Christa M. Stoscheck

Localization of immunoreactive epidermal growth factor receptors in human nervous system.

Epidermal growth factor is a well-defined peptide which stimulates cell growth and elicits cell responses in a variety of tissues by binding to specific receptors, EGF-R. A specific antiserum against the EGF receptor, which has previously been used to characterize EGF-R in human skin, fibroblasts, and smooth muscle, was used to survey the distribution of EGF-R in human nervous system. Portions of formalin-fixed, paraffin-embedded autopsy specimens were examined by use of immunohistochemical staining (PAP technique) with EGF-R antiserum. Many types of nerve cells, e.g., cerebral cortical pyramidal cells, hippocampal pyramidal cells, Purkinje cells, anterior horn cells, and dorsal root ganglion neurons, contained immunoreactive EGF-R. However, immunoreactive EGF-R were not detected in astrocytes, oligodendrogliocytes, and other small neurons such as granule cells. Intense immunostaining for EGF-R was also detected in ependymal cells from choroidal and extrachoroidal locations. Although immunoreactive EGF-R is widely distributed in human nervous system, the functional role of EGF and its receptor in the nervous system remains unknown.

Increased uniformity in the response of the Coomassie blue G protein assay to different proteins.

Coomassie blue G dye-based protein assays are exceptionally convenient because of their simplicity, sensitivity, speed, and resistance to interfering chemicals, notably reducing agents and most buffers. A major problem with the assay is the variation in response to different proteins. The addition of NaOH to the protein assay reagent reduced the variation in the response of this assay to different proteins. In addition, the sensitivity of the assay is increased. The NaOH can be added either in a separate step to solubilize cells or membranes or directly to the reagent. Linear standard curves were obtained when the log of the absorbance was plotted against the log of the protein quantity.

A rapid fluorometric method for the estimation of DNA in cultured cells

Abstract We present here a procedure for the rapid measurement of both DNA and protein from the same aliquot of cell lysate. DNA estimates obtained by this method were compared to replicate determinations using the method of Kissane and Robins (1). The optimal range for the estimation of DNA (1 to 20 μg) is well suited for use with portions of extracts from individual cell cultures; the remainder of the extract remains available for enzyme assays or other parallel determinations.

Protein assay sensitive at nanogram levels

A simple, fast protein assay which utilizes the affinity of colloidal gold for proteins is described. This assay is sensitive at the 20-ng level when a visible light spectrophotometer is used to measure absorbance. Few chemicals interfere with the assay. Interfering reagents include those that are strongly alkaline, contain high levels of salt, or contain sodium dodecyl sulfate. The problem of alkaline interference can be overcome by acidifying the protein solution before performing the assay. Purified proteins have different capacities to interact with the colloidal gold but this variability is not greater than that seen with the Bradford protein assay.

Immunoreactive Epidermal Growth Factor Receptors in Neuritic Plaques from Patients with Alzheimer's Disease

Alzheimers disease (AD) is characterized neuropathologically by the presence of neuritic plaques (NP) in cerebral cortex and hippocampus, as well as intraneuronal neurofibrillary tangles and granulovacuolar degeneration. The etiology of plaque formation has remained obscure, but morphologically NPare known to contain amyloid cores surrounded by astrocytes and degenerating neurons. Although growth factors are important in growth, differentiation and regrowth in response to injury, studies relating growth factors to ADhave been lacking. Epidermal growth factor (EGF) plays an important role outside the central nervous system (CNS) through interaction with its specific receptor, EGF-R. Using an antibody to EGF-R(threestep immunoperoxidase staining) in conjunction with fluorescence staining, we found that the majority of NPfrom patients with pathologically confirmed AD as well as those few NPin the normal aging brain showed intense EGF-Rimmunoreactivity. Specific staining was seen at the periphery of plaques but not in the central amyloid core. Tissue sections from ADcases were also reacted with antibodies to both glial fibrillary acidic protein (GFAP) and paired helical filaments (PHF) in an attempt to identify which component of the NPwas reactive for EGF-R.The antibody to PHFdensely stained the periphery of NP but not the central core in a majority of NP. The antibody to GFAPstained a few reactive astrocytes that bordered plaques in only a small proportion of all plaques present. We conclude that the neuron and its processes although not exclusively may be the site of EGF-Rimmunoreactivity. An EGF/EGF-Rsystem within the CNS may play an important part in scar formation in response to neuronal injury and death or it may function as a trophic factor important in axonal or dendritic sprouting. It is also possible that EGFcould serve as a neurotransmitter/neuromodulator in the CNS

EPIDERMAL GROWTH FACTOR

During the course of purifying nerve growth factor from the submaxillary gland of the mouse, Cohen (1960) and Levi-Montalcini and Cohen (1960) noticed that daily injections of certain gland extract fractions into newborn mice produced developmental changes that could not be ascribed to nerve growth factor. These changes included precocious opening of the eyelids (7 days compared to the usual 14 days) and a similar early eruption of the incisors. Using these gross anatomical changes as an assay, Cohen (1962) proceeded to isolate the active factor — a polypeptide which he termed epidermal growth factor (EGF).

