Announcement

Recently we described a saturable, high-affinity binding site for vesicular stomatitis virus (VSV) on the surface of Vero cells that appears to mediate viral infectivity. To isolate this binding site, we have extracted Vero cells with the detergent, octyl-β-d-glucopyranoside. The dialyzed detergent extract specifically inhibits the saturable, high-affinity binding of ³⁵S-methionine-labeled VSV to Vero cells. The inhibitory activity is resistant to protease, neuraminidase and heating to 100°C. It is soluble in chloroform-methanol and inactivated by phospholipase C, suggesting that it is a phospholipid. Of various puriifed lipids tested, only phosphatidylserine was capable of totally inhibiting the high-affinity binding of VSV. The half-maximal inhibitory concentration for phosphatidylserine was 1 μM. Phosphatidylserine also inhibited VSV plaque formation by 80%–90%; Herpes simplex virus plaque formation was unaffected. Centrifugation and electron microscopy studies have shown that phosphatidylserine-containing liposomes bind to VSV. The finding that phosphatidylserine directly binds to VSV and inhibits VSV attachment and infectivity suggests that plasma membrane phosphatidylserine could function as a binding site or portion of a binding site for VSV.     

Characterization of cyclic AMP-resistant Chinese hamster ovary cell mutants lacking type I protein kinase

A group of three mutants of Chinese hamster ovary cells (10260, 10265, and 10223) which are resistant to cyclic AMP (Gottesman, M. M., LeCam, A., Bukowski, M., and Pastan I. (1980) Somatic Cell Genet. 6, 45-61) have been characterized in this work. By genetic analysis, these mutants are all recessive and fall into two complementation groups. Cycl AMP-stimulated protein kinase activity in crude extracts of these mutants using histone as a substrate is decreased to 10 and 7% (complementation group I), and 31% (complementation group II), respectively, of the activity found in wild type extracts. The binding of cyclic [3H]AMP by extracts of all of these mutants is decreased to 30 to 50% of the binding found in wild type extracts. We have used the photoaffinity label 8-azidoadenosine 3':5'-[32P]monophosphate to label the regulatory subunits of type I and type II protein kinase in wild type and mutant extracts analyzed by DEAE-cellulose and Sephadex chromatography. We find that all three mutants lack type I cyclic AMP-dependent protein kinase and have reduced amounts of type II kinase activity. The regulatory subunits of type I and type II kinase are present in both complementation groups. We conclude that type I protein kinase is not needed for normal growth of Chinese hamster ovary cells. The defect in both classes of mutants appears to be in the failure of the catalytic subunit to associate normally with its regulatory subunits.     

Analysis of the Fusion of XC Cells Induced by Homogenates of Murine Leukemia Virus-Infected Cells and by Purified Murine Leukemia Virus

The fusion of XC cells induced by murine leukemia virus (MuLV)-infected cells is also induced by homogenates prepared from the infected cells and by purified MuLV. The fusion-inducing factor appears to contain a heat-labile lipoprotein. No synthesis of specific macromolecules by the XC cells is necessary to obtain fusion. The results suggest that specific components of the viral particle are the activators for the fusion process and they may also be present in the membranes of infected cells.     

Barnase toxin: a new chimeric toxin composed of pseudomonas exotoxin A and barnase

We have constructed a chimeric toxin composed of Pseudomonas exotoxin A (PE) and the extracellular ribonuclease of Bacillus amyloliquefaciens, barnase. The chimeric protein, termed PE-Bar, reacted with both anti-PE and anti-barnase antisera and had both ADP ribosylation and ribonuclease activities. The chimeric toxin was cytotoxic to the murine fibroblast cell line L929 and to a murine hybridoma resistant to PE. A mutant form of PE-Bar lacking ADP-ribosylating activity was still cytotoxic to L929 cells. Because treatment of cells prelabeld with [3H]uridine resulted in a decrease in their RNA content, we conclude that this cytotoxic effect was due to the ribonuclease activity of barnase molecules that had been translocated to the cytosol. It is now possible to construct chimeric toxins with two or more enzymatic activities that can be delivered to the cytosol of the target cells.     

Pseudomonas exotoxin fusion proteins are potent immunogens for raising antibodies against P-glycoprotein.

Antibodies to specific regions of human P-glycoprotein have been difficult to obtain. We developed a method to express in E. coli fusions between Pseudomonas exotoxin and specific regions of human P-glycoprotein. We used the polymerase chain reaction to amplify the desired regions of MDR1 cDNA and to introduce appropriate restriction sites. These fragments were cloned into the 3' end of the Pseudomonas exotoxin gene. With this system we produced large amounts of fusion proteins for immunizations, and we obtained positive rabbit antiserum against P-glycoprotein with most of these antigens. We now have a comprehensive panel of polyclonal antibodies against P-glycoprotein. This system should be generally useful to raise antibodies against other eukaryotic proteins that are difficult to prepare in large quantities.     

Morphologic demonstration of clathrin-coated pits in frog and turkey erythrocytes

We have examined nucleated erythrocytes of frog and turkey for the presence of clathrin-coated structures using electron microscopy and immunocytochemistry. By electron microscopy, coated pits were found on the plasma membrane of peripheral blood erythrocytes of both species. These structures had an appearance similar to coated pits seen in non-erythroid mammalian cells. Using immunofluorescence with anti-(bovine) clathrin antibody, erythrocytes of both species showed punctate membrane fluorescence similar to the pattern of coated pits seen in other cells. By both methods, frog erythrocytes showed considerable heterogeneity, such that only about 50% of the cells showed significant numbers of coated pits, usually fewer than 20-50 per cell. In contrast, the vast majority of turkey erythrocytes showed no detectable coated pits, but occasional cells (less than 10%) showed large numbers of coated structures. These results suggest that a functional endocytic system may be present in a subpopulation of these nucleated erythrocytes. These findings may be of significance in understanding the ligand-induced loss of some receptors from the surface of these cells, and may serve as an indication of morphologic differentiation.     

Inhibition of phosphatidylcholine synthesis does not alter uptake of transferrin by LM fibroblasts

The receptor-mediated endocytosis of diferric transferrin by LM fibroblasts has been examined to determine if de novo synthesis of phosphatidylcholine is required for this process. To test this possibility, LM cells were allowed to internalize [125I]transferrin in the presence or absence of exogenous choline. Under conditions in which de novo synthesis of phosphatidylcholine was reduced from 51% of the total to less than 10%, the initial rate of transferrin uptake was unaffected. These data suggest that the process of receptor-mediated endocytosis of transferrin can proceed normally in the absence of de novo phosphatidylcholine synthesis.