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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacteriophage lambda genome is one of the classical model replicons in studies on the regulation of DNA replication. Moreover, since genes coding for Shiga toxins are located in genomes of lambdoid phages, understanding of mechanisms controlling lambda DNA replication may be of bio-medical importance. During lytic development of bacteriophage lambda, its genome is replicated according to the theta (circle-to-circle) mode early after infection, and then it is switched to the sigma (rolling circle) mode. Two mechanisms of regulation of this switch were proposed recently and both suggested a crucial role for directionality of lambda DNA replication. Whereas one hypothesis assumed transient impairment of ClpP/ClpX-mediated proteolysis of the lambdaO initiator protein, another suggested a crucial role for transcriptional activation of the orilambda region and factors involved in the control of the p (R) promoter activity. Here we demonstrate that mutations in clpP and clpX genes had little influence on both directionality of lambda DNA replication and appearance of sigma replication intermediates. On the other hand, regulators affecting activity of the p (R) promoter (responsible for initiation of transcription, which activates orilambda) directly or indirectly influenced directionality of lambda DNA replication to various extents. Therefore, we conclude that regulation of the efficiency of transcriptional activation of orilambda, rather than transient impairment of the lambdaO proteolysis, is responsible for the control of the switch from theta to sigma replication, and propose a model for this control.
Mol Genet Genomics 2007 Jul
PMID:Switch from theta to sigma replication of bacteriophage lambda DNA: factors involved in the process and a model for its regulation. 1737 19

A multiplex PCR assay was developed to identify the six clinically important enterohemorrhagic Escherichia coli (EHEC) serotypes classified in seropathotypes A and B and to differentiate these from Shiga toxigenic E. coli. The assay simultaneously detects genes for Shiga toxin (stx) and intimin (eae), including allelic variants of both genes, 16S internal amplification control, as well as unique sequences in the wzx genes that are specific for serotypes O157, O26, O111, O103, O121 and O145. PCR analysis of 40 representative strains showed that the assay correctly identified the virulence genes, if present, and the respective O antigen type of all the strains, including some atypical EHEC, as well as enteropathogenic E. coli and E. coli strains examined.
Mol Cell Probes 2007 Aug
PMID:Identification of Shiga toxigenic Escherichia coli seropathotypes A and B by multiplex PCR. 1738 54

Retrograde transport pathways from early/recycling endosomes to the trans-Golgi network (TGN) are poorly defined. We have investigated the role of TGN golgins in retrograde trafficking. Of the four TGN golgins, p230/golgin-245, golgin-97, GCC185, and GCC88, we show that GCC88 defines a retrograde transport pathway from early endosomes to the TGN. Depletion of GCC88 in HeLa cells by interference RNA resulted in a block in plasma membrane-TGN recycling of two cargo proteins, TGN38 and a CD8 mannose-6-phosphate receptor cytoplasmic tail fusion protein. In GCC88-depleted cells, cargo recycling was blocked in the early endosome. Depletion of GCC88 dramatically altered the TGN localization of the t-SNARE syntaxin 6, a syntaxin required for endosome to TGN transport. Furthermore, the transport block in GCC88-depleted cells was rescued by syntaxin 6 overexpression. Internalized Shiga toxin was efficiently transported from endosomes to the Golgi of GCC88-depleted cells, indicating that Shiga toxin and TGN38 are internalized by distinct retrograde transport pathways. These findings have identified an essential role for GCC88 in the localization of TGN fusion machinery for transport from early endosomes to the TGN, and they have allowed the identification of a retrograde pathway which differentially selects TGN38 and mannose-6-phosphate receptor from Shiga toxin.
Mol Biol Cell 2007 Dec
PMID:The golgin GCC88 is required for efficient retrograde transport of cargo from the early endosomes to the trans-Golgi network. 1791 56

