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Biomedical subjects

M Thelestam

Publications and source records attributed to M Thelestam.

At least 37 records · Page 2Linked to original sources
PubMed1997

Staphylococcus aureus alpha-toxin: characterization of protein/lipid interactions, 2D crystallization on lipid monolayers, and 3D structure.

Staphylococcus aureus alpha-toxin was characterized with respect to surface activity and its interaction with lipid monolayers. The protein alone had a detergent-like behavior at the air/water interface. Its affinity was higher for negatively charged than for neutral phospholipids. The interaction was pH dependent, showing a maximum increase at pH 7.0. Only a small part of the protein oligomer appeared to be inserted into the monolayers. Crystalline sheets of alpha-toxin were formed using negatively charged phospholipids. Electron microscopy of such areas, at different tilt angles, allowed reconstruction of a three-dimensional model following image processing. The sheets analyzed consisted of two protein layers arranged on a tetragonal lattice. Under the conditions used to grow the crystals the toxin formed 90-A-wide cylinders with a height of 70 A. One of the imposed fourfold axes running perpendicular to the plane of the crystalline layer is positioned at a protein-deficient region which forms a 25-A-wide pore through the oligomer.

Air↗
PubMed1997

Development of immunoassays for determination of circulating venom antigens during envenomations by coral snakes (Micrurus species).

A reverse agglutination assay and two capture enzyme-linked immunoassays (ELISAs) for the quantitative determination of Micrurus nigrocinctus nigrocinctus venom antigens in fluids were developed using affinity-purified polyclonal antibodies and a cocktail of three monoclonal antibodies. The lower detection limit was 0.3 mg/ml for the reverse agglutination assay and 4 ng/ml for the capture ELISAs. The optical densities of both ELISAs correlated very well with venom concentrations in the range 4-333 ng/ml (r = 0.99). The ability of these assays to detect venoms of several medically important Micrurus species was studied. Besides detecting homologous venom, both ELISAs were also useful to quantitate venom from M. fulvius, M. dumerilii carinicauda and M. alleni. Using biotinylated polyclonal antibodies, M. n. nigrocinctus venom antigens were detected in sera or plasma from rabbits and mice during experimental envenomations with lethal and sublethal venom doses. The assays described in this work are promising tests to estimate the severity of poisoning in envenomations by the most important coral snakes of North and Central America.

Agglutination Tests↗
PubMed1996

Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation.

Lethal toxin (LT) from Clostridium sordellii is one of the high molecular mass clostridial cytotoxins. On cultured cells, it causes a rounding of cell bodies and a disruption of actin stress fibers. We demonstrate that LT is a glucosyltransferase that uses UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in vitro and in vivo. LT glucosylates Ras, Rap, and Rac. In Ras, threonine at position 35 was identified as the target amino acid glucosylated by LT. Other related members of the Ras GTPase superfamily, including RhoA, Cdc42, and Rab6, were not modified by LT. Incubation of serum-starved Swiss 3T3 cells with LT prevents the epidermal growth factor-induced phosphorylation of mitogen-activated protein kinases ERK1 and ERK2, indicating that the toxin blocks Ras function in vivo. We also demonstrate that LT acts inside the cell and that the glucosylation reaction is required to observe its dramatic effect on cell morphology. LT is thus a powerful tool to inhibit Ras function in vivo.

3T3 Cells↗
PubMed1996

UDP-glucose deficiency in a mutant cell line protects against glucosyltransferase toxins from Clostridium difficile and Clostridium sordellii.

We have previously isolated a fibroblast mutant cell with high resistance to the two Rho-modifying glucosyltransferase toxins A and B of Clostridium difficile. We demonstrate here a low level of UDP-glucose in the mutant, which explains its toxin resistance since: (i) to obtain a detectable toxin B-mediated Rho modification in lysates of mutant cells, addition of UDP-glucose was required, and it promoted the Rho modification dose-dependently; (ii) high pressure liquid chromatography analysis of nucleotide extracts of cells indicated that the level of UDP-glucose in the mutant (0.8 nmol/10(6) cells) was lower than in the wild type (3.7 nmol/10(6) cells); and (iii) sensitivity to toxin B was restored upon microinjection of UDP-glucose. Using the mutant as indicator cell we also found that the related Clostridium sordellii lethal toxin is a glucosyltransferase which requires UDP-glucose as a cofactor. Like toxin B it glucosylated 21-23-kDa proteins in cell lysates, but Rho was not a substrate for lethal toxin.

