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Biomedical subjects

A Messing

Publications and source records attributed to A Messing.

At least 73 records · Page 4Linked to original sources
PubMed1990

Immortalized retinal neurons derived from SV40 T-antigen-induced tumors in transgenic mice.

Immortalized retinal neurons have been established in tissue culture from retinal tumors arising in transgenic mice. The mice carry the SV40 T-antigen under the control of 5' flanking sequences from the human phenylethanolamine N-methyltransferase (PNMT) gene in order to target oncogene expression to adrenergic cell types. The retinal cultures contain a proliferation population of T-antigen-positive cells with a neuronal morphology that includes formation of extensive neuritic processes. We identified the cells as amacrine-derived neurons by immunofluorescence using the cell-specific monoclonal antibodies VC1.1 and HPC-1. The cells also express all three neurofilament subunits and GAP-43. These results indicate that CNS neurons can be transformed in transgenic animals to generate cultured cells with many properties of mature neurons.

Animals↗
PubMed1989

Peripheral neuropathy associated with functional islet cell adenomas in SV40 transgenic mice.

A line of SV40 transgenic mice (SV-202) developed a generalized peripheral neuropathy, islet cell adenomas of the pancreas, and hepatocellular carcinomas. The neuropathy was not directly associated with T-antigen expression in the nervous system. This study was designed to characterize the morphologic appearance and distribution of the neuropathologic lesions in SV-202 mice, and to relate the temporal development of peripheral nerve lesions to transgene-induced tumorigenesis in pancreatic islet cells. SV-202 mice developed an acute axonal degeneration that preferentially affected large diameter myelinated fibers. The onset of the neuropathy is closely correlated with the development of the hyperinsulinemia and hypoglycemia resulting from the islet cell adenomas.

Adenoma↗
PubMed1989

Metal-inducible pathology in the liver, pancreas, and kidney of transgenic mice expressing SV40 early region genes.

Transgenic mice (SV-202) that carry the SV40 early region genes under the control of an inverted metallothionein promoter developed islet cell adenomas, hepatocellular carcinomas, and a generalized peripheral neuropathy. Both the islet cell adenomas and the hepatocellular carcinomas developed from the proliferation of T antigen-positive cells. However, T antigen expression was not seen in either the peripheral or central nervous systems. Stimulation of the metallothionein promoter with heavy metals altered the temporal onset of hepatic expression and broadened the distribution of oncogene expression to include exocrine pancreas and renal tubular epithelium. Although solid tumors were not seen in the exocrine pancreas or kidneys of SV-202 mice, all immunopositive tissues developed histologic changes. These results indicate that metallothionein-directed T antigen expression can induce abnormal cellular growth in a variety of tissues, and the distribution of these tissues can be manipulated with heavy metals.

Animals↗
PubMed1988

Transgenic mice express the human phenylethanolamine N-methyltransferase gene in adrenal medulla and retina.

The human gene for phenylethanolamine N-methyltransferase (hPNMT), responsible for the conversion of norepinephrine to epinephrine, has been cloned and the complete nucleotide sequence has been determined. The structural gene consists of three exons and two introns spanning approximately equal to 2100 base pairs. Transgenic mice containing the hPNMT gene with either 2 or 8 kilobases of 5'-flanking sequence were produced and resulted in expression of hPNMT mRNA in the adrenal gland and eye. A chimeric gene consisting of 2 kilobases of the hPNMT 5'-flanking region fused to the simian virus 40 early region also resulted in tumor (T) antigen mRNA expression in adrenal glands and eyes; furthermore, immunocytochemistry showed that tumor antigen was localized in nuclei of adrenal medullary cells and cells of the inner nuclear cell layer of the retina, prominent sites of epinephrine synthesis. These results indicate that the enhancer(s) for appropriate expression of the hPNMT gene in these cell types is in the 2-kilobase 5'-flanking region of the human gene.

Adrenal Medulla↗
PubMed1988

Heart and bone tumors in transgenic mice.

Tissue-specific tumorigenesis can be induced in transgenic mice by the directed expression of simian virus 40 (SV40) large tumor (T) antigen. In an attempt to determine the susceptibility of haploid, round spermatids to neoplastic transformation by this oncogene, transgenic mice were generated that harbored a chimeric gene composed of the SV40 T-antigen genes fused to the 5' and 3' flanking sequences of the mouse protamine 1 gene. The transgene was expressed in round spermatids and, surprisingly, in the heart and temporal bone as well. Expression in the heart resulted in rhabdomyosarcomas that always appeared in the right atrium. Bilateral osteosarcomas developed within the petrous portion of the temporal bone. No testicular pathology was observed. T-antigen immunostaining was readily detected in tumor tissue but not in the testis. In addition, SV40 transcripts were processed differently in testis and tumor tissue. Transgenic mouse lines were established that routinely develop these tumors, and they should provide a valuable resource for studies involving cardiac and bone physiology and neoplasia. The atrial tumor cells can be maintained in vitro and some continue to display a cardiac muscle phenotype.

