Defects in Galactose Metabolism and Glycoconjugate Biosynthesis in a UDP-Glucose Pyrophosphorylase-Deficient Cell Line Are Reversed by Adding Galactose to the Growth Medium.

UDP-glucose (UDP-Glc) is synthesized by -encoded UDP-Glc pyrophosphorylase (UGP) and is required for glycoconjugate biosynthesis and galactose metabolism because it is a uridyl donor for galactose-1-P (Gal1P) uridyltransferase. Chinese hamster lung fibroblasts harboring a hypomrphic UGP(G116D) variant display reduced UDP-Glc levels and cannot grow if galactose is the sole carbon source. Here, these cells were cultivated with glucose in either the absence or presence of galactose in order to investigate glycoconjugate biosynthesis and galactose metabolism.

Is there a risk of cancer development after Campylobacter infection?

Objective: All Campylobacter jejuni species produce a genotoxin, which induce DNA double strand breaks, could lead to an increased risk of cancer especially in the gastro-intestinal tract.

Material And Methods: All individuals in Stockholm County who tested positive with C. jejuni between 1989 and 2006 were included.

Myc is required for activation of the ATM-dependent checkpoints in response to DNA damage.

Background: The MYC protein controls cellular functions such as differentiation, proliferation, and apoptosis. In response to genotoxic agents, cells overexpressing MYC undergo apoptosis. However, the MYC-regulated effectors acting upstream of the mitochondrial apoptotic pathway are still unknown.

A novel mode of translocation for cytolethal distending toxin.

Thermal instability in the toxin catalytic subunit may be a common property of toxins that exit the endoplasmic reticulum (ER) by exploiting the mechanism of ER-associated degradation (ERAD). The Haemophilus ducreyi cytolethal distending toxin (HdCDT) does not utilize ERAD to exit the ER, so we predicted the structural properties of its catalytic subunit (HdCdtB) would differ from other ER-translocating toxins. Here, we document the heat-stable properties of HdCdtB which distinguish it from other ER-translocating toxins.

A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage.

Background: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown.

Principal Findings: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR.

Increased infectivity of Staphylococcus aureus in an experimental model of snake venom-induced tissue damage.

Soft-tissue infection is commonly found in patients bitten by Latin American Bothrops snakes. Staphylococcus aureus, which is not present in the mouth of the snake, is frequently isolated from these infections. The effects of B.

Effects of Clostridium perfringens phospholipase C in mammalian cells.

Clostridium perfringens phospholipase C (Cp-PLC), the major virulence factor in the pathogenesis of gas gangrene, is a Zn(2+) metalloenzyme with lecithinase and sphingomyelinase activities. Its structure shows an N-terminal domain containing the active site, and a C-terminal Ca(2+) binding domain required for membrane interaction. Although the knowledge of the structure of Cp-PLC and its interaction with aggregated phospholipids has advanced significantly, an understanding of the effects of Cp-PLC in mammalian cells is still incomplete.

Inhibition of hippocampal synaptic transmission by impairment of Ral function.

Large clostridial cytotoxins and protein overexpression were used to probe for involvement of Ras-related GTPases (guanosine triphosphate) in synaptic transmission in cultured rat hippocampal neurons. The toxins TcdA-10463 (inactivates Rho, Rac, Cdc42, Rap) and TcsL-1522 (inactivates Ral, Rac, Ras, R-Ras, Rap) both inhibited autaptic responses. In a proportion of the neurons (25%, TcdA-10463; 54%, TcsL-1522), the inhibition was associated with a shift from activity-dependent depression to facilitation, indicating that the synaptic release probability was reduced.

Cellular internalization of cytolethal distending toxin: a new end to a known pathway.

The cytolethal distending toxins (CDTs) are unique in their ability to induce DNA damage, activate checkpoint responses and cause cell cycle arrest or apoptosis in intoxicated cells. However, little is known about their cellular internalization pathway. We demonstrate that binding of the Haemophilus ducreyi CDT (HdCDT) on the plasma membrane of sensitive cells was abolished by cholesterol extraction with methyl-beta-cyclodextrin.

A cellular deficiency of gangliosides causes hypersensitivity to Clostridium perfringens phospholipase C.

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied.

Revised nomenclature of Clostridium difficile toxins and associated genes.

Several different nomenclatures have been applied to the Clostridium difficile toxins and their associated genes. This paper summarizes the new nomenclature that has been agreed to by the research groups currently active in the field. The revised nomenclature includes C.

Membrane independent activation of fibroblast proMMP-2 by snake venom: novel roles for venom proteinases.

ProMMP-2 activation by Bothrops asper venom was investigated in mouse gastrocnemius muscle, mammalian cell culture and a cell-free system. Zymography revealed an increment of latent and activated forms of MMP-2 in muscle homogenates 1-3 days after venom injection. To clarify if venom can induce expression and activation of MMP-2, independently of the inflammatory response, venom was added to cultured human fibroblasts, endothelial and skeletal muscle cells, which expressed proMMP-2 constitutively.

Cytolethal distending toxins.

The cytolethal distending toxins (CDTs) constitute the most recently discovered family of bacterial protein toxins. CDTs are unique among bacterial toxins as they have the ability to induce DNA double strand breaks (DSBs) in both proliferating and nonproliferating cells, thereby causing irreversible cell cycle arrest or death of the target cells. CDTs are encoded by three linked genes ( cdtA, cdtB and cdtC) which have been identified among a variety of Gram-negative pathogenic bacteria.

