsin with activation of guanosine 3',5'-monophosphate phosphodiesterase, was determined with the aid of a specific antiserum. alpha-Transducin was found in rod photoreceptor cells but was not detected in cones. These results show that rods and cones differ with respect to alpha-transducin content and suggest that the processes of phototransduction may differ correspondingly in rods and cones.

Animals↗
PubMed1986

Identification of the chick neural retina cell surface N-acetylgalactosaminyltransferase using monoclonal antibodies.

Intact embryonic chick neural retina cells have at their surface an N-acetylgalactosaminyltransferase which catalyzes the incorporation of N-acetylgalactosamine from UDP-N-acetylgalactosamine into endogenous macromolecular acceptors. The enzyme along with its endogenous acceptors can be isolated as a particulate complex following treatment of membrane-enriched fractions with Triton X-100. In this paper we report on two separate fusions generating monoclonal antibodies: one using as immunogen the particulate complex and the second using as immunogen a soluble N-acetylgalactosaminyltransferase found in tissue-culture-conditioned medium which lacks endogenous acceptor activity. Antibodies from both fusions recognize an antigen which is tightly associated with the particulate transferase/acceptor complex and a soluble antigen having N-acetylgalactosaminyltransferase activity toward exogenously added acceptors. The antibodies recognize a component of ca Mr 220,000, which shows N-acetylgalactosaminyltransferase activity after SDS-gel electrophoresis and transfer to nitrocellulose. This component comigrates on two-dimensional gel electrophoresis with an iodinatable cell surface component whose presence at the cell surface correlates with endogenous transferase activity. We conclude that the antibodies recognize the transferase enzyme itself. Immunohistochemical analysis shows that the enzyme is initially localized throughout the embryonic neural retina in a pattern indicative of a cell surface disposition but becomes restricted to the outer plexiform layer and to outer segments in the adult.

Animals↗
PubMed1986

Anti-neurofilament antibodies in the sera of patients with small cell carcinoma of the lung and with visual paraneoplastic syndrome.

The sera of patients with small cell carcinoma of the lung (SCCL) and an associated visual paraneoplastic syndrome (VPNS) have high titer immunoglobulins that react with retinal ganglion cells and with cloned lines of the SCCL. The immunoglobulins in the sera of two patients with SCCL and VPNS reacted with at least one common antigen shared by neural cells and cloned lines of the SCCL. The molecular weights of the predominant neural and tumor antigens were 205,000, 145,000, 65,000, and 20,000-24,000 as determined by Western blots. Three of the antigens from neural tissue copurify and comigrate electrophoretically with neurofilament proteins. Polyclonal antibodies prepared against authentic neurofilament proteins react with antigens having molecular weights identical to those of proteins that react with immunoglobulins from the SCCL-VPNS patients. Polyclonal antibodies that were prepared against isolated retinal ganglion cells and that were shown previously to cause the immunoablation of the ganglion cells in vivo reacted most intensely with the Mr 205,000 antigen and weakly with the Mr 145,000 and Mr 70,000 antigens. Treatment of the Western blots with alkaline phosphatase from Escherichia coli did not affect the immunoreactivity between the immunoglobulins and the purified neurofilament proteins. It is proposed that the immunoglobulins in the sera of patients with SCCL-VPNS may be involved etiologically in the development of the VPNS.

Animals↗
PubMed1985

Autoimmune basis for visual paraneoplastic syndrome in patients with small-cell lung carcinoma.

Unexplained neurological disorders sometimes develop in patients with cancer, even when the primary tumour or its metastases have not invaded the central nervous system. Visual dysfunction in patients with small-cell carcinoma of the lung is one of these paraneoplastic syndromes and is associated with serum auto-antibodies which react with specific subsets of retinal neurons. These sera have now been shown to react with cell lines derived from small-cell carcinoma. The antibodies recognise a small number of antigens shared by retinal and tumour cells, suggesting that an autoimmune response may be triggered by the tumour.

Aged↗
PubMed1985

Monoclonal antibody 18B8 detects gangliosides associated with neuronal differentiation and synapse formation.