Characteristics of antibodies to the epidermal growth factor receptor-kinase

Polyclonal antibodies to different antigenic forms of the epidermal growth factor (EGF) receptor-kinase from human A-431 cells have been produced, and their properties have been characterized and compared. Biochemically active receptor-kinase purified by affinity chromatography was employed as one type of antigen. Denatured receptor-kinase prepared by sodium dodecyl sulfate-gel electrophoresis of the affinity-purified receptor was used as the second type of antigen. Animals immunized with either type of antigen produced antibody capable of immunoprecipitating the receptor-kinase molecule. Antibodies produced in response to the biochemically active antigenic form of the receptor-kinase are capable of blocking 125I-EGF binding to the receptor and inhibited EGF-stimulated biological responses. These antisera are not species specific in their ability to inhibit growth-factor binding to the EGF receptor of various mammalian cells. However, these rabbit antisera were unable to inhibit 125I-EGF binding to rabbit cells. Although antisera produced in response to the denatured receptor-kinase molecule are not able to block 125I-EGF binding or EGF-stimulated biological responses, they are particularly efficient for the immunoprecipitation of solubilized 125I-EGF:receptor complexes. None of the antisera contain antibodies capable of interfering with basal receptor-kinase phosphorylation activity. Although each of the antisera immunoprecipitated this kinase activity, none of the antisera contained antibody which served as a phosphorylation substrate for the EGF receptor-kinase in contrast to the immunoglobulins present antisera to the src gene product of the Rous sarcoma virus.

Epidermal growth factor binding and receptor distribution in term human placenta

Human placenta has a large number of epidermal growth factor (EGF) receptors when measured either by [125I]iodoEGF binding or by protein yield after purification. To localize EGF receptors in situ in normal human term placenta, two different light microscopic methods were used. To detect unoccupied, accessible EGF binding sites on the extracellular surface of placental cells in intact blocks of tissue, samples were incubated with [125I]iodoEGF, sectioned and autoradiography performed. To detect the total pool of intracellular and extracellular EGF receptors, placental tissue was sectioned, treated with detergent, and then anti-EGF receptor antibody was localized by immunohistoperoxidase techniques. Both [125I]iodoEGF and anti-EGF receptor antibody methods showed that EGF receptors were primarily present on syncytiotrophoblast cells of placental villi. Smooth muscle cells of placental blood vessels also contained EGF receptors. Neither connective tissue cells within the core of terminal chorionic villi nor endothelium of fetal blood vessels had detectable [125I]iodoEGF binding or immunoreactive EGF receptors. Since the quantity of placental smooth muscle cells is only a small fraction compared to trophoblast cells, we conclude that syncytiotrophoblast cells are primarily responsible for the high levels of EGF receptors found in extracts prepared from human term placenta.

Characterization of binding and receptors for epidermal growth factor in smooth muscle

SummaryThe mitogenic and differentiation-inducing activities of epidermal growth factor (EGF) in epithelial tissues have been well described. Since non-mitogenic effects of EGF, especially in mesenchymal tissues such as smooth muscle are not well-known (Nanney et al. 1984), we have examined EGF-binding and receptors in smooth muscle from many sites. Specific EGF binding sites were detected by incubating small pieces of tissue with 125I-EGF; immunoreactive EGF receptors were detected by immunohistochemistry. In-situ localization of 125I-EGF binding sites and immunoreactive EGF receptors of smooth muscle cells in intact mammalian tissues were identical using either 125I-EGF autoradiography or anti-EGF receptor antibody in an immunoperoxidase method. Cultured rat aortic smooth muscle also contained specific EGF receptors as detected by their biological response to EGF-binding and internalization of 125I-EGF, as well as EGF-stimulated phosphorylation of a 170K protein. The presence of EGF receptors in a well-differentiated smooth muscle cell indicates that EGF may play a physiological, but non-mitogenic role in mammalian tissues in vivo.

Epidermal Growpi Factor: Its Relevance to Dermatology

AbstractSince epidermal growth factor (EGF) and related growth factors have been cloned and are available for clinical trials, the field of peptide growth factors is of general clinical interest. All known effects of EGF are mediated by its binding and activating specific membrane receptors, which contain a tyrosine protein kinase intrinsic to the EGF receptor. In psoriasis and other proliferative skin conditions, levels of EGF receptors are increased because of persistent expression proportional to the degree of abnormal differentiation. What factors cause these EGF receptor abnormalities is unknown, but factors other than EGF, such as transforming growth factors (TGFs), may regulate EGF receptors. For example, in a patient with malignant melanoma the sign of Leser-Trelat, malignant acanthosis nigricans and eruptive achrochordons, was due to changes in EGF/TGFa metabolism. Therefore, growth factor interactions may play a controlling role in proliferative skin diseases, wound healing, cancers, and paraneo...