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G Rex

Publications and source records attributed to G Rex.

10 recordsLinked to original sources
PubMed1996

Difference in protein substrate specificity between hemorrhagic toxin and lethal toxin from Clostridium sordellii.

The hemorrhagic toxin (HT) from Clostridium sordellii is pharmacologically related to Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin which have been recently identified as mono-glucosyl-transferases. Here we report that HT, which is coexpressed with lethal toxin, is also a glucosyltransferase. Whereas lethal toxin glucosylates the Rho subfamily proteins Rac and Cdc42 and the Ras subfamily proteins H-Ras and Rap, the substrate specificity of HT is strictly confined to the Rho subfamily proteins Rho, Rac and Cdc42. Comparable to lethal toxin, transferase activity of HT is stimulated by Mn2+. Acceptor amino acid in Rho was identified by mutagenesis as threonine-37. C. sordellii HT is a novel member of the family of clostridial mono-glucosyl-transferases, a family which modifies the Rho and Ras GTPases.

3T3 Cells↗
PubMed1996

Clostridium novyi alpha-toxin-catalyzed incorporation of GlcNAc into Rho subfamily proteins.

The lethal and edema-inducing alpha-toxin from Clostridium novyi causes rounding up of cultured cell lines by redistribution of the actin cytoskeleton. alpha-Toxin belongs to the family of large clostridial cytotoxins that encompasses Clostridium difficile toxin A and B and the lethal toxin from Clostridium sordellii. Toxin A and toxin B have been recently identified as monoglucosyltransferases to modify the low molecular mass GTPases of the Rho subfamily (Just, I., Selzer, J., Wilm, M., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503 and Just, I., Wilm, M., Selzer, J., Rex, G., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) J. Biol. Chem. 270, 13932-13936). We report here the identification of the alpha-toxin-catalyzed modification of Rho. Using electrospray mass spectrometry, the mass of the modification was determined as 203 Da, consistent with a N-acetyl-hexosamine moiety. UDP-N-acetyl-glucosamine selectively served as cosubstrate for alpha-toxin-catalyzed modification into the Rho subfamily proteins Rho, Rac, Cdc42, and RhoG. The acceptor amino acid of N-acetyl-glucosaminylation was identified by mutagenesis as Thr-37 in Rho (equivalent to Thr-35 in Rac/Cdc42), which is located in the effector domain of the GTPases. C. novyi alpha-toxin seems to mediate its cytotoxic effects on cells by mimicking endogenous post-translational modification of cellular proteins.

3T3 Cells↗
PubMed1996

The ras-related protein Ral is monoglucosylated by Clostridium sordellii lethal toxin.

Clostridium sordellii lethal toxin (LT), a cytotoxin which causes preferential destruction of the actin cytoskeleton, has been recently identified as glucosyltransferase to modify the low molecular mass GTPases Rac, Ras and Rap. We report here on LT produced by C. sordellii strain 6018 which glucosylates in addition to Rac, Ras and Rap the Ral protein. LT from strain VPI9048 however does not glucosylate Ral. Besides recombinant Ral, cellular Ral is also substrate. In the GDP-bound form, Ral is a superior substrate to the GTP form. Acceptor amino acid for glucose is threonine-46 which is equivalent to threonine-35 in H-Ras located in the effector region. The Ral-glucosylating toxin is a novel isoform of Ras-modifying clostridial cytotoxins.

Animals↗
PubMed1995

The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins.