Shiga toxin (Stx) binds to the cell, and it is transported via endosomes and the Golgi apparatus to the endoplasmic reticulum and cytosol, where it exerts its toxic effect. We have recently shown that Stx activates the tyrosine kinase Syk, which in turn induces clathrin phosphorylation and up-regulates Stx uptake. Here, we show that toxin-induced signaling can also regulate another step in intracellular Stx transport. We demonstrate that transport of Stx to the Golgi apparatus is dependent on the mitogen-activated protein kinase p38. Treatment of cells with chemical inhibitors or small interfering RNA targeting p38 inhibited Stx transport to the Golgi and reduced Stx toxicity. This p38 dependence is specific to Stx, because transport of the related toxin ricin was not affected by p38 inhibition. Stx rapidly activated p38, and recruited it to early endosomes in a Ca(2+)-dependent manner. Furthermore, agonist-induced oscillations in cytosolic Ca(2+) levels were inhibited upon Stx stimulation, possibly reflecting Stx-dependent local alterations in cytosolic Ca(2+) levels. Intracellular transport of Stx is Ca(2+) dependent, and we provide evidence that Stx activates a signaling cascade involving cross talk between Ca(2+) and p38, to regulate its trafficking to the Golgi apparatus.
Mol Biol Cell 2008 Jan
PMID:The Mitogen-activated protein kinase p38 links Shiga Toxin-dependent signaling and trafficking. 1795 27

Shiga-like toxin 1 (SLT-1) is a type II ribosome-inactivating protein; its A(1) domain blocks protein synthesis in eukaryotic cells by catalyzing the depurination of a single adenine base in 28 S rRNA. The molecular mechanism leading to this site-specific depurination event is thought to involve interactions with eukaryotic ribosomal proteins. Here, we present evidence that the A(1) chain of SLT-1 binds to the ribosomal proteins P0, P1, and P2. These proteins were identified from a HeLa cell lysate by tandem mass spectrometry, and subsequently confirmed to bind to SLT-1 A(1) chain by yeast-two-hybrid and pull-down experiments using candidate full-length proteins. Moreover, the removal of the last 17 amino acids of either protein P1 or P2 abolishes the interaction with the A(1) chain, whereas P0, lacking this common C terminus, still binds to the A(1) domain. In vitro pull-down experiments using fusion protein-tagged C-terminal peptides corresponding to the common 7, 11, and 17 terminal residues of P1 and P2 confirmed that the A(1) chain of SLT-1 as well as the A chain of ricin bind to this shared C-terminal peptide motif. More importantly, a synthetic peptide corresponding to the 17 amino acid C terminus of P1 and P2 was shown to inhibit the ribosome-inactivating function of the A(1) chain of SLT-1 in an in vitro transcription and translation-coupled assay. These results suggest a role for the ribosomal stalk in aiding the A(1) chain of SLT-1 and other type II ribosome-inactivating proteins in localizing its catalytic domain near the site of depurination in the 28 S rRNA.
J Mol Biol 2008 Apr 25
PMID:The catalytic subunit of shiga-like toxin 1 interacts with ribosomal stalk proteins and is inhibited by their conserved C-terminal domain. 1835 91

The biosynthetic sorting of acid hydrolases to lysosomes relies on transmembrane, mannose 6-phosphate receptors (MPRs) that cycle between the TGN and endosomes. Herein we report that maintenance of this cycling requires the function of the mammalian Golgi-associated retrograde protein (GARP) complex. Depletion of any of the three GARP subunits, Vps52, Vps53, or Vps54, by RNAi impairs sorting of the precursor of the acid hydrolase, cathepsin D, to lysosomes and leads to its secretion into the culture medium. As a consequence, lysosomes become swollen, likely due to a buildup of undegraded materials. Missorting of cathepsin D in GARP-depleted cells results from accumulation of recycling MPRs in a population of light, small vesicles downstream of endosomes. These vesicles might correspond to intermediates in retrograde transport from endosomes to the TGN. Depletion of GARP subunits also blocks the retrograde transport of the TGN protein, TGN46, and the B subunit of Shiga toxin. These observations indicate that the mammalian GARP complex plays a general role in the delivery of retrograde cargo into the TGN. We also report that a Vps54 mutant protein in the Wobbler mouse strain is active in retrograde transport, thus explaining the viability of these mutant mice.
Mol Biol Cell 2008 Jun
PMID:Requirement of the human GARP complex for mannose 6-phosphate-receptor-dependent sorting of cathepsin D to lysosomes. 1836 45

