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Biomedical subjects

I Just

Publications and source records attributed to I Just.

At least 73 records · Page 4Linked to original sources
PubMed1994

Interaction of Clostridium botulinum C2 toxin with lipid bilayer membranes. Formation of cation-selective channels and inhibition of channel function by chloroquine.

Lipid bilayer experiments were performed with the C2-II binding component of the ADP-ribosylating C2 toxin from Clostridium botulinum. The trypsin-activated but not the nonactivated form of the protein was able to increase the specific conductance of artificial lipid bilayer membranes by the formation of ion-permeable channels. The channels had on average a single-channel conductance of 55 pS in 0.1 M KCl and were found to be cation-selective and voltage-dependent. The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated point charge effects on the channel properties. Incubation of the activated C2-II binding component with antibodies against C2-II or with C2-I toxin inhibited channel formation to a large extent. Addition of chloroquine, a known inhibitor of endocytosis in cells, led to a dose-dependent decrease of the C2-II-induced membrane conductance. This result suggested that the activated C2-II component contains a binding site for chloroquine inside the channel. It is discussed that the channels formed by C2-II component are involved in the translocation of C2-I toxin across the target cell membrane.

Alkanes↗
PubMed1994

Cysteine-specific ADP-ribosylation of actin.

Incubation of lysate from human polymorphonucleated neutrophils and human platelets with [32P]NAD resulted in the labeling of a 42-kDa protein. Phosphodiesterase (Crotalus durissus) released 5'-AMP from the radiolabeled protein. The 42-kDa protein was identified as actin by binding to DNAse-I, two-dimensional gel electrophoresis and partial proteolysis. The rate of ADP-ribosylation was greater with [32P]ADP-ribose than with [32P]NAD, indicating a non-enzymic modification. ADP-ribose also modified actin in the actin-DNAase-I complex, but denatured actin was not modified by ADP-ribose. Only cytoplasmic beta/gamma-actin isoforms were non-enzymically ADP-ribosylated but not muscle alpha-actin. The acceptor amino acid was identified as a cysteine residue whereas the bacterial ADP-ribosyltransferase C. perfringens iota toxin catalyzes incorporation of ADP-ribose to Arg177 of actin. Alkylation of cysteine residues of actin with N-ethylmaleimide prevented subsequent non-enzymic ADP-ribosylation but not the toxin catalyzed modification. Non-enzymically ADP-ribosylated actin was further modified by C. perfringens iota toxin. The F-actin stabilizing mycotoxin phalloidin blocked the non-enzymatic ADP-ribosylation and, conversely, ADP-ribosylation inhibited the phalloidin-induced polymerization of ADP-ribosylated actin. The data indicate that cytoplasmic actin is non-enzymically ADP-ribosylated by ADP-ribose at a cysteine residue to inhibit actin polymerization.

Actins↗
PubMed1994

Clostridium difficile toxin B acts on the GTP-binding protein Rho.

Clostridium difficile toxin B exhibits cytotoxic activity that is characterized by the disruption of the microfilamental cytoskeleton. Here we studied whether the GTP-binding Rho protein, which reportedly participates in the regulation of the actin cytoskeleton, is involved in the toxin action. Toxin B treatment of Chinese hamster ovary cells reveals a time- and concentration-dependent decrease in the ADP-ribosylation of Rho by Clostridium botulinum C3 exoenzyme in the cell lysate. Disruption of the microfilament system induced by C. botulinum C2 toxin or cytochalasin D does not cause impaired ADP-ribosylation of Rho. Toxin B exhibits its effects on Rho not only in intact cells but also when added to cell lysates. Besides endogenous Rho, RhoA-glutathione S-transferase (Rho-GST) fusion protein added to cell lysate showed decreased ADP-ribosylation after toxin B treatment. Immunoblot analysis reveals identical amounts of Rho-GST and no change in molecular mass after toxin B treatment compared with controls. ADP-ribosylation of Rho-GST purified from toxin B-treated cell lysate is inhibited, indicating a modification of Rho itself. Finally, transfection of rhoA DNA under the control of a strong promoter into cells protects them from the activity of toxin B. Altogether, the data indicate that C. difficile toxin B acts directly or indirectly on Rho proteins to inhibit ADP-ribosylation and suggest that the cytotoxic effect of toxin B involves Rho.

3T3 Cells↗
PubMed1994

Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets.

