David W. Leung

Cloning, expression during development, and evidence for release of a trophic factor for ciliary ganglion neurons

Ciliary ganglion (CG) neurons undergo a period of cell death during development that may be regulated by the limited availability of trophic factor produced by their target tissues. We have previously reported the purification of a ciliary neurotrophic factor from adult chick sciatic nerve that we called growth promoting activity (GPA). Here we demonstrate that GPA can be purified and cloned from embryonic day 15 (E15) chick eyes, which contain all the target tissues of the CG. Our studies show the following: GPA mRNA is induced in embryonic chick eyes during the period of CG neuron cell death; GPA mRNA is expressed specifically in the layer of the eye that contains the targets of the CG and in primary cultures of smooth muscle cells isolated from the choroid layer of the eye; and biologically active GPA is released from cells transfected with a GPA cDNA.

Molecular Characterization and Distribution of Vascular Endothelial Growth Factor

The cardiovascular system is the first organ system to develop and reach a functional state in an embryo. This precocity reflects the fundamental role that blood circulation plays in the delivery of nutrients and the disposal of catabolic products in a multicellular organism. Angiogenesis, the growth of new blood vessels, begins in the extraembryonic mesoderm of the yolk sac, the connecting stalk and the chorion as early as day 13 in the human (1). Nests of initially isolated endothelial cell cords develop a lumen. Primitive embryonic vessels form by confluence of lumina of separate cords about 2 d later (2,3). Further angiogenesis requires enzymatic degradation of the basement membrane of a local venule (4), endothelial cell Chemotaxis (5) and proliferation (6). Angiogenesis also is required in postnatal life for a wide variety of fundamental physiological processes such as somatic growth, wound healing, tissue and organ regeneration, cyclical growth of the corpus luteum and endometrium. Conversely, uncontrolled angiogenesis is now recognized as an important pathogenic component of a variety of conditions, including rheumatoid arthritis, retinopathies, psoriasis and retrolental fibroplasia (7–9). Also, a major factor that determines potential for rapid growth and propensity to metastasize in tumors is the ability of tumor cells to induce a neovascular response (10). This allows the tumor to establish contact with the vascular bed of the host and to be nourished.

Growth hormone receptor and binding protein

Publisher Summary The purification and cloning of the growth hormone (GH) receptor and the binding protein have contributed to our understanding of hormonally controlled growth in mammals. This chapter discusses the purification and cloning of growth hormone receptor and binding protein. The GH and prolactin receptors constitute a new family of signaling proteins with a single transmembrane domain. The ability of GH to promote whole-body growth has been demonstrated clinically in the treatment of GH-deficient children and in animals. The lack of easily demonstrated GH effects in cell-based assay systems has led to the somatomedin hypothesis according to which GH produced by the pituitary acts on the liver to induce synthesis and secretion into the circulation of insulin-like growth factor I (IGF-I), which is responsible for skeletal and tissue growth. The availability of the cloned receptors allows elucidation of the intracellular signaling mechanism for these hormones. In situ hybridization work suggests that the GH receptor is widely distributed on many tissues, although it is most abundant in the liver. This wide distribution suggests that direct actions of GH could play a role in the growth process instead of the indirect action suggested by the somatomedin hypothesis. The availability of the GH receptor expressed by recombinant DNA techniques have been useful in determining the amino acids in GH required for binding to the receptor.

GROWTH HORMONE RECEPTORS AND BINDING PROTEINS

Growth hormone receptors are found in a wide variety of tissues and are thought to mediate the various actions of growth hormone. Recently, a growth hormone binding protein was demonstrated in serum and shown to have antigenic identity with the liver growth hormone receptor by use of a panel of monoclonal antibodies to the receptor. Here we describe the purification, part sequence and cloning of the rabbit liver growth hormone receptor. Purification and N terminal sequence analysis of the rabbit serum binding protein for GH showed it to be identical to the extracellular region of the rabbit liver GH receptor. Rabbit liver receptor and human binding protein sequences were expressed in COS-7 cells and shown to display predicted hormone specificity and antigenic characteristics. Finally, the rabbit mammary gland prolactin receptor was purified and part sequenced. This showed 34% homology with the rabbit liver GH receptor, and therefore constitutes the second member of a new class of transmembrane receptors regulating growth and lactation.

ISOLATION AND CHARACTERIZATION OF HUMAN IMMUNE INTERFERON (IFN-γ) MESSENGER RNA

Publisher Summary This chapter describes an experiment where messenger RNA (mRNA) was isolated from human peripheral blood lymphocytes stimulated with the mitogen Staphylococcal enterotoxin β. Sucrose-gradient centrifugation and acid-urea gel electrophoresis were used to fractionate mRNA. Following fractionation, mRNA was recovered and injected into Xenopus oocytes, and the translated fractions were assayed for antiviral activity in a cytopathic-effect inhibition assay. Two activity peaks were observed in the sucrose-gradient-fractionated RNA. One peak sedimented with a calculated size of 12S and contained 100–400 units/ml of antiviral activity per microgram of injected RNA. The other peak of activity sedimented as 16S in size and contained approximately half the activity of the slower sedimenting peak. Only one peak of activity was observed in the gel-fractionated mRNA. The size discrepancies among activity peaks observed on the sucrose gradients and acid-urea agarose gels may be the result of fractionation under conditions that do not totally denature the mRNA.