The enterotoxin from Clostridium difficile (ToxA) is one of the causative agents of the antibiotic-associated pseudomembranous colitis. In cultured monolayer cells ToxA exhibits cytotoxic activity to induce disassembly of the actin cytoskeleton, which is accompanied by morphological changes. ToxA-induced depolymerization of actin filaments is correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins (Just, I., Selzer, J., von Eichel-Streiber, C., and Aktories, K. (1995) J. Clin. Invest. 95, 1026-1031). Here we report on the identification of the ToxA-induced modification of Rho. Applying electrospray mass spectrometry, the mass of the modification was determined as 162 Da, which is consistent with the incorporation of a hexose into Rho. From several hexoses tested UDP-glucose selectively served as cosubstrate for ToxA-catalyzed modification. The acceptor amino acid of glucosylation was identified from a Lys-C-generated peptide by tandem mass spectrometry as Thr-37. Mutation of Thr-37 to Ala completely abolished glucosylation. The members of the Rho family (RhoA, Rac1, and Cdc42Hs) were substrates for ToxA, whereas H-Ras, Rab5, and Arf1 were not glucosylated. ToxA-catalyzed glucosylation of lysates from ToxA-pretreated rat basophilic leukemia (RBL) cells resulted in a decreased incorporation of [14C]glucose, indicating previous glucosylation in the intact cell. Glucosylation of the Rho subtype proteins appears to be the molecular mechanism by which C. difficile ToxA mediates its cytotoxic effects on cells.

Amino Acids↗
PubMed1994

The mechanism of translational coupling in Escherichia coli. Higher order structure in the atpHA mRNA acts as a conformational switch regulating the access of de novo initiating ribosomes.

Bacterial genes are commonly transcribed to form polycistronic mRNAs bearing reading frames whose respective translational efficiencies are not independently determined. As in many bacterial operons, expression of the atp genes of Escherichia coli is strongly influenced by translational coupling. The gene pair atpHA is tightly coupled, whereby atpA is translated at least three times more efficiently than atpH. However, there is no fixed stoichiometry of coupling: mutations in atpH lead to increases in the translation ratio (atpA/atpH) of up to approximately 40-fold. We have demonstrated that secondary structure sequestering the atpA translational initiation region (TIR) is important to the coupling mechanism in that it inhibits de novo translational initiation at the atpA start codon. Genetic and structural analyses indicate that this inhibitory structure can be induced to refold into a less inhibitory conformation either by introducing two single-base substitutions or as a result of ribosomes translating atpH. We propose a model in which the secondary structure of the atpA TIR acts analogously to a "gating device" in that it restricts de novo ribosomal initiation until it is "switched" into a more open conformation. This contrasts with the function of a stem-loop structure located immediately downstream of atpA and upstream of the Shine-Dalgarno region of atpG, which was found to inhibit translation, but not to mediate tight coupling. Results obtained using the "specialized" ribosome system of Hui and de Boer ((1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4762-4766) indicate that primarily ribosomes reinitiating after termination on atpH are responsible for inducing refolding of the atpA TIR. The principle of alternative mRNA conformations with different functional properties embodied in the model presented here can only be fulfilled by certain types of structure. It is likely to operate in several steps of prokaryotic gene expression, underlying a range of regulatory events including transcriptional attenuation and translational activation.

Base Sequence↗
PubMed1993

Primary health care in support of community development.

A community development approach has been adopted in the outreach component of the work of the Alexandra Health Centre in South Africa. The importance of local township organizations has been recognized and the Centre is seen not only as providing technical solutions but also as helping people to achieve improved living conditions. This requires clear motivation, rigorous management, purposeful action by teams of health staff, and planning in conjunction with the community.

Community Health Centers↗
PubMed1991

Translational coupling varying in efficiency between different pairs of genes in the central region of the atp operon of Escherichia coli.

A series of atp::lacZ fusions has been constructed for use in a study of translational coupling in the central region of the Escherichia coli atp operon. Five genes, atpE, atpF, atpH, atpA and atpG, were shown to be translationally coupled to various degrees of tightness. A new lac promoter vector, compatible with the atp::lacZ fusion vectors, was used to express individual atp genes in the same hosts as the fusion genes. The H(+)-ATPase subunits thus synthesized exercised no significant trans-regulation on the expression of the atp::lacZ fusions, indicating that the coupling is primarily cis. The mechanism of this coupling was investigated using in vitro mutagenesis. At least in the case of the pair atpHA, coupling seems to involve facilitated binding of fresh ribosomes to the atpA translational initiation regions.

Amino Acid Sequence↗