To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time- and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by approximately 80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G- and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of microfilaments with C2 toxin, most notably during the first phase. This effect was, however, diminished, and the second phase became slightly inhibited when the islets were degranulated. These results indicate an important role for AFs in insulin secretion. In the poorly granulated HIT-T15 cells actin-myosin interactions may participate in the recruitment of secretory granules to the releasable pool. In native islet beta-cells the predominant function of AFs appears to be the limitation of the access of granules to the plasma membrane.

Actin Cytoskeleton↗
PubMed1994

Probing the action of Clostridium difficile toxin B in Xenopus laevis oocytes.

Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme caused comparable morphological alteration of CHO cells, which was accompanied by disaggregation of the microfilamental cytoskeleton. The cytotoxic effect of toxin B was correlated with a decrease in C3-catalyzed ADP-ribosylation of the low-molecular-mass GTP-binding protein Rho, which is involved in the regulation of the actin cytoskeleton. We used Xenopus laevis oocytes as a model to study the toxin effect on Rho in more detail. Toxin B treatment of oocytes caused a decrease in subsequent ADP-ribosylation of cytoplasmic Rho by C3. This decrease was observed when toxin B was applied externally or after microinjection. Besides endogenous Rho, microinjected recombinant Rho-glutathione S-transferase fusion protein was affected. Impaired ADP-ribosylation of Rho was neither due to altered guanine nucleotide binding nor to complexation with the guanine nucleotide dissociation inhibitor, which is known to inactivate Rho and to prevent Rho modification by C3. Proteolytical degradation of Rho was excluded by immunoblot analysis. In intact oocytes toxin B caused neither ADP-ribosylation nor phosphorylation of Rho. The data indicate that C. difficile toxin B acts on Rho proteins in Xenopus oocytes to inhibit ADP-ribosylation by C3. It is suggested that toxin B mediates its cytotoxic effect via functional inactivation of Rho.

ADP Ribose Transferases↗
PubMed1994

Adenine nucleotides regulate ADP-ribosylation of membrane-bound actin and actin-binding to membranes.

Adenine nucleotides were shown to increase the ADP-ribosylation of actin by Clostridium perfringens iota and Clostridium botulinum C2 toxin in membranes from human platelets, hamster fat cells, rat liver- and rat brain cells. ATP and ATP gamma S were the most effective agents with ATP showing half-maximal and maximal effects at about 2 and 10 microM, respectively. The rank order of various adenine nucleotides were ATP = ATP gamma S > ADP > AMP-PNP >> AMP = cAMP. Guanine nucleotides showed the same rank order of potencies but were less effective than adenine nucleotides. Adenine nucleotides which increased ADP-ribosylation were able to release actin from membranes. [32P]ADP-ribosylated rabbit skeletal muscle actin, which is unable to polymerize, was used as tool to study membrane-binding of actin. The [32P]ADP-ribosylated actin bound to stripped rat liver membranes in a saturable, time- and temperature-dependent manner. ATP inhibited the binding of ADP-ribosylated actin with a half-maximal and maximal inhibitory concentration at about 50 and 300 microM, respectively. The data indicate that actin-binding to membranes is prevented or reversed in the presence of adenine nucleotides thereby increasing the accessibility of actin for ADP-ribosylation by toxins.

ADP Ribose Transferases↗
PubMed1993

ADP-ribosylation of Rho proteins inhibits sperm motility.

The highly homologous Rho proteins RhoA, RhoB and RhoC are low-molecular-mass GTP-binding proteins. They are selectively ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase C3 (C3 exoenzyme). The biological function of the Rho proteins is still unclear; there is evidence that they are involved in the regulation of the filamental network of cells. Here we report that C3 exoenzyme-like toxins ADP-ribosylate small GTP-binding proteins in bovine spermatozoa and inhibit sperm motility. These findings indicate that Rho proteins which reportedly regulate the microfilament system are basically involved in sperm motility.

ADP Ribose Transferases↗
PubMed1993

NAD-binding site of the C3-like ADP-ribosyltransferase from Clostridium limosum.

Treatment of the Rho-ADP-ribosylating C3-like transferase from Clostridium limosum by ultraviolet irradiation in the presence of [carbonyl-14C]NAD incorporated 1 mol of label/mol of exoenzyme. Concomitantly, the transferase and NAD glycohydrolase activity was impaired. A peptide containing the radiolabel was obtained by proteolysis with either staphylococcal protease V8 or trypsin. Their amino acid sequences were Ala/Asp-Gly-Tyr-Ile-Glu-Pro-Ile-Ser-Thr-Phe-Lys-Gly-Gln-Leu-X-Val-Leu-Le u-Pro- Arg and Gly-Gln-Leu-X-Val-Leu-Leu-Pro-Arg, respectively. These sequences correspond with regions Ala-160 through Arg-179 and Gly-171 through Arg-179, respectively, of the very similar Clostridium botulinum C3 transferase, with X being Glu in the unlabeled enzyme. This identifies the glutamic acid residue that corresponds to Glu-174 of C. botulinum C3 transferase as part of the NAD-binding site of the catalytic center of the C. limosum exoenzyme.