Shiga toxin-producing Escherichia coli (STEC) strains are pathogens that cause severe infections, especially in children. Although early data suggested that the STEC phenotype was linked to the O157 serotype, subsequent studies indicated no such strict phenotype-serotype correlation. Thus, it seems that only a test detecting stx1 and stx2 genes (responsible for Shiga toxin production) can provide unambiguous results in STEC identification. Here, we present a system for the rapid detection of the presence of o157, stx1, and stx2 genes, on the basis of an asymmetric polymerase chain reaction and subsequent detection of the amplification products on electric biochips. The whole test lasts less than 100 minutes, and allows for the accurate identification of the stx1 and stx2 genes and the presence of pO157 plasmid in a bacterial colony or in a liquid bacterial culture. The procedure is fully automated, and the only required manual activity is a chip exchange.
Diagn Mol Pathol 2008 Sep
PMID:Rapid identification of shiga toxin-producing Escherichia coli (STEC) using electric biochips. 1838 65

The targeting of solid tumors requires delivery tools that resist intracellular and extracellular inactivation, and that are taken up specifically by tumor cells. We have shown previously that the recombinant nontoxic B-subunit of Shiga toxin (STxB) can serve as a delivery tool to target digestive tumors in animal models. The aim of this study was to expand these experiments to human colorectal cancer. Tissue samples of normal colon, benign adenomas, colorectal carcinomas, and liver metastases from 111 patients were obtained for the quantification of the expression of the cellular STxB receptor, the glycosphingolipid globotriaosyl ceramide (Gb(3) or CD77). We found that compared with normal tissue, the expression of Gb(3) was strongly increased in colorectal adenocarcinomas and their metastases, but not in benign adenomas. Short-term primary cultures were prepared from samples of 43 patients, and STxB uptake was studied by immunofluorescence microscopy. Of a given tumor sample, on average, 80% of the cells could visibly bind STxB, and upon incubation at 37 degrees C, STxB was transported to the Golgi apparatus, following the retrograde route. This STxB-specific intracellular targeting allows the molecule to avoid recycling and degradation, and STxB could consequently be detected on tumor cells even 5 days after initial uptake. In conclusion, the targeting properties of STxB could be diverted for the delivery of contrast agents to human colorectal tumors and their metastases, whose early detection and specific targeting remains one of the principal challenges in oncology.
Mol Cancer Ther 2008 Aug
PMID:Human colorectal tumors and metastases express Gb3 and can be targeted by an intestinal pathogen-based delivery tool. 1868 97

Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the alpha-sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the alpha-sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild-type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the DeltaP1 and DeltaP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild-type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.
Mol Microbiol 2008 Dec
PMID:The ribosomal stalk is required for ribosome binding, depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae. 1901 45

Delivery of drugs to the appropriate target cells would improve efficacy and reduce potential side effects. The nontoxic B-subunit of the intestinal pathogen-produced Shiga toxin (STxB) binds specifically to the glycosphingolipid Gb3, overexpressed in membranes of certain tumor cells, and enters these cells through the retrograde pathway. Therefore, STxB binding to Gb3 receptors may be useful for cell-specific vectorization or imaging purposes. Here we labeled STxB with a fluorophore to evaluate its potential as an in vivo cell-specific targeting reagent in two different models of human colorectal carcinoma. Fluorescent STxB was administered systemically to xenografted nude mice, and its biodistribution was studied by optical imaging. The use of fluorescent STxB allowed the combination of the macroscopic observations with analyses at the cellular level using confocal microscopy. After administration, the fluorescent STxB was slowly eliminated by renal excretion. However, it accumulated in the tumor area. Furthermore, STxB was demonstrated to enter the Gb3-expressing tumoral cells, as well as the epithelial cells of the neovascularization and the monocytes and macrophages surrounding the xenografts.
Mol Imaging
PMID:In vivo tumor targeting by the B-subunit of shiga toxin. 1912 94


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