Endonuclease of high specific activity in a purified mouse interferon preparation

We find an endonuclease of high specific activity in a purified mouse interferon preparation. The interferon was purified from Ehrlich ascites tumor cultures which were induced with Newcastle disease virus. It has a higher specific activity (1.5 × 10⁹ NIH mouse reference standard interferon units/mg protein) than reported for any interferon preparation but is not homogeneous. We do not know if the endonuclease activity is due to a contaminating protein or to interferon. The endonuclease does not degrade in our conditions polyuridylic acid or double stranded reovirus RNA and does not inactivate the tRNA₂^Gln species from $\text{E. coli}$, or tRNA^Val species or polysomes from mouse L cells. Endonuclease in as little as 0.5 ng protein of the interferon preparation degrades μg quantities of messenger RNA from mouse L cells, of encephalomyocarditis virus RNA and of $\text{in vitro}$-synthesized reo-virus messenger RNA at 37° in 1 hour. Further characteristics of the endonuclease and its possible relationship (if any) to interferon remain to be established.     

Blocked and non-blocked ricin immunotoxins against the CD4 antigen exhibit higher cytotoxic potency than a ricin A chain immunotoxin potentiated with ricin B chain or with a ricin B chain immunotoxin

Summary An immunotoxin consisting of ricin A chain linked to the monoclonal antibody M-T151, recognising the CD4 antigen, was weakly toxic to the human T-lymphoblastoid cell line CEM in tissue culture. The incorporation of [³H]leucine by CEM cells was inhibited by 50% at an M-T151-ricin-A-chain concentration (IC₅₀) of 4.6 nM compared with an IC₅₀ of 1.0 pM for ricin. In contrast, immunotoxins made by linking intact ricin to M-T151 in such a way that the galactose-binding sites of the B chain subunit were either blocked sterically by the antibody component or were left unblocked, were both powerfully cytotoxic with IC₅₀ values of 20–30 pM. The addition of ricin B chain to CEM cells treated with M-T151—ricin-A-chain enhanced cytotoxicity by only eight-fold indicating that isolated B chain potentiated the action of the A chain less effectively than it did as an integral component of an intact ricin immunotoxin. Ricin B chain linked to goat anti-(mouse immunoglobulin) also potentiated weakly.Lactose completely inhibited the ability of isolated ricin B chain to potentiate the cytotoxicity of M-T151—ricin-A-chain and partially (3- to 4-fold) inhibited the cytotoxicity of the blocked and non-blocked ricin immunotoxins. Thus, in this system, the galactose-binding sites of the B chain contributed to cell killing regardless of whether isolated B chain was associated with the A chain immunotoxin or was present in blocked or non-blocked form as part of an intact ricin immunotoxin. The findings suggest that the blocked ricin immunotoxin may become unblocked after binding to the target antigen to re-expose the cryptic galactose-binding sites. However, the unblocking cannot be complete because the maximal inhibition of [³H]leucine incorporation by the blocked immunotoxin was only 80% compared with greater than 99% inhibition by the non-blocked immunotoxin.     

Expression of Mutant Dynamin Inhibits Toxicity and Transport of Endocytosed Ricin to the Golgi Apparatus

Endocytosis and intracellular transport of ricin were studied in stable transfected HeLa cells where overexpression of wild-type (WT) or mutant dynamin is regulated by tetracycline. Overexpression of the temperature-sensitive mutant dyn^G273D at the nonpermissive temperature or the dyn^K44A mutant inhibits clathrin-dependent endocytosis (Damke, H., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131: 69–80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915–934). Under these conditions, ricin was endocytosed at a normal level. Surprisingly, overexpression of both mutants made the cells less sensitive to ricin. Butyric acid and trichostatin A treatment enhanced dynamin overexpression and increased the difference in toxin sensitivity between cells with normal and mutant dynamin. Intoxication with ricin seems to require toxin transport to the Golgi apparatus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949–966), and this process was monitored by measuring the incorporation of radioactive sulfate into a modified ricin molecule containing a tyrosine sulfation site. The sulfation of ricin was much greater in cells expressing dyn^WT than in cells expressing dyn^K44A. Ultrastructural analysis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dyn^K44A. In contrast, ricin transport to lysosomes as measured by degradation of ¹²⁵I-ricin was essentially unchanged in cells expressing dyn^K44A. These data demonstrate that although ricin is internalized by clathrin-independent endocytosis in cells expressing mutant dynamin, there is a strong and apparently selective inhibition of ricin transport to the Golgi apparatus. Also, in cells with mutant dynamin, there is a redistribution of the mannose-6-phosphate receptor.     

