A sensitive method to introduce membrane-bound proteins into recipient cells based on affinity enrichment of lipid vesicles to the recipient cell prior to fusion

A sensitive analytical procedure for studying membrane-bound structures has been developed. Membrane glycoproteins inserted into liposomes were transferred to recipient cells by use of a lectin, concanavalin A, bound to the cells as a bridge to generate proximity between the recipient cell and the glycoprotein-containing liposome, prior to exposure to the fusing agent, poly(ethylene glycol). Partially purified histocompatibility antigen from rats was introduced into the membrane of human lymphocytes. After treating the cells with poly(ethylene glycol) under fusion conditions, some of the antigen present in the preparation could not be eluted with alpha-methyl mannoside and EDTA, indicating that incorporation in the cell membrane had taken place. This antigen remained exposed on the lymphocyte surface for approximately 1 h as demonstrated by sensitivity of the lymphocytes to the lytic effect of an antiserum to the histocompatibility antigen in the presence of complement. Some of the lectin molecules seemed to be internalized in the cells but no induction of cell mitosis was observed. The described method gives an opportunity to work with small amounts of membrane proteins inserted into liposomes, introducing them into recipient cells for analysis of their biological activities.     

Binding to erythrocyte membrane is the physiological mechanism for activation of Ca2+-dependent neutral proteinase

In the presence of micromolar concentrations of Ca2+ the catalytic 80 kDa subunit of human erythrocyte procalpain binds to the cytosolic surface of the erythrocyte membrane. Binding is rapid, highly specific and is reversed by the removal of Ca2+. In the bound form the 80 kDa catalytic subunit undergoes a rapid conversion to calpain, the active 75 kDa Ca2+-requiring proteinase. The activated proteinase produces extensive degradation of membrane components, particularly of band 4.1 and 2.1 proteins. Binding to membranes may represent an obligatory physiological mechanism for the conversion of procalpain to calpain.     

Interferon-gamma modulates protein kinase C activity in murine peritoneal macrophages

The content of Ca2+-, phospholipid-dependent protein kinase activity (protein kinase C) in murine peritoneal macrophages treated with recombinant interferon-gamma (IFN-gamma) has been investigated. Protein kinase C activity was solubilized by nonionic detergent extraction of sonicated cells and separated by high performance liquid chromatography on a TSK 4000 SW gel filtration column. The enzyme eluted from the column in a molecular weight range of 60-80 X 10(3) and was identified by virtue of Ca2+ and phospholipid requirements. Macrophages treated with recombinant IFN-gamma exhibited a substantial increase in total protein kinase activity which could be accounted for entirely by increased protein kinase C activity. This activity was enhanced as much as 5-fold over that seen in untreated macrophages and was specific for IFN-gamma in that other agents known to signal changes in macrophage function had no effect. The time required for the elevation of kinase activity was identical to that required for induction of other functions by IFN-gamma in macrophages. These observations suggest that protein kinase C may be a focus of regulatory action in IFN-gamma-mediated macrophage activation.     

Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C

The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated.     

Cell cycle traverse and protein metabolism in human NHIK 3025 cells: the role of anchorage

We have studied the effect of cell anchorage on the human cell line NHIK 3025 in vitro, to see whether the growth regulating effect of cell anchorage primarily affected DNA division cycle or mass growth cycle. It was found that cell to cell anchorage had the same effect on cell cycle progression as anchorage to a solid surface, which indicates that it is anchorage per se and not cell shape that is important for growth control in NHIK 3025 cells. When NHIK 3025 cells were grown without attachment to a solid surface, both G1 and cell cycle duration was prolonged by 6 h, which means that the prolonged cell cycle was due to a prolonged G1. During the first part of the cell cycle the rate of protein synthesis and degradation was constant, and at the same level in cells grown with and without attachment. This means that the prolonged G1 was not due to a reduced protein accumulation or mass growth. Towards the end of the cell cycle protein accumulation was reduced. This effect was either due to a size control before cell division or a secondary effect of the prolonged G1. We therefore conclude that cell anchorage as a growth regulator primarily affects the DNA/cell division cycle.     

Carbohydrate metabolism in transforming lymphocytes from the aged

There is an age-related decline in immune capacity which has been linked to a decreased response of lymphocytes to mitogens in vitro. During transformation, lymphocytes require a marked increase in energy production and biosynthesis which is supplied primarily by glycolysis. In the elderly, the glycolytic enzymes increase significantly in transforming lymphocytes at least 24 hr later than in the young and then at significantly reduced levels. Glucose utilization is also impaired in stimulated lymphocytes from the elderly but follows the impairment of glycolysis. In stimulated cells from the young, increases in glycolytic enzyme activity levels accompany sharp increases in blastogenesis while a delayed increase in glycolytic enzyme activity in the elderly is accompanied by a delay in blastogenesis. Maximal glycolytic enzyme activity levels are significantly reduced in transformed lymphocytes from the elderly though the number of transformed cells is also significantly reduced. However, glycolytic enzyme activity levels are significantly lower in the elderly than in the young even on a per transformed cell basis. Thus, this reduction cannot be attributed to the lower number of transformed cells that are present in the elderly. This defect in the increase of glycolysis in stimulated cells from the elderly suggests an intracellular mechanism which could be related to the impaired lymphocyte stimulation in vitro in the aged.     