ADP Ribose Transferases↗
PubMed1993

Gelsolin-actin complex is target for ADP-ribosylation by Clostridium botulinum C2 toxin in intact human neutrophils.

The gelsolin-actin complex was ADP-ribosylated by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin in lysates of human platelets and human neutrophils. When [32P]orthophosphate-labelled human neutrophils were treated with C. botulinum C2 toxin, [32P]ADP-ribosylated gelsolin-actin was precipitated with anti-gelsolin antibody. Stimulation of neutrophils by formyl-methionine-leucine-phenylalanine decreased the interaction of gelsolin with ADP-ribosylated actin in toxin-treated cells. The data indicate that the gelsolin-actin complex is a pathophysiological substrate for actin-ADP-ribosylating toxins.

Actins↗
PubMed1993

ADP-ribosylation of Drosophila indirect-flight-muscle actin and arthrin by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin.

Purified Drosophila indirect-flight-muscle actin and arthrin, an actin-ubiquitin conjugate, were ADP-ribosylated by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. Phalloidin treatment inhibited the ADP-ribosylation of Drosophila actin and arthrin. Like actin, the ADP-ribose-arthrin linkage was sensitive towards hydroxylamine treatment, indicating arginine as the amino acid acceptor. Actin translated in vitro from the indirect-flight-muscle-specific gene Act88F was ADP-ribosylated by C. botulinum C2 toxin and C. perfringens iota toxin. Actin from the R177Q mutant of Act88F translated in vivo was not ADP-ribosylated confirming Arg-177 as the ADP-ribose acceptor. Mutant L176M actin was modified by both toxins, indicating that amino acid 176 of actin does not define the substrate specificity of C. botulinum C2 toxin. Whereas the gene products of various C-terminal mutants of Act88F translated in vitro (E334K, V339I, E364K, G368E, R372H) were substrates for ADP-ribosylation by C. botulinum C2 toxin and by C. perfringens iota toxin, neither toxin modified the N-terminal O-12 deletion mutant.

ADP Ribose Transferases↗
PubMed1993

Enhancement of Clostridium botulinum C3-catalysed ADP-ribosylation of recombinant rhoA by sodium dodecyl sulfate.

The influence of sodium dodecyl sulfate (SDS) on ADP-ribosylation by Clostridium botulinum C3 exoenzyme (C3) was studied. SDS increased the ADP-ribosylation of recombinant rhoA and human platelet cytosolic proteins maximally at 0.01% whereas higher concentrations of the detergent (> 0.01%) inhibited the ADP-ribosylation. In contrast, ADP-ribosylation of human platelet membranes and of recombinant rhoB was inhibited by the detergent. The Km for NAD of the ADP-ribosylation of rhoA was decreased by SDS from about 10 to 0.6 microM. Whereas in the absence of SDS, the C3-induced ADP-ribosylation of recombinant rhoA is not affected by the amphiphilic wasp venom mastoparan, in the presence of SDS (0.01%) mastoparan (100 microM) inhibited the ADP-ribosylation. C3-associated NAD-glycohydrolase activity was maximally and half-maximally inhibited by 0.1 and 0.013% SDS, respectively. Inhibition of NAD-glycohydrolase activity was reversed by diluting out SDS indicating that C3 was not irreversibly denatured by SDS treatment. SDS (0.01%) completely inhibited the [3H]GTP binding of rhoA whereas the release of previously bound nucleotide was not affected. The data indicate that changes in the lipophilicity of rhoA protein largely affect its ability to serve as a substrate for C3-like ADP-ribosyltransferases.

ADP Ribose Transferases↗
PubMed1992

Posttranslational isoprenylation of rho protein is a prerequisite for its interaction with mastoparan and other amphiphilic agents.

The amphiphilic agents melittin, compound 48/80 and mastoparan inhibit ADP-ribosylation of porcine brain rho protein by Clostridium botulinum exoenzyme C3. However, ADP-ribosylation of recombinant rhoA expressed in E.coli was not inhibited by these agents. Accordingly, steady state GTP hydrolysis by recombinant rhoA was not stimulated by mastoparan, whereas GTP hydrolysis by porcine brain rho was stimulated 2.5-fold in the presence of this wasp venom. After microinjection of recombinant rhoA into Xenopus laevis oocytes the inhibitory effect of mastoparan on C3 ADP-ribosylation was restored. The data suggest that the amphiphilic agents tested are only active at the posttranslationally processed form of rho and that they exert their effects via the C-terminal end.