Animals↗
PubMed1988

Developmental study of SV40 large T antigen expression in transgenic mice with choroid plexus neoplasia.

Expression of SV40 large T antigen was correlated with the development of pathology in three lines of transgenic mice. Immunohistochemical detection of large T revealed positive cells only in subsets of choroid plexus epithelium, thymus and renal tubular epithelium, which was first observed during the first month of post-natal life. Morphologic abnormalities became apparent several weeks later. Only some of the choroid plexus cells which expressed T antigen progressed to form neoplasms, suggesting the need for one or more secondary events to effect transformation. Tumors did not develop in thymus and kidney.

Animals↗
PubMed1987

Pancreatic neoplasia induced by SV40 T-antigen expression in acinar cells of transgenic mice.

Three lines of transgenic mice were produced that develop pancreatic neoplasms as a consequence of expression of an elastase I-SV40 T-antigen fusion gene in the acinar cells. A developmental analysis suggests at least a two-stage process in the ontogeny of this disease. The first stage is a T antigen-induced, preneoplastic state characterized by a progression from hyperplasia to dysplasia of the exocrine pancreas, by an increased percentage of tetraploid cells, and by an arrest in acinar cell differentiation. The second stage is characterized by the formation of tumor nodules that appear to be monoclonal, because they have discrete aneuploid DNA contents. The cells within the nodules as compared to normal pancreatic tissue have less total RNA by a factor of 5, less pancreas-specific messenger RNA by a factor of about 50, and increased levels of T-antigen messenger RNA. A tumor cell line has been derived that retains both pancreatic and neoplastic properties.

Animals↗
PubMed1985

Resolution of diaminobenzidine for the detection of horseradish peroxidase on surfaces of cultured cells.

The resolution of indirect immunoperoxidase methods for localizing antigens on the surface of plasma membranes of cultured cells was tested using dissociated monolayer cultures of ciliary ganglion neurons prelabeled with cationic ferritin. Clusters of ferritin were produced on the cell surface by warming the cells to 37 degrees C after the ferritin, rabbit anti-ferritin, and goat anti-rabbit immunoglobulin coupled to horseradish peroxidase had all been applied. Intense 3,3'-diaminobenzidine tetrahydrochloride (DAB) staining was limited to the regions immediately surrounding the ferritin clusters. The lateral spread of the DAB reaction product beyond the outer ferritin particles in each cluster averaged 54-81 nm in four experiments. A second type of increased density, coinciding with the thickness of the plasma membrane, was also seen. These stained plasma membranes extended 161-339 nm from the ferritin clusters.

3,3'-Diaminobenzidine↗
PubMed1984

Concanavalin A inhibits nicotinic acetylcholine receptor function in cultured chick ciliary ganglion neurons.

The effects of various lectins and toxins on neuronal nicotinic acetylcholine receptor function have been studied in primary cultures of chick ciliary ganglion neurons. Neuronal response to acetylcholine receptor activation was measured by a cation flux method at 4 degrees C in a high potassium-low sodium medium designed to stabilize membrane potential near zero, with acetylcholine as the agonist and cesium-137 as the tracer ion. Exposure to 1 mM acetylcholine for 30 s produced a 5-10-fold stimulation of cesium-137 influx. Acetylcholine-stimulated influx was inhibited more than 95% by 10 microM D-tubocurarine, but was insensitive to both 1 microM tetrodotoxin and 1 microM alpha-bungarotoxin. Concanavalin A (50 micrograms/ml) inhibited agonist-induced ion flux by 80% at 4 degrees C. Succinyl-concanavalin A was ineffective at concentrations up to 250 micrograms/ml, and could not protect against the concanavalin A inhibition. However, inhibition by concanavalin A was eliminated by prior incubation of the lectin with 0.2 M alpha-methyl-D-mannoside and subsequent co-incubation with the sugar. Wheat germ agglutinin, lentil lectin, cholera toxin and tetanus toxin were without effect at either 4 degrees C or 37 degrees C. These results suggest a specific interaction between concanavalin A and neuronal nicotinic acetylcholine receptors.

Acetylcholine↗
PubMed1984

Transgenic mice harboring SV40 T-antigen genes develop characteristic brain tumors.

A high percentage of transgenic mice developing from eggs microinjected with plasmids containing the SV40 early region genes and a metallothionein fusion gene develop tumors within the choroid plexus. A line of mice has been established in which nearly every affected animal succumbs to this brain tumor. Thymic hypertrophy and kidney pathology are also observed in some mice. SV40 T-antigen mRNA and protein are readily detected in affected tissues; however, SV40 T-antigen gene expression is barely detectable in unaffected tissues or in susceptible tissues prior to overt pathology, suggesting that tumorigenesis depends upon activation of the SV40 genes. Comparison of DNA from tumor tissue (or cell lines derived from tumors) with DNA from unaffected tissues reveals structural rearrangements as well as changes in DNA methylation of the foreign DNA. The SV40 genes are frequently amplified in tumor tissue, which further indicates that their expression is intimately involved in tumorigenesis in transgenic mice.

