Three-dimensional reconstruction of mammalian 40 S ribosomal subunit.

The small (40 S) subunit from rabbit reticulocyte ribosomes has been reconstructed from electron micrographs of a negatively stained single-particle specimen to a resolution of 3.85 nm. The reconstruction reveals a morphology consisting of a broad wedge-shaped head structure set atop a quasi-cylindrical body. Distinctive features recognized in two-dimensional projections, such as the beak, back lobes, and feet, can now be localized in three dimensions. By reference to a recent reconstruction of the monomeric 80 S ribosome we can identify the interface and exterior surfaces of the subunit, thus enabling more detailed functional interpretations.

Shiga toxin binds to activated platelets.

Hemolytic uremic syndrome (HUS) is associated with acute renal failure in children and can be caused by Shiga toxin (Stx)-producing Escherichia coli. Thrombocytopenia and formation of renal thrombi are characteristic of HUS, suggesting that platelet activation is involved in its pathogenesis. However, whether Shiga toxin directly activates platelets is controversial. The present study evaluates if potential platelet sensitization during isolation by different procedures influences platelet interaction with Shiga toxin. Platelets isolated from sodium citrate anticoagulated blood were exposed during washing to EDTA and higher g forces than platelets prepared from acid-citrate-dextrose (ACD) plasma. Platelet binding of Stx was significantly higher in EDTA-washed preparations relative to ACD-derived platelets. Binding of Stx was also increased with ACD-derived platelets when activated with thrombin (1 U mL-1) and exposure of the Gb3 Stx receptor was detected only on platelets subjected to EDTA, higher g forces or thrombin. EDTA-exposed platelets lost their normal discoid shape and were larger. P-selectin (CD62P) exposure was significantly increased in EDTA-washed preparations relative to ACD-derived platelets, suggesting platelet activation. Taken together, these results suggest that direct binding of Stx occurs only on 'activated' platelets rather than on resting platelets. The ability of Stx to interact with previously activated platelets may be an important element in understanding the pathogenesis of HUS.

Shiga toxin mode of action in E. coli O157:H7 disease.

Shiga toxins (Stx) are virulence factors produced by selected bacteria pathogenic for humans. These multicomponent protein complexes are among the more potent toxins known. As inhibitors of eukaryotic protein synthesis, these toxins selectively inactivate ribosomes in an enzymatic manner. Specificity of cell targeting is determined by the high-affinity binding of Stx to its receptor, a glycosphingolipid (Gb3) located in the plasma membrane or some eukaryotic cells. Elaborated by food-borne E. coli O157:H7 bacteria, isotypes of Stx (Stx1 & Stx2) are required for the ensuing vascular changes in humans, including hemorrhagic colitis and renal hemolytic uremic syndrome. Experimental therapeutic intervention of Stx-associated disease includes the Stx receptor immobilized on biologically inert particles designed for oral presentation.

Toxins that inhibit host protein synthesis.

Methods have been described that are sufficient to determine if a bacterial protein toxin is a selective inhibitor of eukaryotic protein synthesis, and, if so, which part of the overall process is affected. More defined assays are presented for studying the steps of peptide elongation as this is where such toxins have been shown to act.

Human platelet aggregation is not altered by Shiga toxins 1 or 2.

The hemolytic uremic syndrome involves the presence of Shiga toxin producing strains of Escherichia coli and is associated with thrombocytopenia, platelet activation, and microthrombi formation. We have, therefore, investigated the ability of Shiga toxin isotypes 1 and 2 to cause or enhance platelet aggregation under resting or arterial-flow conditions using a sensitive quenched-flow system and single-particle counting. Incubation of platelets with Shiga toxins 1 or 2 at 10(-10) M or 10(-9) M for 0.5-2 hours failed to induce platelet aggregation under static or physiological flow conditions, either by themselves or in the presence of ADP or thrombin. Thus, these Shiga toxins do not appear to be able to influence platelet function directly, and their ability to cause platelet thrombi in vivo must result from indirect mechanisms.

The effect of metals on protein biosynthesis in rabbit reticulocyte lysates and whole cells.

Six metal chlorides were tested as inhibitors of the protein synthesis in reticulocyte lysate systems supplemented with ATP, GTP, hemin and amino acids. At concentrations of less than 10(-4) M, HgCl2 and CuCl2 were effective in reducing initial rates of [3H]leucine incorporation into protein while CdCl2, NiCl2, PbCl2 and ZnCl2 were poor inhibitors. It is concluded that HgCl2 and CuCl2 are potent inhibitors of the process of eukaryotic protein biosynthesis.

Human renal microvascular endothelial cells as a potential target in the development of the hemolytic uremic syndrome as related to fibrinolysis factor expression, in vitro.

