The CD4-binding domain of human immunodeficiency virus type 1 (HIV-1) gp120 elicits antibodies that are present in infected human sera. Monoclonal antibodies that recognize the HIV-1 gp120 CD4-binding domain have been isolated. Some of these antibodies can neutralize laboratory-adapted strains of HIV-1 and probably mediate neutralization by interfering with virus binding to its cellular CD4 receptor. However, most anti-CD4 binding domain antibodies do not neutralize primary HIV-1 isolates. We used primary HIV-1 isolates in an infectivity reduction assay to test the uniquely derived anti-CD4 binding domain recombinant human monoclonal antibody, IgG1b12. All of the tested HIV-1 isolates were neutralized by this antibody. Additional studies indicated that neutralization of a primary isolate with MAb IgG1b12 did not require continuous exposure of human peripheral blood mononuclear cell cultures to the antibody. Finally, a complete IgG1 molecule of an in vitro-selected b12 FAb mutant with a > 400-fold increase in affinity was assembled, expressed in mammalian cells, and evaluated in the infectivity reduction assay in comparative studies with the parent IgG1b12 antibody. The mutant did not retain the level of primary isolate neutralization potency that was a property of the parent molecule. Thus, we confirm that recombinant IgG1b12 has a unique specificity, and that it can neutralize all primary isolates tested in human PBMC cultures in vitro.

This report describes the cloning, expression and characterization of two members of a novel human gene family of proteins, HBNF and MK, which exhibit neurite outgrowth-promoting activity. The HBNF cDNA gene codes for a 168-residue protein which is a precursor for a previously described brain-derived heparin-binding protein of 136 amino acids. The second human gene identified in this study, called MK, codes for a 143-residue protein (including a 22-amino acid signal sequence) which is 46% homologous with HBNF. Complementary DNA constructs coding for the mature HBNF and MK proteins were expressed in bacteria and purified by heparin affinity chromatography. These recombinant proteins exhibited neurite-outgrowth promoting activity, but lacked mitogenic activity. The HBNF gene is expressed in the brain of adult mice and rats, but only minimal expression of MK was observed in this tissue. Different patterns of developmental expression were observed in the embryonic mouse, with MK expression peaking in the brain between days E12 and E14 and diminishing to minimal levels in the adult, while expression of HBNF mRNA was observed to gradually increase during embryogenesis, reaching a maximal level at birth and maintaining this level into adulthood. Expression of these genes was also observed in the human embryonal carcinoma cell line, NT2/D1. Retinoic acid induced the expression of HBNF and MK 6- and 11-fold, respectively, in this cell line. Our studies indicate that HBNF and MK are members of a new family of highly conserved, developmentally regulated genes that may play a role in nervous tissue development and/or maintenance.

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Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measured by using [32P]phosphorylase a as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at %5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-i phosphatase. Similar effects on phQsphatase activity were seen when epidermal growth factor aid platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase.Insulin triggers a complex array of metabolic processes in a variety of mammalian cell types. These effects range from increased uptake of ions and nutrients, altered flux pattern of carbon in synthetic and degradative pathways of glucose, lipid, and protein metabolism to changes in specific gene expression (for reviews, see refs. 1 and 2). Although these biochemical events have been under intense investigation for many years, the detailed signaling mechanisms by which insulin functions remain to be elucidated. A major mode of action of insulin involves changes of the phosphorylation state of various proteins. The first event initiated upon binding of the hormone to its cell-surface receptor is the induction ofan intrinsic protein-tyrosine kinase activity in the cytoplasmic domain of the receptor. Based on site-specific mutagenesis studies of the human insulin receptor, it has been suggested that this kinase activity is essential for at least some of the metabolic actions of insulin, such as an increase in ribosomal protein S6 phosphorylation and in glycogen synthesis (3). These and many other intracellular enzyme systems are known to be regulated through phosphorylation cascades involving serine/threonine residues, but the steps connecting the target proteins to tyrosine phosphorylation by the receptor are still unknown.In glycogen metabolism, the rate-limiting enzymes of breakdown and synthesis are regulated by phosphorylation/ dephosphorylation mechanisms. (7,8). From studies carried out with purified enzymes, various mechanisms for regulating protein phosphatase activities in vitro have been identified; however, to date little is known about the regulation of these enzymes in vivo.Our finding that insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) activate glycogen synthase (11, 12) in cultured 3T3 cells has prompted an investigation of the effect of these polypeptide factors on phosphoprotein phosphatase activity. Since glycogen synthase is activated by dephosphorylation (6, 13, 14), it seemed probable that phosphatases would have a critical role in the regulation of this enzyme. In this initi...

Recombinant rat growth hormone (rrGH) and recombinant mouse growth hormone (rmGH) were developed to evaluate the potential carcinogenicity of each biologically active growth hormone (GH) as assessed in the respective species. Biological activities of rrGH and rmGH were demonstrated by showing an increase in body weight gain and serum levels of insulin-like growth factor-1 (IGF-1) in hypophysectomized rats receiving daily sc injections for 6 days. With the exception of pharmacologically mediated weight gain, rrGH and rmGH had no adverse effects in 5-week oral toxicity studies and no production of anti-recombinant GH antibodies. The high doses selected for the carcinogenicity studies provided systemic exposures of GH up to approximately 10-fold over basal levels. In the 105-week mouse carcinogenicity study, daily sc injections of rmGH at 0.1, 0.2, or 0.5 mg/kg/day were well tolerated and had no effects on survival or incidence of tumors. In the 106-week rat carcinogenicity study, daily sc injections of rrGH at 0.2, 0.4, or 0.8 mg/kg/day had a favorable effect on longevity in female rats administered 0.4 or 0.8 mg/kg/day, an increased weight gain in females and males, and no increase in the incidence of tumors. The absence of carcinogenic effects of recombinant GH administered daily for 2 years to rodents was consistent with publications of clinical experience, indicating a lack of convincing evidence for an increased risk of cancer in children receiving human recombinant GH replacement therapy.