Animals↗
PubMed1996

Characterization of multiple nicotinic acetylcholine receptor-binding proteins and phospholipases A2 from the venom of the coral snake Micrurus nigrocinctus.

The presence of multiple nicotinic acetylcholine receptor (AchR)-binding proteins and phospholipases A2 was detected in the venom of a member of the Elapinae subfamily, Micrurus nigrocinctus nigrocinctus. Multi-step chromatographies were used to isolate four AchR-binding proteins (Mnn-9, Mnn-4, Mnn-3C and Mnn-1A) and five basic PLA2s (nigroxins A, B, C1, C2 and C3). The Micrurus AchR-binding proteins are antigenically and structurally related to short- and long-chain alpha-neurotoxins from Naja. The nigroxins are antigenically similar and constitute a new antigenic subclass of PLA(2)s. Nigroxins A and B are class I PLA(2)s, structurally more related to enzymes from Bungarinae than to those from Hydrophinae/Laticaudinae. These data contribute to clarify the relationships between Micrurus venom proteins and other elapid toxins and may be useful to improve the neutralizing efficiency of antivenoms.

Amino Acid Sequence↗
PubMed1996

Large clostridial cytotoxins--a family of glycosyltransferases modifying small GTP-binding proteins.

Some Clostridium species produce ABX-type protein cytotoxins of high molecular weight. These toxins constitute the group of large clostridial cytotoxins (LCTs), which have homologous protein sequences, exert glycosyltransferase activity and modify GTP-binding proteins of the Ras-superfamily. These characteristics render the LCTs valuable tools for developmental and cell biologists.

Bacterial Toxins↗
PubMed1994

Immunochemical characterization of Micrurus nigrocinctus nigrocinctus venom with monoclonal and polyclonal antibodies.

Eleven murine monoclonal antibodies (MAbs) against Micrurus nigrocinctus nigrocinctus venom were produced and partially characterized. When M. n. nigrocinctus venom proteins were separated by SDS-PAGE under non-reducing conditions four sharp and three diffuse bands were observed. The sharp bands had migration rates comparable to reduced standards of 10, 12, 50 and 72 kDa. The diffuse bands migrate in the range of reduced standards from 14.5 to 32 kDa. When venom proteins were separated under reducing conditions the same sharp bands and an additional prominent 14.5 kDa band were observed. Three antibodies (MAbs 4, 21 and 28) recognized the diffuse bands in western blots of non-reducing SDS-PAGE, whereas MAbs 7G, 22 and 26 reacted with only the 72 kDa protein. MAbs 21 and 28 reacted with the 14.5 kDa band whereas MAb 7G recognized the 72 kDa band in blots of reducing SDS-PAGE. Two M. nigrocinctus antivenoms cross-reacted by ELISA against nine neurotoxic snake venoms, as well as with gamma-toxin from Naja nigricollis and notexin. One antibody (MAb 9A) was used to affinity purify a fraction (called nigroxin) from M. n. nigrocinctus venom. Nigroxin showed phospholipase and myotoxic activities and appeared as a single 15 kDa band in SDS-PAGE under reducing conditions. However, three bands with slight differences in charge were resolved by urea-PAGE, representing isoforms named nigroxin a, b, and c. Nigroxin induced a dose-dependent release of peroxidase trapped in negatively charged liposomes. Nigroxin induced myonecrosis and increased the plasma creatine kinase levels in mice, when injected intramuscularly. The plasma membrane of cultured L6 myoblasts was permeabilized by nigroxin, as evidenced by the release of 3H-uridine nucleotides from prelabelled cells. This effect was completely abolished after preincubation with MAb 9A, although this antibody failed to neutralize the enzymatic activity of nigroxin. Nigroxin was also recognized by MAbs 4, 7H, 21, 27 and 28. Additionally, the epitope recognized by MAb 27 is also present in notexin and beta-bungarotoxin.

Animals↗
PubMed1994

Electrophoretic and immunochemical studies of Micrurus snake venoms.