Intracellular site of prolactin synthesis in rat pituitary cells in culture

Free and membrane-bound polyribosomes were isolated from control and thyrotropin releasing hormone-treated GH₃ cells. The two polysome fractions were used to direct {³H}leucine incorporation into prolactin in both heterologous and homologous cell-free protein-synthesizing systems. Prolactin was measured by immunoprecipitation and SDS-disc gel electrophoresis of the reaction products. Only membrane-bound polysomes directed incorporation of {³H}leucine into labeled prolactin. In additon, intact cells were pulselabeled with {³H}leucine, free and membrane-bound polysomes were isolated, and newly synthesized prolactin associated with each polysome fraction was measured. In control cells, {³H}prolactin represented about 0.4 and 4.2% of total acid-insoluble radioactivity in free and membrane-bound polysomes, respectively; whereas, in thyrotropin releasing hormone-treated cells, these values were about 1 and 20%, respectively. Added {³H}prolactin did not associate nonspecifically with membrane-bound polysomes. We conclude that prolactin is synthesized predominantly on membrane-bound polysomes in GH₃ cells.     

Blocked ricin-conjugated T cell immunotoxins: Effect of anti-CD6-blocked ricin on normal T cell function

Summary The biological properties of an immunotoxin composed of an anti-CD6 monoclonal antibody conjugated to whole ricin, which had been modified so that the galactose-binding sites of the B chain were blocked (blocked ricin), were examined. Treatment of peripheral blood lymphocytes with anti-CD6-blocked ricin for a 24-h period prevented T cell proliferation induced by phytohemagglutinin in a dose-dependent manner with concentrations causing 50% inhibition (IC₅₀) ranging from 5 pM to 30 pM. In contrast, treatment with either blocked ricin alone or with a control immunotoxin prepared with a B-cell-lineage-restricted monoclonal antibody gave IC₅₀ values of approximately 2 nM. Although shortening the duration of the anti-CD6-blocked ricin treatment to as little as 3 h had little significant effect on the observed inhibition, T cell viability experiments demonstrated that the magnitude of immunotoxin-induced killing after a given time period is significantly higher when the target cells become activated. Thus, from the initial concentration of cells treated with anti-CD6-blocked ricin placed in culture, 40%–45% viable cells remained after 2 days yet only 3%–9% remained if phorbol ester and Ca²⁺ ionophore were added; activation of T cells after mock treatment using blocked ricin plus nonconjugated anti-CD6 demonstrated that this effect was not the result of activation alone. The toxicity of anti-CD6-blocked ricin was also measured by inhibition of PHA-induced clonogenic growth of normal T cells. Continuous treatment of the cells using anti-CD6-blocked ricin at 0.1 nM resulted in a surviving fraction of about 3.5 × 10^–3; when immunotoxin treatment was for 24 h or less, the surviving fraction was only about 10^–1. As an indication of the unique specificity of anti-CD6-blocked ricin, immunotoxin pretreatment of potential responder cells prevented the generation of allogeneic cytolytic T lymphocytes in mixed lymphocyte cultures yet had little effect on the generation of interleukin-2-induced lymphokine-activated killer cell activity. We conclude that anti-CD6-blocked ricin demonstrates a cellular specificity and potency that make it a highly promising anti-T cell reagent.     

Selective determination of mRNA specific radioactivity in HeLa cells without the use of inhibitors

Polysomes from (³H)-uridine pulse-labeled HeLa cells were isolated and the specific radioactivity of polysome-associated mRNA was determined by selective enzymic hydrolysis at 0°C of the $\text{inter}$ribosomal mRNA sections. $\text{Intra}$ribosomal mRNA protected from hydrolysis during ribonuclease treatment and subsequently isolated by the proteinase K method (1) exhibited the same specific radioactivity as the interribosomal mRNA split products.When labeled polysomes were subjected to ribonuclease treatment at 25°C instead of 0°C a higher specific radioactivity of the interribosomal split products resulted, while intraribosomal sections still exhibited the same values as after 0°C treatment. The labeled polysomes used as substrate exhibited one single A₂₆₀ and radioactivity peak in CsCl density gradients. No RNP material banding at ? = 1.35 ? 1.45 could be detected. However, the radioactivity maximum banded at slightly lower densities than the A₂₆₀ peak (? = 1.55 versus 1.57). The shift appears to be caused by a contaminant RNA. These findings as well as the radioactivity pattern of pulse-labeled polysomes in sucrose gradients may indicate the presence of newly synthesized mRNA associated with monosomes (and oligosomes) protected from ribonuclease action at 0°C by (transport?) proteins.     