Renal glomerular microvascular endothelial cell damage is characteristic of Shiga toxin-associated hemolytic uremic syndrome (HUS). An impaired renal fibrinolysis may be responsible for renal microvascular fibrin accumulation during the course of HUS disease. This study examined the effect of Shiga toxin, bacterial lipopolysaccharide (LPS, endotoxin), and tumor necrosis factor (TNF) on the expression of fibrinolysis factors by human renal glomerular microvascular endothelial cells (HRMEC) in vitro. The results were compared to a previously better-characterized endothelial cell type, human umbilical vein endothelial cells (HUVEC). In HUVEC, the ratio of fibrinolysis antigens was antifibrinolytic, consisting of 55-fold more plasminogen activator inhibitor type 1 (PAI-1) than tissue-type plasminogen activator (tPA). Treatment of HUVEC with LPS or TNF accentuated this ratio by decreasing tPA and increasing PAI-1 expression. In contrast, HRMEC produced urokinase-type plasminogen activator (uPA) in a 24-fold excess to PAI-1 and were thereby profibrinolytic with regard to fibrinolysis antigen expression. LPS and TNF further decreased PAI-1 antigen expression by HRMEC. These results argue against a role for LPS or TNF in decreasing renal fibrinolysis at the level of fibrinolysis factor expression by renal endothelial cells. Nevertheless, HUVEC and HRMEC were responsive to the same LPS analogs in the same order of potency. Shiga toxin decreased fibrinolysis factor expression to a greater extent in HRMEC than in HUVEC. Since HRMEC fibrinolysis antigen expression was profibrinolytic, the Shiga toxin-mediated decrease in renal endothelial uPA synthesis may predispose renal microvasculature to thrombosis and may have implications for the development of HUS.

Shiga toxin-associated hemolytic uremic syndrome: combined cytotoxic effects of shiga toxin and lipopolysaccharide (endotoxin) on human vascular endothelial cells in vitro.

This study explores the in vitro relationship between Shiga toxin-producing Shigella spp. and Escherichia coli and the development of vascular complications in humans following bacillary dysentery. We propose that lipopolysaccharide (LPS; endotoxin) may combine with Shiga toxin to facilitate vascular damage characteristic of hemolytic uremic syndrome. We have examined the direct cytotoxic effects of Shiga toxin and LPS on human umbilical vein endothelial cells (HUVEC) in culture. Shiga toxin alone was cytotoxic to HUVEC, whereas LPS was noncytotoxic at concentrations at or below 10 micrograms/ml. Combinations of LPS with Shiga toxin resulted in a synergistic cytotoxic effect. The synergistic cytotoxic response of HUVEC to Shiga toxin plus LPS was dose dependent for both agents and was maximal at 24 h of exposure. This synergistic response was enhanced by preincubation of HUVEC with LPS. LPS (1 micrograms/ml) alone depressed HUVEC protein synthesis in a transient manner and enhanced the protein synthesis-inhibiting activity of Shiga toxin. The synergistic cytotoxic activity of LPS analogs was as follows, in decreasing order: complete LPS = diphosphoryl lipid A greater than monophosphoryl lipid A greater than deacylated LPS. These results are consistent with a role for Shiga toxin and LPS in the development of hemolytic uremic syndrome at the level of the vascular endothelium in humans.

Shiga toxin-associated hemolytic-uremic syndrome: combined cytotoxic effects of Shiga toxin, interleukin-1 beta, and tumor necrosis factor alpha on human vascular endothelial cells in vitro.

This study explores the relationship between Shiga toxin-producing Shigella or Escherichia coli strains and the development of vascular complications in humans following bacillary dysentery. We propose that endotoxin-elicited interleukin-1 or tumor necrosis factor alpha (TNF) may combine with Shiga toxin to facilitate vascular damage characteristic of hemolytic-uremic syndrome. This study examines the cytotoxic effects of Shiga toxin, interleukin-1, and TNF on cultured human umbilical vein endothelial cells (HUVEC). Both Shiga toxin and TNF were cytotoxic to HUVEC, although HUVEC obtained from individual umbilical cords differed in their sensitivities to these agents. With Shiga toxin-sensitive HUVEC, combinations of TNF with Shiga toxin resulted in a synergistic cytotoxic effect. In contrast, interleukin-1 was not cytotoxic to HUVEC, nor did it enhance cell death in combination with Shiga toxin. The synergistic cytotoxic response of HUVEC to Shiga toxin and TNF was dose and time dependent for both agents and could be neutralized by monoclonal antibodies directed against either Shiga toxin or TNF. This synergistic response was delayed, being maximal on day 2. Preincubation (24 h) of HUVEC with TNF sensitized the cells to Shiga toxin. TNF alone had no effect on HUVEC protein synthesis but enhanced the inhibitory activity of Shiga toxin. These results are consistent with a role for Shiga toxin in the development of hemolytic-uremic syndrome at the level of the vascular endothelium in humans.