Mouse monoclonal antibody 18B8 detects developmentally regulated antigens in chicken retina and brain. The antigens detected by immunofluorescence appear initially on cell bodies in retinas of 6-13 day embryos. In older embryos during synapse formation and in adults, the antigen is localized in discrete laminae within the inner synaptic layer of retina and also is present in the outer synaptic layer and the outer segments of photoreceptor cells. The antigens from retina and brain were purified partially and were shown to be gangliosides of unknown structure that contain at least two sialic acid residues. Gangliosides that are recognized by antibody 18B8 change both qualitatively and quantitatively during neuronal development. These changes were correlated with the spatial and temporal changes in antigen expression detected histochemically.

Animals↗
PubMed1982

Enzymic dissection of embryonic cell adhesive mechanisms. III. Immunological identification of a component of the calcium-dependent adhesive system of embryonic chick neural retina cells.

Immunological probes and cell-surface biochemical analysis are combined to identify a cell-surface glycoprotein, which appears to be involved in the formation of calcium-dependent adhesions among embryonic chick neural retina cells. Comparison of radioiodinated cells shows that adhesive-competent cells have at their surface two glycoproteins not present on non-adhesive cells. These two components (with Mr 130 x 10(3), pI 4.8 and Mr 70 x 10(3), pI 4.8) are immunoprecipitable with adhesion-blocking antisera. A fraction derived from retina-conditioned medium, containing one major immunoprecipitable component, neutralizes the inhibitory activity of aggregation-inhibiting Fab' fragments and competes in immunoprecipitation for the two glycoprotein components. The evidence suggests that one or both of these components is involved in the formation of calcium-dependent adhesions among embryonic chick neural retina cells.

Animals↗
PubMed1980

Enzymatic dissection of embryonic cell adhesive mechanisms.

In this paper we describe a kinetic assay for cell adhesion which measures the formation of cell clusters. Cluster formation is dependent on both calcium and protein synthesis, two parameters essential for the formation of histotypic aggregates. We also describe modifications of the stndard method for trypsinization of tissues which result in populations of single cells that appear to bear intact and functional cell surface adhesive systems. These modifications involve the use of chymotrypsin and the inclusion of calcium during enzyme digestion of tissues with trypsin and chymotrypsin. Using the cluster formation assay and the modified tissue dissociation techniques, we demonstrate the presence of two functionally distinct adhesive systems operating among embryonic chick neural retina cells. These two systems differ in proteolytic sensitivity, protection by calcium against proteolysis, dependence on calcium for function and morphogenetic potential. Cells possessing one of these intact adhesive systems are capable of extensive morphogenetic interactions in the absence of protein synthesis.

Animals↗
PubMed1977

The effects of N-ethylmaleimide on active amino acid transport in Escherichia coli.

N-Ethylmaleimide (MalNEt) binds covalently and without specificity to accessible sulfhydryl residues in proteins. In some cases specificity has been imposed on this reaction by manipulating reaction conditions, yielding information concerning both enzyme mechanism and the identity of specific proteins (for example C.F. Fox and E.P. Kennedy (1965) Proc. Natl. Acad. Sci. u.s. 54, 891-899) and R.E. McCarty and J. Fagan (1973) Biochemistry 12, 1503-1507). We have examined the effects of MalNEt on the active accumulation of nine amino acids by Escherichia coli strains ML 308-225 and DL 54. Whole cells have been used in order that transport systems both dependent on and independent of periplasmic binding proteins could be studied under various conditions of energy supply for transport. Our results suggest that the systems transporting ornithine, phenylalanine and proline are those most likely to undergo inactivation by direct reaction of MalNEt with the transport apparatus, rather than merely via side effects such as interruption of their energy supply. The inhibition of proline transport is specifically enhanced by the presence of proline, competitive inhibitors of proline transport, or carbonylcyanide p-trifluoromethyoxyphenylhydrazone during MalNEt treatment. The other eight systems tested showed no analogous effects.

Amino Acids↗