ISOLATION OF TWO DISTINCT CLASSES OF POLYSOMES FROM A NUCLEAR FRACTION OF RAT LIVER

Isolated rat liver nuclei were washed with Triton-X-100 in the presence of liver cell sap. This treatment liberated a fraction of polysomes which were isolated by differential centrifugation and were designated "outer membrane polysomes." The outer membrane polysomes synthesized protein in vivo. Shortly after injection of orotic acid-¹⁴C, the RNA of outer membrane polysomes had a higher specific activity than that of cytoplasmic polysomes. It was postulated that outer membrane polysomes may be an intermediate in the transfer of newly synthesized RNA from the nucleus to the cytoplasm. In other experiments, Triton-washed rat liver nuclei were lysed in the presence of deoxycholate and deoxyribonuclease. A ribonucleoprotein fraction was isolated from the lysate by differential centrifugation. This fraction contained "intranuclear ribosomes," which sedimented like partially degraded polysomes in sucrose gradients. This degradation could be partially prevented if intranuclear ribosomes were purified by sedimentation through heavy sucrose. The resulting pellets were termed "intranuclear polysomes" because they contained some undergraded polysomes. Intranuclear polysomes were highly radioactive after a brief pulse with orotic acid-¹⁴C, but did not appear to synthesize protein rapidly in vivo. Intranuclear polysomes may represent the initial stage of assembly of polyribosomes in the nucleus.     

Differential effects of ca2+ and Mg2+ on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

In autodigestion assays, endonucleaw activity in non-apoptotic HL-60 promydocytic leukemia cell nuclei cleaved the chromatin of he autologous cells to an oligonucleosomal length pattern. Both EGTA and EDTA inhibited the activation of endonuclease activity in isolated HL-60 cell nuclei. The inhibition by EDTA could be reversed by exogenous Ca²⁺. but not by exogenous Mg²⁺. In Ca²⁺/Mg²⁺-free nuclei digation buffer, addition of Ca^2→ (1-10 mmol/L) induced endonuclease activity in the isolated nuclei, while addition of Mg²⁺ had no effect. In the presence of Ca²⁺(0.1 mmol/L), endonuclease activity was enhanced by exogenous Mg²⁺ (0.1-10mmol/L). These results suggest that the endonuclease responsible for internucleosomal DNA fragmentation in HL-60 cells during apoptosis is activated by Ca²⁺ and further modulated by Mg²⁺ in the presence of ca²⁺.     

On the mechanism of protein-synthesis inhibition by abrin and ricin. Inhibition of the GTP-hydrolysis site on the 60-S ribosomal subunit.

The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell-free systems from rabbit reticulocytes and Krebs II ascites cells. In crude systems abrin and ricin strongly inhibited protein synthesis from added aminoacyl-tRNA, demonstrating that the toxins act at some point after the charging of tRNA. Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti-toxins were added. Recombination experiments between toxin-treated ribosomes and untreated supernatant factors and vice versa showed that the toxin-treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis. Recombination experiments between toxin-treated isolated 40-S subunits and untreated 60-S subunits and vice versa showed that only when the 60-S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited. Both the EF-1-catalyzed and the EF-2-catalyzed ability of the ribosomes to hydrolyze [gamma-32P]GTP was inhibited by abrin and ricin. An 8-S complex released from the 60-S subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8-S complex were inhibited by abrin and ricin. The present data indicate that there is a common site on the 60-S subunits for EF-1- and EF-2- stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.     