Immunocytochemical studies of relaxin in ovaries of pregnant and cycling mice

Immunocytochemical staining for relaxin in ovarian sections of pregnant mice from day 11 through day 18 of gestation revealed that only corpora lutea (CL) of pregnancy are stained. Evaluation of serial sections of ovaries from a day 16 pregnant mouse revealed that the only luteal structures present are CL of pregnancy. The number of CL present in each ovary equaled the number of implantation sites in each related horn (7 on the right side and 8 on the left side). These large CL varied in shape, being round in some profiles to very elongate in others. All CL were immunochemically stained for relaxin using the peroxidase-antiperoxidase method of L. Sternberger (Immunocytochemistry, 2nd ed. Wiley, New York, 1979). The intensity of the strain varied from cell to cell within each CL. Small luteal structures that were observed to be immunochemically stained for relaxin were demonstrated to represent the periphery of CL of pregnancy. No luteinized follicles were observed and interstitial cells and follicles were not immunochemically stained in any of the day 16 serial ovarian sections or in any of the ovarian sections from pregnant mice on the other days of gestation studied. CL of previous cycles were not observed to be present in the ovaries at days 15, 16, or 18 of gestation. However on day 14 and before, CL of previous cycles were observed and they did not exhibit any relaxin immunostaining. Immunocytochemical studies using the biotin-avidin system revealed that no relaxin immunostaining could be demonstrated in the ovaries of cycling mice at any stage of the estrous cycle. In conclusion, this study revealed that the only ovarian structures demonstrating relaxin immunocytochemical staining in the mouse were CL of pregnancy.     

Reconstitution of in vivo cell-mediated lympholysis responses in aged mice with interleukin 2

Advancing age is accompanied by declining immune potential. Both humoral and cellular immune responses are diminished in aged humans and experimental animals. A major lesion preventing effective T cell-mediated responses is the lack of interleukin 2 (IL 2) synthesis in aged mice. Addition of IL 2 can effectively reconstitute in vitro T cell-dependent humoral and cell-mediated immunity. These results have been extended, demonstrating that IL 2-containing lymphokine preparations when administered with antigen can restore the ability of aged mice to generate cytotoxic T lymphocytes in vivo. Furthermore, IL 2 has been identified as the active component of these lymphokine preparations through the use of purified human IL 2 prepared by recombinant DNA and gene cloning technology. The cloned IL 2 is highly effective in enhancing the immune responses of aged animals both in vitro and in vivo.     

In vivo administration of purified human interleukin 2. I. Half-life and immunologic effects of the Jurkat cell line-derived interleukin 2

A total of 12 patients with cancer or the acquired immunodeficiency syndrome have been treated with Jurkat-derived purified human interleukin 2 (IL 2). The toxicity was dose-related and consisted primarily of fever, chills, malaise, and mild reversible hepatic dysfunction. No evidence of clinical efficacy was seen when IL 2 was administered at doses of up to 2000 micrograms by bolus or continuous infusion once a week for 4 wk. No significant chronic immunologic effects (changes in mitogen responsiveness of induction of cytotoxic cells) were demonstrated. IL 2 was measured in the serum of patients, and a half-life of approximately 5 to 7 min was demonstrated with a second component of clearance of 30 to 120 min. Heating the serum at 56 degrees C for 30 min allowed for detection of smaller quantities of IL 2 by removing a serum inhibitor whose effect was seen at dilutions of up to 1/80 in our biologic assay. Sustained levels of IL 2 could be maintained by continuous infusion. Acute effects of IL 2 administration included a rapid decrease in peripheral mononuclear cells with a shift to cells of macrophage lineage and a rapid decrease in total T lymphocytes and T lymphocyte subsets. IL 2 responsiveness of peripheral mononuclear cells decreased within 15 min of IL 2 administration, with a concurrent decrease in the ability to generate lymphokine-activated killer cells. These changes did not recover until 48 hr after IL 2 administration. A rise in serum ACTH and cortisol levels was seen after the administration of 1 to 2 mg of IL 2. Future studies will evaluate the role of larger quantities of recombinant IL 2 given alone or in conjunction with in vitro-generated lymphokine-activated killer cells.