The electrophoretic mobilities of venom components from 15 Micrurus species were studied by polyacrylamide gel electrophoresis. The venoms showed species-specific protein patterns under native (PAGE) or denaturing (urea-PAGE) conditions. However, electrophoretic patterns obtained by SDS-PAGE under reducing conditions were similar. The proteins of all venoms had mol. wts either in the range of 45 to 75 kDa or lower than 14.5 kDa. PAGE and urea-PAGE of single extraction venom samples from 22 M. nigrocinctus nigrocinctus specimens revealed some proteins completely conserved, whereas others exhibited intraspecies variation. Based on ELISA cross-reactivity studies with 11 monoclonal antibodies against M. n. nigrocinctus venom, venoms from M. n. nigrocinctus, M. nigrocinctus mosquitensis, M. fulvius fulvius, M. dumerilii carnicauda and M. albicinctus were included in the same antigenic group, whereas M. frontalis frontalis and M. frontalis braziliensis venoms constituted a second group. Micrurus alleni and M. spixii spixii showed reactivity patterns similar to groups 1 and 2, respectively. Venoms from M. surinamensis surinamensis, M. corallinus, M. ibiboboca, M. hemiprichii ortoni, M. lemniscatus helleri and M. mipartitus had unique cross-reactivity patterns with monoclonal antibodies against M. n. nigrocinctus venom.

Animals↗
PubMed1993

The projection structure of perfringolysin O (Clostridium perfringens theta-toxin).

The cytolysin Perfringolysin O was applied to lipid layers and the obtained ring-shaped oligomers analyzed by electron microscopy and image processing. The final result shows the periodic repeat of 2.4 nm along the outer rim of the ring. The asymmetric protein unit, corresponding to one monomer, spans the ring from the convex to the concave surface. It shows a clear protein peak close to the outer radius and less density in the middle of the oligomer. The number of monomers in the average ring is 50, and the inner radius of the aggregate is approximately 15 nm.

Bacterial Toxins↗
PubMed1993

Signal transduction pathways and cellular intoxication with Clostridium difficile toxins.

In cultured cells the cytopathic effects (CPE) of Clostridium difficile toxins A and B are superficially similar. The irreversible CPEs involve a reorganization of the cytoskeleton, but the molecular details of the mechanism(s) of action are unknown. As part of the work to elucidate the events leading to the CPE, cultured cells were preincubated with agents known to either stimulate or inhibit some major signal transduction pathways, whereupon toxin was added and the development of the CPE was followed. Both toxin-induced CPEs were enhanced by phorbol esters and mezerein, which stimulate protein kinase C, while they were inhibited by the phospholipase A2 inhibitors quinacrine and 4-bromophenacylbromide. Agents affecting certain G-proteins, cGMP and cAMP levels, phosphatases, prostacyclin, lipoxygenase, and phospholipase C did not affect the development of the CPE of either toxin. Thus, the cytoskeletal effect induced by toxins A or B appears to require PLA2 activity and involves at least part of a protein kinase C-dependent pathway, but not pertussis toxin-sensitive G-proteins, cyclic nucleotides, eicosanoid metabolites, or phospholipase C activity. In addition, both toxins were shown to activate phospholipase A2.

Bacterial Proteins↗
PubMed1993

Activation of cellular phospholipase A2 by Clostridium difficile toxin B.

C. difficile toxin B is a potent cytotoxin known to disrupt the microfilaments of cultured cells. We have recently shown also increased phospholipase A2 activity in cells treated with toxin B. The activity was detected as a toxin-induced, dose-dependent release of 14C-arachidonic acid from prelabeled fibroblasts. Here is shown that the toxin elicited a 14C-arachidonic acid release in a cell mutant resistant to the toxin B effect on the microfilaments. The toxin-induced release was further characterized using fibroblasts. Within 20 min high doses of toxin B (6 micrograms/ml) elicited a release which increased exponentially with time. Of the major membrane phospholipids the lipase activity affected mainly phosphatidyl ethanolamine. Neither cycloheximide nor pertussis toxin treatment or target cells inhibited the toxin-induced release, while it could be increased with 12-O-tetradecanoylphorbol-13-acetate. Our results also suggest a toxin-mediated increase in phospholipase C activity occurring at a later stage than the phospholipase A2 activation. We conclude that the ability of toxin B to induce phospholipase activation represents a hitherto unrecognized toxin B effect which is neither a cause nor a consequence of toxin-induced microfilament disorganization.

Animals↗
PubMed1993

Dithiothreitol generates an activated 250,000 mol. wt form of Clostridium difficile toxin B.

The potent cytotoxin of Clostridium difficile, toxin B, is internalized by endocytosis and activated intracellularly by an unidentified mechanism. Here it is shown that dithiothreitol treatment of toxin B resulted in (1) a mol. wt of 250,000 which is the smallest species of this toxin shown to be cytotoxic; (2) an increased endpoint titre; and (3) translocation of plasma membrane-bound toxin across the membrane at pH 4.5. Treatment with dithiothreitol can thus mimic intracellular activation of the toxin. Radiolabelling of highly purified toxin with retained activity, as well as the 32 N-terminal amino acids and the amino acid composition, is also presented.