Adenosine Diphosphate Ribose↗
PubMed1992

Purification and characterization of an ADP-ribosyltransferase produced by Clostridium limosum.

We purified a novel ADP-ribosyltransferase produced by a Clostridium limosum strain isolated from a lung abscess and compared the exoenzyme with Clostridium botulinum ADP-ribosyltransferase C3. The C. limosum exoenzyme has a molecular weight of about 25,000 and a pI of 10.3. The specific activity of the ADP-ribosyltransferase is 3.1 nmol/mg/min with a Km for NAD of 0.3 microM. Partial amino acid sequence analysis of the tryptic peptides revealed about 70% homology with C3. The novel exoenzyme modifies selectively the small GTP-binding proteins of the rho family in human platelet membranes presumably at the same amino acid (asparagine 41) as known for C3. Recombinant rhoA and rhoB serve as substrates for C3 and the C. limosum exoenzyme. Whereas recombinant rac1 protein is only marginally ADP-ribosylated by C3 or by the C. limosum exoenzyme in the absence of detergent, in the presence of 0.01% sodium dodecyl sulfate rac1 is modified by C3 but not by the C. limosum exoenzyme. Recombinant CDC42Hs protein is a poor substrate for C. limosum exoenzyme and is even less modified by C3. The C. limosum exoenzyme is auto-ADP-ribosylated in the presence of 0.01% sodium dodecyl sulfate by forming an ADP-ribose protein bond highly stable toward hydroxylamine. The data indicate that ADP-ribosylation of small GTP-binding proteins of the rho family is not unique to C. botulinum C3 ADP-ribosyltransferase but is also catalyzed by a C3-related exoenzyme from C. limosum.

ADP Ribose Transferases↗
PubMed1992

ADP-ribosylation of rho proteins is inhibited by melittin, mast cell degranulating peptide and compound 48/80.

The amphiphilic agents melittin, mast cell degranulating peptide and compound 48/80 inhibit the ADP-ribosylation of the small GTP-binding proteins rho by Clostridium botulinum exoenzyme C3. Half-maximal and maximal inhibition (greater than 90%) of ADP-ribosylation occurred at about 8 and 25 micrograms/ml for compound 48/80, at 10 and 45 microM for mast cell degranulating peptide and at 15 and 50 microM for melittin, respectively. In addition, these compounds increase the steady state GTP hydrolysis and the association and dissociation rate of GTP-binding of rho proteins through an increase of GDP/GTP exchange. The data suggest that the amphiphilic agents tested interact with small GTP-binding proteins of the rho protein family.

ADP Ribose Transferases↗
PubMed1992

ADP-ribosylation of small GTP-binding proteins by Bacillus cereus.

A human pathogenic strain of Bacillus cereus produces an exoenzyme which selectively ADP-ribosylates 20-25 kDa GTP-binding proteins in platelet membranes. Pre-ADP-ribosylation of rho proteins of human platelet membranes with Clostridium botulinum exoenzyme C3 or Clostridium limosum exoenzyme inhibits subsequent ADP-ribosylation by the exoenzyme from B. cereus indicating similar substrate specificity of the transferases. The ADP-ribosyltransferase from B. cereus reveals no immunological cross-reactivity with C. botulinum C3 and C. limosum exoenzyme.

ADP Ribose Transferases↗
PubMed1992

ADP-ribosylation by Clostridium botulinum C3 exoenzyme increases steady-state GTPase activities of recombinant rhoA and rhoB proteins.

ADP-ribosylation of recombinant rhoA and rhoB proteins by Clostridium botulinum C3 exoenzyme increased steady-state GTP hydrolysis by 50 to 80%. ADP-ribosylation and increase in GTP hydrolysis occurred at similar concentrations of C3, depended on the presence of NAD and were prevented by anti-C3 antibody or heat inactivation of C3. In contrast, GTP hydrolysis by Ile-41 rhoA or Ha-ras, which are no substrates for the transferase, were not affected by C3. ADP-ribosylation facilitated the [3H]GDP release and subsequently, the binding of [3H]GTP to rhoA. The data indicate that the increase in the steady-state GTPase activity by ADP-ribosylation is caused by increasing the rate of GDP release which is suggested to be the rate limiting step of the GTPase cycle of the small GTP-binding proteins.

ADP Ribose Transferases↗