Specific interaction of Escherichia coli O157:H7-derived Shiga-like toxin II with human renal endothelial cells.

In Escherichia coli O157:H7 foodborne infections of humans, the Shiga-like toxins (SLTs) are thought to be the cause of life-threatening vascular complications, including acute renal disease known as hemolytic uremic syndrome or HUS. As virtually all E. coli O157:H7 isolates from HUS patients produce SLT-II (vs. SLT-I), the possible preferential interaction of SLT-II with human renal microvascular endothelial cells (HRMEC), the putative target of the SLTs in the development of HUS, was studied. SLT-II was 1000 times more potent a cytotoxic agent than SLT-I toward HRMEC. Toxin binding studies showed that this occurred although HRMEC could bind 10 times more SLT-I than SLT-II. This preferential action of SLT-II was specific for renal endothelial cells, as human umbilical vein endothelial cells were almost equally affected by SLT-I and SLT-II.

In vitro protein synthesis and aging in Rhizoctania solani.

A study was made of the ability of cell-free protein synthesis systems from vegetative cells of different age of the fungus Rhizoctonia solani to produce polyphenylalanine. Polyuridylic acid-directed phenylalanine incorporation into peptides decreased linearly with cell age. The 105,000 x g supernatant fluid and ribosomal fractions were equally responsible for the total loss of synthetic activity of the older cells. Initial rates of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase activity decreased with increasing cell age, which accounted for the defect of the supernatant fraction. An accelerated degradation of soluble phenylalanyl-RNA was associated with the ribosomes of the older cells. In vitro systems from cells of different age transferred phenylalanine from phenylalanyl-tRNA to polyphenylalanine at similar rates. Of the 15 specific aminoacyl-tRNA synthetases assayed, 5 increased and 5 decreased in specific activity with increased age; 3 others did not change during aging and 2 were below acceptable detectable levels.

Effect of chartreusin on macromolecular synthesis.

Chartreusin (CT), a natural compound derived from Streptomyces chartreusis which exhibits antitumor activity, was demonstrated to be an equally potent inhibitor of DNA and RNA synthesis in cultured human KB cells and in isolated nuclei. Polymerization of nucleic acids in nuclei prepared from adenovirus-infected KB cells responded to CT in a similar manner. In contrast, protein biosynthesis was not inhibited by up to 30 microgram/ml CT in whole KB and rabbit reticulocyte cells as well as in their cell free lysates.

Recent advances in understanding the pathogenesis of the hemolytic uremic syndromes.

One of the requirements for an agent to cause hemolytic uremic syndrome (HUS) is its ability to injure endothelial cells. Shiga-like toxin (SLT) can do this. SLT is produced by Escherichia coli and Shigella dysenteriae serotype 1; both have been implicated as causes of typical HUS. Endothelial cells have receptors (GB3) for SLT and the toxin can inhibit eukaryotic protein synthesis, thereby causing cell death. Glomerular endothelial cell injury or death results in a decreased glomerular filtration rate and many of the perturbations seen in HUS. It is no longer certain that hemolysis is the result of a microangiopathy. Cell injury results in release of von Willebrand multimers; if these are ultra-large, thrombosis may ensue. There is also increasing evidence that neutrophils have a role in the pathogenesis of typical HUS. Streptococcus pneumoniae can also cause HUS and care must be taken to avoid giving plasma to patients with S. pneumoniae-associated HUS. There is compelling evidence that types of HUS are inherited by autosomal recessive and autosomal dominant modes. Patients with autosomal recessive HUS may have recurrent episodes. Mortality and morbidity rates are high for the inherited forms.

Ribonuclease activity associated with the 60S ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin.

All purified preparations of the ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin were shown to exhibit ribonuclease activity with 5S or 5.8S rRNA substrates. These toxin species generated reproducible patterns of RNA fragments distinct for each toxin species while multiple preparations of a single toxin species yielded similar RNA fragment patterns. The heat inactivation profile of Shiga toxin was identical for its RNase and protein synthesis inhibitory activities. These data are the first to indicate that the ribosome-inactivating catalytic toxins, in addition to alpha-sarcin, exhibit RNase activity. These results suggest RNase activity may be responsible for ribosome-inactivation catalyzed by ricin, phytolaccin and Shiga toxin proteins.

The mode of action of Shiga toxin on peptide elongation of eukaryotic protein synthesis.