Amino Acid Sequence↗
PubMed1993

Effects of Bothrops asper (terciopelo) myotoxin III, a basic phospholipase A2, on liposomes and mouse gastrocnemius muscle.

The action of Bothrops asper myotoxin III, a basic phospholipase A2 which induces acute muscle damage, was studied in mouse gastrocnemius muscle in vivo and in vitro and in multilamellar liposomes. Myonecrosis occurred rapidly after myotoxin injection, as indicated by histological alterations and by a drastic increment in plasma creatine kinase levels. A dose-dependent release of creatine kinase from mouse gastrocnemius muscle incubated in vitro with the toxin was observed. Myotoxin III affected negatively charged multilamellar liposomes, as evidenced by the release of peroxidase trapped in the vesicles. In contrast, very little effect was observed on positively charged vesicles. When gastrocnemius muscle or liposomes were incubated at 4 degrees C there was no membrane-disruptive effect. Inhibition of phospholipase A2 activity, by elimination of calcium and addition of EDTA, resulted in a significant, but not total, reduction in both muscle-damaging and liposomal disrupting effects. It is proposed that B. asper myotoxin III affects cellular and artificial membranes inducing prominent alterations in membrane permeability to ions and macromolecules. Membrane-disrupting activity is probably related to a molecular region different from the catalytic site, although enzymatic activity greatly enhances myotoxin action.

Animals↗
PubMed1992

Effect of Bothrops asper (Fer-De-Lance) snake venom on erythrocyte membrane. A comparative study.

1. The effect of Bothrops asper venom on erythrocytes of different species, sheep erythrocyte ghost vesicles and liposomes made of phospholipids from sheep erythrocytes was studied. 2. B. asper venom had a direct hemolytic effect on mouse erythrocytes, but no lytic activity on goat, rabbit, horse, toad and sheep erythrocytes. Human erythrocytes were lysed only in the presence of bovine serum albumin and Ca2+. 3. Although the phospholipase A2 activity in the venom might be involved in the lytic effect on mouse erythrocytes, there is also evidence of direct lytic factor(s) acting in the absence of calcium ions. 4. Sheep erythrocytes were modified in order to study the basis of their resistance. Enhancement of membrane fluidity and variation of pH were of no consequence for the resistance of sheep erythrocytes to the action of venom. 5. The reorganization of membranes that takes place during ghost or liposome preparation made them susceptible to B. asper venom-induced disruption. Thus the resistance of sheep erythrocytes seems related to the accessibility of the target.

Animals↗
PubMed1992

Oligomer formation of staphylococcal alpha-toxin analyzed by electron microscopy and image processing.

The 12S oligomeric form of Staphylococcus aureus alpha-toxin has been studied with electron microscopy after incubation of the toxin with membrane preparations or liposomes. The target material originated from human platelets. Different electron microscopic preparation techniques were used including negative staining, freeze-fracture and vitrification in liquid ethane. Analysis of micrographs with image processing methods revealed two groups of ring-like structures corresponding to alpha-toxin oligomers. One form measured 75 A in diameter and had a high stain density in the central protein deficient part while the other was larger with a diameter of 100 A and less stain accumulation in the center. The conditions under which the latter were formed suggest that this corresponds to an inactive loosely-bound form of the toxin. The high stain density in the smaller particle is consistent with the presence of a penetrating pore in this structure.

Bacterial Toxins↗
PubMed1992

Clostridium difficile toxin A induces multinucleation in the human leukemic T cell line JURKAT.

Clostridium difficile toxin A is a cytotoxic enterotoxin known to be active on all mammalian cell lines tested up to now. It induces a disruption of the cytoskeleton, particularly the microfilament system, leading to inhibition of cell proliferation. Here, we describe some effects of toxin A on the leukemic T cell line JURKAT. Cells exposed to the toxin did not divide, as cell numbers remained constant for 3 days in the presence of 0.5 to 1.0 micrograms/ml of the toxin. However, these cells were found to become multinucleated, a phenomenon which was time- and dose-dependent. After treatment for 72 h with 0.5 micrograms/ml toxin A, 95% of the cells were multinucleated and had a considerably increased cell diameter. These effects in JURKAT cells were partially reversible upon removal of the toxin within 12 h after the beginning of toxin exposure, but irreversible after 24 h of toxin treatment. These results suggest a continuing nuclear division in the absence of cytoplasmic division, i.e., an effect of toxin A on contractile ring formation. The JURKAT cell is the first cell type reported to respond to toxin A with multinucleation.

Bacterial Toxins↗