Jerry L. Phillips

Specific binding of zinc transferrin to human lymphocytes.

Summary Radioiodinated zinc transferrin has been found to interact with human lymphocytes in a manner characterized by rapid binding, saturability, reversibility, and specificity. Each lymphocyte can bind a maximum of approximately 61,000 molecules of zinc transferrin with an association constant of 4 × 10 7 M −1 . Binding is strongly inhibited by cyanide and fluoride and only moderately inhibited by sulfhydryl reagents.

In Vitro Exposure to Electromagnetic Fields: Changes in Tumour Cell Properties

Two human colon cancer cell lines, Colo 205 and Colo 320 DM, have been studied for their responses to 60 Hz-generated electromagnetic fields (EMF) using soft agar cloning and monoclonal antibody binding assays to assess exposure-induced changes. Cellular responses have been studied after 24 h continuous exposure of cells concurrently to four experimental conditions; i.e. no EMF (E-M-), magnetic field only (M+, 1.0 G rms), electric field only (E+, 300 mA/m2 rms), and combined electric plus magnetic fields at these intensities (E+M+). Under these conditions, both cell lines demonstrated significantly increased colony formation in soft agar and increased expression of tumor associated antigens after exposure to E+M+ and to M+ as compared to unexposed controls.

Uptake of transferrin-bound zinc by human lymphocytes.

Abstract Uptake of transferrin-bound zinc was stimulated in phytohemagglutinin-treated human lymphocytes as compared to untreated lymphocytes. Stimulation of zinc uptake depended upon the concentration of phytohemagglutinin and was maximal for the first hour after addition of phytohemagglutinin to lymphocyte cultures. Thereafter, increased zinc uptake declined until approximately basal levels were reached 5 hr after addition of phytohemagglutinin. Stimulation of zinc uptake was insensitive to sulfhydryl reagents, but was decreased by KCN, actinomycin D, aurin tricarboxylic acid, and by lowering the incubation temperature. Two compounds, NaF and poly- l -ornithine, were found to markedly increase zinc uptake over that seen with only phytohemagglutinin. Additionally, compounds known to increase cellular levels of cyclic AMP, such as epinephrine, histamine, serotonin, glucagon, and prostaglandin E 1 , as well as 8-bromo-cyclic AMP and dibutyryl-cyclic AMP, also increased uptake of transferrin-bound zinc by phytohemagglutinin-stimulated lymphocytes.

Specific binding of radioiodinated transferrin to polypropylene culture tubes.

Abstract Radioiodinated human serum transferrin has been found to interact with polypropylene culture tubes in a manner that mimics the specific binding of a protein to a cellular receptor. The magnitude of this transferrin-culture tube interaction is such that any true binding of the protein to cells present is masked. Characteristics of this non-specific “binding” process as well as kinetic and thermodynamic parameters are compared with previously reported values for the interaction of transferrin with both reticulocytes and chinese hamster fibroblasts.

Zinc-induced synthesis of low molecular weight zinc-binding protein by human lymphocytes

Incubation of human peripheral blood lymphocytes with zinc transferrin (with or without phytohemagglutinin) induces the synthesis of protein that elutes from a Sephadex G-75 column at aVe/Vo value corresponding to a molecular weight of 6600. Synthesis depends on the concentration of zinc transferrin in the medium and is sensitive to actinomycin D. Detectable synthesis occurs 5h after initiation of lymphocyte culture and plateaus at 24–30h. Polyacrylamide gel electrophoresis of the zinc-induced protein showed two closely moving bands, both of which show immunologic identity to rat liver metallothionein. Partial characterization of this protein yielded the following results: absorbance maximum at 220 nm; zinc content of 5.8 mol/6600 daltons; sulfhydryl content of 20.2 mol/6600 daltons. Additionally, synthesis of zinc-induced protein is altered in both chronic lymphocytic leukemic and acute lymphoblastic leukemic lymphocytes.

Molecular distribution of cytoplasmic zinc in phytohemagglutinin-stimulated and unstimulated human lymphocytes

Human peripheral blood lymphocytes were incubated with [65Zn] zinc transferrin and with and without phytohemagglutinin for 1, 2, 4, 10, 24, 48, 72, and 96h. Gel filtration of cytoplasmic fractions obtained from these lymphocytes was then performed to determine the molecular distribution of incorporated zinc as a function of time in culture. The data obtained indicated that: (1) transferrin-bound zinc incorporated by human lymphocytes is associated with a variety of soluble molecules whose molecular weights range from less than 5,000 to greater than 70,000 daltons; (2) there is a time-dependent change in the distribution of cytoplasmic zinc for both phytohemagglutinin-stimulated and unstimulated lymphocytes; and (3) for all times studied, there is a difference in the elution profiles obtained for phytohemagglutinin-stimulated and unstimulated lymphocytes. Furthermore, lymphocytes from a donor with untreated hairy cell leukemia exhibited a totally different pattern of cytoplasmic zinc distribution than did lymphocytes from apparently healthy donors.

Subcellular distribution of transferrin-bound zinc incorporated by phytohemagglutinin-stimulated and unstimulated human lymphocytes

The distribution of transferrin-bound zinc incorporated by phytohemagglutinin-stimulated and unstimulated human lymphocytes has been studied as a function of time in four subcellular fractions (nuclei, mitochondria, microsomes, and soluble). In untreated lymphocytes, the percent of total incorporated zinc in each fraction remains relatively constant over 72 h in culture. However, there is a time-dependent change in the percent of total incorporated zinc in all fractions isolated from phytohemagglutinin-stimulated cells, and this change is most apparent for the nuclear and soluble fractions. Apparently some sustained production of energy is required for this change in subcellular distribution of zinc to occur. Additionally, the uptake of cytoplasmic zinc by purified lymphocyte nuclei has been studied. Uptake is rapid and occurs maximally under conditions known to be optimal for stimulation of nuclear adenylate cyclase.

Effects of iron transferrin and zinc transferrin on ferritin synthesis by human lymphocytes

Using a single-incubation two-site immunoradiometric assay, ferritin levels in human peripheral blood lymphocytes have been quantitated up to 72 h in culture. Ferritin levels were found to increase markedly when the cells were treated with phytohemagglutinin or with iron transferrin (in the presence or absence of phytohemagglutinin). On the other hand, zinc transferrin, with or without iron transferrin in the culture medium and in the presence and absence of phytohemagglutinin, produced a dose-dependent decrease in cellular ferritin compared to controls. Additionally, the increase in lymphocyte ferritin produced by phytohemagglutinin and iron transferrin appears to require transcriptional and translation events.

Lymphocyte zinc metabolism in disease : Paroxysmal noctural hemoglobinuria.

Lymphocytes were obtained from two patients with paroxysmal nocturnal hemoglobinuria as well as from apparently healthy controls and from patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia. Subsequently, several aspects of zinc metabolism were studied in vitro in short-term cultures of these lymphocytes in order to assess lymphocyte functional capacity. The results of mitogen stimulation and zinc uptake studies for lymphocytes from donors with paroxysmal nocturnal hemoglobinuria were similar to those obtained for leukemic lymphocytes. The results of studies to determine the inducibility of the low molecular weight zinc-binding protein, metallothionein, by zinc were complicated by the decrease in overall protein synthesis in lymphocytes from donors in the paroxysmal nocturnal hemoglobinuria. It is proposed that paroxysmal nocturnal hemoglobinuria is indeed a clonal disorder and the relationship between lymphocytes in this disorder and leukemic lymphocytes is discussed.