The effect of Shiga toxin, from Shigella dysenteriae 1, on the component reactions of peptide elongation were investigated. Enzymic binding of [3H]phenylalanine-tRNA to reticulocyte ribosomes was inhibited by 50% at 7 nM toxin. Elongation factor 1 (eEF-1)-dependent GTPase activity was also inhibited. Both reactions were not restored by addition of excess eEF-1 protein. In contrast, toxin concentrations of 200 nM were required to inhibit by 50% the elongation factor 2 (eEF-2)-dependent translocation of aminoacyl-tRNA on ribosomes. Addition of excess eEF-2 restored translocation activity. The eEF-2-dependent GTPase activity was unaffected at toxin concentrations below 100 nM, and Shiga-toxin concentrations of up to 1,000 nM did not affect either GTP.eEF-2.ribosome complex-formation or peptidyltransferase activity. Thus Shiga toxin closely resembles alpha-sarcin in action, both being primary inhibitors of eEF-1-dependent reactions. In contrast, the 60 S ribosome inactivators ricin and phytolaccin are primary inhibitors of eEF-2-dependent reactions of peptide elongation.

Sensitization of human umbilical vein endothelial cells to Shiga toxin: involvement of protein kinase C and NF-kappaB.

Infection of humans with Shiga toxin-producing Escherichia coli O157:H7 and Shigella dysenteriae 1 is strongly associated with vascular endothelial cell damage and the development of hemolytic-uremic syndrome. The cytotoxic effect of Shiga toxins on vascular endothelial cells in vitro is enhanced by prior exposure to bacterial lipopolysaccharide (LPS) or either of the host cytokines tumor necrosis factor alpha (TNF) and interleukin-1beta (IL-1). The purpose of this study was to examine individual signal transduction components involved in the sensitization of human umbilical vein endothelial cells (HUVEC) to Shiga toxin 1. The results demonstrate that class I and II protein kinase C (PKC) isozymes are required for sensitization of HUVEC to Shiga toxin by phorbol myristate acetate (PMA) or LPS but not by TNF or IL-1. Thus, the specific competitive inhibitor of class I/II PKC, 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG), prevented only the action of PMA and LPS on HUVEC. Additional data obtained with ATP binding site inhibitors which affect all PKCs (i.e., classes I, II, and III) suggest that TNF may utilize class III PKC isozymes in the Shiga toxin sensitization of HUVEC. Transcriptional activator NF-kappaB did not appear to be involved in the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA. Thus, the specific serine protease inhibitor L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) did not inhibit the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA despite its ability to inhibit NF-kappaB activation and the induction of the NF-kappaB-dependent tissue factor gene by these agents. Finally, all-trans retinoic acid partially inhibited the sensitization of HUVEC to Shiga toxin, by unknown mechanisms which also appeared to be independent of NF-kappaB activation. These results indicate that PKC plays a role in the sensitization of HUVEC to Shiga toxin in response to some, but not all, sensitizing agents. In contrast, NF-kappaB activation appears not to be involved in the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA.

Shiga toxin-associated hemolytic uremic syndrome: interleukin-1 beta enhancement of Shiga toxin cytotoxicity toward human vascular endothelial cells in vitro.

Development of hemolytic uremic syndrome (HUS) after infection by Shigella dysenteriae 1 or enterohemorrhagic Escherichia coli has been associated with the production of Shiga toxins (verotoxins). The putative target of Shiga toxins in HUS is the renal microvascular endothelium. This report shows that preincubation of human umbilical vein endothelial cells (HUVEC) with interleukin-1 beta (IL-1 beta) enhances the cytotoxic potency of Shiga toxin toward HUVEC. A preincubation of HUVEC with IL-1 beta is required for sensitization of HUVEC to Shiga toxin. Sensitization of HUVEC to Shiga toxin is IL-1 beta dose dependent. Development of the IL-1 beta response is time dependent, beginning within 2 h of IL-1 beta preincubation and increasing over the next 24 h. That these responses were due to IL-1 beta was demonstrated by heat inactivation of IL-1 beta, by neutralization of IL-1 beta by specific antibody, and by the ability of an IL-1 beta receptor antagonist to inhibit the effect of IL-1 beta. Shiga toxin-related inhibition of HUVEC protein synthesis preceded loss of cell viability. IL-1 beta incubation with HUVEC induced the receptor for Shiga toxin, globotriaosylceramide. Lipopolysaccharide included during IL-1 beta preincubation with HUVEC increased sensitivity to Shiga toxin in an additive manner. We conclude that IL-1 beta may induce Shiga toxin sensitivity in endothelial cells and contribute to the development of HUS.