The specific binding of ricin and its polypeptide chains to rat liver ribosomes and ribosomal subunits

Ricin from Ricinus communis was isolated and the binding of ³H-reductively alkylated or ¹²⁵I-iodinated ricin was studied by incubating the toxic protein with ribosomes and isolating the ricin-ribosome complex by centrifugation. Neither of the labeled ricin derivatives nor ³H-labeled A chain bound Escherichia coli ribosomes, but both bound rat liver ribosomes in a reproducible manner. ³H-labeled ricin bound in a ratio of 1 mol/mol of ribosomes with a dissociation constant of 3 μm as calculated from a Scatchard plot. Similarly, ³H-labeled B chain isolated from ricin also bound in a one-to-one complex with a dissociation constant of 1 μm. The binding of ricin and ricin B chain was sensitive to lactose, while the binding of reduced ricin or ricin A chain was not prevented by lactose. Reduced ¹²⁵I-labeled ricin in the presence of lactose and ³H-labeled A chain bound with a ratio of 2 mol/mol of ribosomes. It was further demonstrated that ³H-labeled ricin A chain bound only to the 60S ribosomal subunit and not to the 40S ribosomal subunit. The dissociation constant for the binding was 2 μm both in the presence and absence of lactose and 2 mol of A chain were bound per mole of 60S ribosomal subunit.     

Isolation of Chinese hamster ovary ribosomal mutants differentially resistant to ricin, abrin, and modeccin

The molecular action of ricin A chain involves cleavage of the N-glycosidic bond between ribose and the adenine 4324 nucleotides from the 5' end of mammalian 28 S rRNA (Endo, Y., and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130). In this paper, four ricin- and abrin-resistant Chinese hamster ovary cell mutants that possess ribosomes resistant to this N-glycosidase action are described. Three of the mutant phenotypes, Lec26, Lec27, and Lec28, were recessive in somatic cell hybrids and define at least two new lectin-resistant complementation groups. The most extensively characterized mutant type, LEC17, was dominant in such hybrids. None of the mutants were cross-resistant to modeccin. Post-mitochondrial supernatants from each of the four mutants were resistant to inhibition of cell-free protein synthesis by ricin, ricin A chain, and abrin. In addition, polysomes isolated from mutant cells were resistant to cleavage of the adenine-ribose N-glycosidic bond by ricin A chain or abrin, as assayed by the release of an approximately 470-nucleotide fragment following aniline treatment of ribosomal RNA extracted from toxin-treated polysomes. The unique lectin-resistance properties of the different mutants suggests that the accessibility of adenine 4324 to each toxin differs. It seems likely that the recessive Chinese hamster ovary ribosomal mutants reflect structural changes in different ribosomal proteins while the dominant phenotype may be due to the modification of protein(s) or rRNA involved in toxin-ribosome interaction. Further analysis of these cell lines should provide new insights into the structure/function relationships of eukaryotic ribosomes.     

Significant role for polysomes associated with the cytoskeleton in the control of protein synthesis during germination of triticale caryopses in the presence of abscisic acid

The influence of abscisic acid (ABA) on the process of polysome formation and synthesis of newly-formed proteins by different polysome populations was studied. Triticale caryopses were germinated in water or various ABA concentrations for 48 hrs, and afterwards they were transferred to a solution of ¹⁴C-amino acids and germinated for an additional 30 min. Embryos were separated from caryopses, and four polysome populations were isolated: the FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). ABA retarded both the process of polysome formation and their activity in forming new proteins in vivo in all studied fractions. Participation of polysomes in total ribosomal materials (sub-units, monosomes and polysomes) of each polysome population in the control sample was as follows: FP — 77; MBP — 72; CBP — 70 and CMBP — 66 %, whereas in sample treated by ABA (100 μM) it was accordingly: 17; 23; 27 and 28%. The largest population made up FP (in control sample 69%), participation of MBP was always lower and ranged from about 19 to 30 %. Participation of polysome populations bound with the cytoskeleton CBP and CMBP, both in control sample as well as in samples treated with 1 and 10 μM ABA solution, was only a few per cent. It should be noted that when the ABA concentration was higher (100 μM) (process of germination was strongly inhibited), participation of those two populations (CBP and CMBP) was much increased in embryos, respectively to about 18 and 20 %. In both the control group and in embryonal tissue treated with ABA increasing incorporation of radioactive precursors to newly-formed proteins in vivo in fractions of polysomes isolated by following buffers: C (FP), C + PTE (MBP), C + Tris (CBP) and buf. U (CMBP) was observed. It should be noted, that the biggest incorporation of ¹⁴C-amino acids into nascent polypeptide chains was found in the last polysome population (CMBP). In the sample treated with ABA (100 μM) the activity of this fraction (CMBP) in forming new proteins is several times, and in the case of FP dozens of times, more intense. Increased participation of CBP and CMBP in embryos of triticale caryopses treated with ABA (100 μM) and the largest incorporation of ¹⁴C-amino acids into nascent polypeptide chains synthesised by CMBP, may indicate the important role of proteins formed by polysomes associated with cytoskeleton in inhibition of germination and seedling growth by ABA.     