Animals↗
PubMed1982

Cholinergic agonist-induced down regulation of neuronal alpha-bungarotoxin receptors.

The ability of cholinergic ligands to regulate neuronal alpha-bungarotoxin receptor number was studied in dissociated monolayer cultures of embryonic chick ciliary ganglion neurons. Carbamylcholine and nicotine, but not D-tubocurarine, caused a loss of 25% of the surface [125I]alpha-bungarotoxin receptors within 1 h at 37 degrees C. This receptor loss occurred without change in affinity for [125I]alpha-bungarotoxin, was temperature-sensitive and was prevented by co-incubation with D-tubocurarine.

Animals↗
PubMed1981

Developments of alpha-bungarotoxin receptors in cultured chick ciliary ganglion neurons.

We have maintained embryonic chick ciliary ganglion neurons in dissociated cell culture and studied the progressive appearance of surface receptors for [125I]alpha-bungarotoxin. Cultures were established from 8-day-old embryos and fed a medium supplemented with 180 micrograms/ml of a soluble protein extract prepared from the eye, the target organ for the ciliary ganglion. Approximately 8064 neurons survived per ganglion and there was no evident loss of neurons through two weeks in culture. Binding of [125I]alpha-bungarotoxin was determined at room temperature in intact cells still attached to their coverslips. Non-specific binding was less than 2% of the total. Specific binding of [125I]alpha-bungarotoxin was saturable with respect to both time of incubation (20-30 min) and concentration of toxin (5-10 nM), with an apparent Kd = 1.0 nM. Binding sites for [125I]alpha-bungarotoxin increased during the first week in culture from 1.8 fmol per 10(4) neurons at 1 day in vitro (DIV) to 8.6 fmol per 10(4) neurons at 7 DIV, after which the number of sites seemed to plateau. Light microscopic autoradiography was performed on cultures at 4 DIV and showed most of the grains associated with the surfaces of neuronal cell bodies, while scattered grains occurred over neuronal processes. When compared with previous reports on the in vivo development of alpha-bungarotoxin receptors in chick ciliary ganglia, the appearance of receptors in these cultured neurons followed a time course similar to, but at lower levels, than, their in vivo counterparts. Nevertheless, this culture system should prove useful for the study of questions concerning the regulation, surface distribution and intracellular pathways of neuronal alpha-bungarotoxin receptors.

Animals↗
PubMed1981

Enrichment of Schwann cell cultures from neonatal rat sciatic nerve by differential adhesion.

A novel method of Schwann cell purification from neonatal rat sciatic nerve has been developed using differential adhesion. After enzymatic and mechanical dissociation, the cell digest is allowed to settle on polylysine-coated glass coverslips for 30 min with intermittent shaking. After an 18-h incubation, bipolar cells comprise greater than 95% of the non-adherent population. Indirect immunofluorescence with the cell-specific markers rabbit anti-galactocerebroside and rabbit anti-bovine-P-2 basic protein antiserum confirmed light microscopic identification of these bipolar cells as Schwann cells. Rabbit anti-human fibronectin specifically labeled fibroblasts which comprised less than 5% of the cell population, but did not bind to Schwann cells. Schwann cells isolated by differential adhesion were injected into a rabbit. When absorbed with cultured rat skin fibroblasts, serum from this rabbit specifically surface labeled greater than 99% of the bipolar and round cells after 18 h and 5 days in vitro and also labeled Schwann cells in fetal rat dorsal root ganglia cultures, but not fibroblasts or neurons.

Animals↗
PubMedDate not supplied

Functional consequences of oncogene-induced horizontal cell degeneration in the retinas of transgenic mice.

Visual function was evaluated in transgenic mice expressing the simian virus 40 early region under the control of the promoter for phenylethanolamine-N-methyltransferase. These transgenic mice undergo a degeneration of the retinal horizontal cells and the outer plexiform layer. Electroretinograms (ERGs) were recorded under stimulus conditions chosen to elicit both receptoral and postreceptoral responses. The dark-adapted a-waves obtained from transgenic mice were not different from control recordings, indicating that the degenerative process does not interfere with function of the rod photoreceptors. In comparison, the ERG b-wave was markedly reduced in transgenic mice under both dark- and light-adapted conditions. Reproducible visual evoked potentials (VEPs) were recorded from transgenic mice in response to both low luminance stimuli that isolate rod function, and to higher luminance stimuli, indicating that retinal activity is transmitted centrally to the visual cortex. However, VEPs were delayed at all stimulus luminances compared to controls. Analysis of luminance-response functions suggests that the VEP delays could reflect the combination of a decrease in synaptic efficacy and an overall loss in visual sensitivity. These functional abnormalities correlate well with the anatomical abnormalities that have been previously observed in the transgenic retina (Hammang et al., 1993), namely a reduced number of synapses between photoreceptors and second-order neurons.

Animals↗