Glycosylation of nascent polypeptide chains in Aspergillus niger

Polysomes were isolated from Aspergillus niger and were characterized on sucrose gradients in several ways. First, they were found to be susceptible to degradation by treatment with RNase or EDTA. Second, they were labeled after treating mycelia with short pulses of [³H]uridine or [³H]leucine prior to polysome isolation. Third, they were capable of stimulating incorporation of [³H]leucine into trichloroacetic acid-precipitable material in a chick reticulocyte cell-free protein-synthesizing system. When isolated [³H]leucine pulse-labeled polysomes were treated with either EDTA-RNase or puromycin, 80–90% of the radioactivity was released, indicating that only the nascent polypeptide chains were labeled. After exposing mycelia for 1 min to [¹⁴C]mannose, the polysomes were exclusively labeled, indicating that initial glycosylation takes place on nascent polypeptide chains. Preincubation of mycelia with 2-deoxyglucose followed by pulse-labeling with [³H]leucine and [¹⁴C]mannose showed that 2-deoxy-d-glucose inhibits both protein synthesis and glycosylation. However, similar preincubation with tunicamycin caused an 80% drop in [¹⁴C]mannose label in the polysomes, but only a 10–20% drop of [³H]leucine label, suggesting that glycosylation of nascent chains in A. niger involves an oligosaccharide-lipid intermediate, since it has been shown that tunicamycin inhibits the synthesis of such an intermediate. When isolated polysomes were placed into an in vitro glycosylating mixture containing Mn²⁺, GDP-[¹⁴C]mannose, and smooth membranes from A. niger nascent chains were labeled. This reaction was shown to be dependent on addition of polysomes to the mixture and was not inhibited by 2-deoxy-d-glucose or tunicamycin. Both in vivo and in vitro glycosylated nascent chains were found to have about the same size range, and so it is suggested that in vitro no new oligosaccharide chains were synthesized, but preexisting chains were extended.     

Inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin.

The mechanism of inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin was studied. No significant disaggregation of polysomes into monosomes was detected in the toxin-treated cells. The activity of the polysomes isolated from the cells treated with the toxin in protein synthesis was remarkably lower than that of the untreated cells, while the activity of the supernatant enzyme fraction was retained. The ribosomes derived from the polysomes of the toxin-treated cells were inactive in poly(U)-dependent polyphenylalanine synthesis. The activity of ribosomes reconstituted by hybridizing subunits derived from the ribosomes of normal and toxin-treated cells were measured in poly(U)-dependent polyphenylalanine synthesis, and the 60 S subunit was revealed to be inactive. These results indicate that the target of action of the toxin towards intact cells is the 60 S ribosomal subunit.     

Cytoplasmic distribution of heat shock proteins in soybean

Previous analyses of the distribution of heat shock (hs) proteins in soybean (Glycine max L. Merr., var Wayne) have demonstrated that a fraction of the low molecular weight hs protein associates with ribosomes during hs. To more specifically characterize the nature of this association, isokinetic centrifugation of ribosomes through sucrose gradients was used to separate monosomes from polysomes. The present analysis demonstrated that hs proteins were bound to polysomes but not monosomes. Treatment of polysomes with puromycin, K⁺, and Mg²⁺, which caused dissociation of ribosomes into 40S and 60S subunits, also caused dissociation of the hs proteins. Using the procedure of Nover et al. (1983, Mol. Cell Biol, 3: 1628-1655), a hs granule fraction was also isolated. As in tomato cells, hs granules from soybean seedlings contained the low molecular weight hs proteins as a primary component and a number of other non-hs proteins of relative molecular mass 30 to 40 kilodaltons and 70 to 90 kilodaltons. On metrizamide gradients they exhibited a buoyant density of 1.20 to 1.21 grams per cubic centimeter, typical of ribonucleoprotein particles. Heat shock granules were characterized as unique cytoplasmic particles based on protein composition and buoyant density. Isopycnic centrifugation of ribosome preparations demonstrated that they contained hs granules, but the hs proteins bound to polysomes were not released by KCI/EDTA treatment. Thus, the polysome-bound hs proteins and the granule-bound hs proteins appear to represent two distinct populations of hs proteins in the cytoplasm. Heat shock granules were not distinguishable from ribosomes at the level of resolution used in transmission electron microscopy.     

Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

Treatment of Ltk^?, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk⁺ clones with a frequency of 10^?6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk⁺ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk^? phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10^?3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.     

Action of ribonuclease upon the polysomes of cultured mouse cells

Summary Pancreatic ribonuclease (RNase) and³H-uridine were used to study certain compositional and ontogenetic features of the polysomes of strain L mouse cells. Growing cells were exposed to the radioactive nucleoside,³H-uridine, for brief defined periods, and the sensitivity of the polysomes to digestion by RNase was determined. The RNase-resistant RNA of polysomes is shown to be primarily ribosomal, and the RNase-sensitive material formed during brief pulse labeling studies is largely messenger RNA. Actinomycin D inhibition of RNA synthesis was used to confirm this identification. The technique described here was used to investigate the effects of hydrocortisone on polysome formation. The hormone (10⁻⁶ m) lessens the extent of the nucleoside incorporation into polysomal and total RNA and delays the appearance of newly synthesized 18 S and 28 S rRNA into cytoplasmic polysomes. This work was partially supported by grants from the United States Public Health Service (GM 10866), from the National Science Foundation (GB 13924), and from The University of Kansas General Research Fund.     

Ricin and alpha-sarcin alter the conformation of 60S ribosomal subunits at neighboring but different sites

The effects of ricin and alpha-sarcin separately or in combination on the conformation of rat liver ribosomes were investigated by measuring the relative accessibility of individual ribosomal proteins to N-ethylmaleimide after 80S ribosomes were treated with these toxins. By using a double-labelling technique in which ribosomes were incubated with the toxins and then treated with 3H-labelled or 14C-labelled N-ethylmaleimide, it was found that labelling of protein L14 was specifically reduced by treatment with ricin, and that of proteins L3 and L4 by treatment with alpha-sarcin, suggesting that the toxins alter the conformation of ribosomes in the vicinity of these proteins. When ribosomes were treated with both ricin and alpha-sarcin, the extent of labelling of protein L3 was reduced compared to that observed after treatment with alpha-sarcin alone. These results are discussed in relation to previous observations showing that these three proteins are neighbours in the 60S ribosomal subunit and probably play important roles in protein biosynthesis, and in the actions of ricin and alpha-sarcin on 28S rRNA.     

Ricin A-Chain and Ricin A-Chain Immunotoxins Rapidly Damage Human Endothelial Cells: Implications for Vascular Leak Syndrome

The results of Phase I/II clinical trials indicate that ricin A-chain-containing immunotoxins cause vascular leak syndrome, characterized by hypoalbuminemia with resultant weight gain and edema. Vascular leak syndrome may be a dose-limiting factor during treatment with ricin A-chain-containing immunotoxins. In this report, we determined the effect of ricin A-chain and ricin A-chain-containing immunotoxins on human umbilical vein endothelial cells with the aim of developing an in vitro model to study vascular leak syndrome. The major findings of our study are: (1) Human umbilical vein endothelial cells undergo rapid and dramatic changes in morphology after treatment with ricin A-chain and ricin A-chain-containing immunotoxins. These changes include rounding of the cells and, eventually, the formation of gaps between them. (2) The permeability of human umbilical vein endothelial cell monolayers to passage of molecules increases after exposure to ricin A-chain or ricin A-chain-containing immunotoxins and this is consistent with the morphologic changes. (3) Human umbilical vein endothelial cells bind ¹²⁵ I-rRTA in a dose-dependent manner but binding is not specific. (4) Human umbilical vein endothelial cells are moderately more sensitive to ricin A-chain-induced inhibition of protein synthesis and proliferation than simian virus-transformed mouse endothelial cells. (5) The morphologic changes are observed 1 h after exposure to the toxins, whereas inhibition of protein synthesis is not detectable until 4 h after a similar exposure. The in vitro model represents a first step in dissecting the complex events which occur in cancer patients who develop vascular leak syndrome after treatment with ricin A-chain-containing immunotoxins.