Glucose starvation results in UDP-glucose deficiency and inactivation of glycogen synthase.

The effects of glucose starvation on glycogen synthase (GS) activity and protein expression were investigated. Fibroblasts were cultured in medium supplemented with either glucose or pyruvate. Pyruvate-cultured cells exhibited UDP-glucose contents that amounted to approximately 10% of those in cells cultured with glucose.

A cellular UDP-glucose deficiency causes overexpression of glucose/oxygen-regulated proteins independent of the endoplasmic reticulum stress elements.

A low level of UDP-Glc occurs in cells exposed to hypoxia or glucose starvation. This work reveals that a 65% reduction in the cellular UDP-Glc level causes up-regulation of the mitochondrial chaperone GRP75 and the endoplasmic reticulum (ER) resident chaperones GRP58, ERp72, GRP78, GRP94, GRP170, and calreticulin. Conditions that cause misfolding of proteins within the ER activate the transcription factors ATF6alpha/beta and induce translation of the transcription factors XBP-1/TREB5 and ATF4/CREB2.

The Haemophilus ducreyi cytolethal distending toxin induces DNA double-strand breaks and promotes ATM-dependent activation of RhoA.

Among bacterial protein toxins, the cytolethal distending toxins (CDTs) are unique in their ability to activate the DNA damage checkpoint responses, causing cell cycle arrest or apoptosis in intoxicated cells. We provide direct evidence that natural intoxication of cells with the Haemophilus ducreyi CDT (HdCDT) holotoxin induces DNA double-strand breaks similarly to ionizing radiation. Upon DNA damage, epithelial cells and fibroblasts promote the formation of actin stress fibres via activation of the small GTPase RhoA.

R-Ras glucosylation and transient RhoA activation determine the cytopathic effect produced by toxin B variants from toxin A-negative strains of Clostridium difficile.

Clostridium difficile induces antibiotic-associated diarrhea through the production of toxin A and toxin B; the former toxin has been assumed to be responsible for the symptoms of the disease. Several toxin A-negative strains from C. difficile have recently been isolated from clinical cases and have been reported to produce toxin B variants eliciting an atypical cytopathic effect.

A point mutation in the UDP-glucose pyrophosphorylase gene results in decreases of UDP-glucose and inactivation of glycogen synthase.

The regulatory role of UDP-glucose in glycogen biogenesis was investigated in fibroblasts containing a point mutation in the UDP-glucose pyrophosphorylase gene and, consequently, chronically low UDP-glucose levels (Qc). Comparisons were made with cells having the intact gene and restored UDP-glucose levels (G3). Glycogen was always very low in Qc cells.

The Haemophilus ducreyi cytolethal distending toxin activates sensors of DNA damage and repair complexes in proliferating and non-proliferating cells.

Cytolethal distending toxins (CDTs) block proliferation of mammalian cells by activating DNA damage-induced checkpoint responses. We demonstrate that the Haemophilus ducreyi CDT (HdCDT) induces phosphorylation of the histone H2AX as early as 1 h after intoxication and re-localization of the DNA repair complex Mre11 in HeLa cells with kinetics similar to those observed upon ionizing radiation. Early phosphorylation of H2AX was dependent on a functional Ataxia Telangiectasia mutated (ATM) kinase.

Cytolethal distending toxins and activation of DNA damage-dependent checkpoint responses.

Cytolethal distending toxins (CDTs) are unique among bacterial protein toxins in their ability to cause DNA damage, due to their functional similarity to the mammalian deoxyribonuclease I (DNase I). The cellular response to CDT intoxication is characterised by activation of DNA damage-induced checkpoint responses, and the final outcome is cell type dependent. Cells of epithelial origin and normal keratinocytes are arrested in the G2 phase of the cell cycle, normal fibroblasts are also arrested in G1, while B cells die of apoptosis.

The cytolethal distending toxins induce DNA damage and cell cycle arrest.

The cytolethal distending toxins (CDTs) are a newly discovered family of bacterial protein toxins with the unique ability to interfere with the cell cycle, causing irreversible cell cycle arrest and consequently death of the target cells. CDTs are encoded by three linked genes (cdtA, cdtB and cdtC) and are produced by a variety of Gram negative bacteria. The mechanism of action of this toxin family only now begins to be elucidated.

GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes: direct activation of Cdc42.

Members of the genus Brucella are intracellular alpha-Proteobacteria responsible for brucellosis, a chronic disease of humans and animals. Little is known about Brucella virulence mechanisms, but the abilities of these bacteria to invade and to survive within cells are decisive factors for causing disease. Transmission electron and fluorescence microscopy of infected nonprofessional phagocytic HeLa cells revealed minor membrane changes accompanied by discrete recruitment of F-actin at the site of Brucella abortus entry.

Venom of the crotaline snake Atropoides nummifer (jumping viper) from Guatemala and Honduras: comparative toxicological characterization, isolation of a myotoxic phospholipase A(2) homologue and neutralization by two antivenoms.

A comparative study was performed on the venoms of the crotaline snake Atropoides nummifer from Guatemala and Honduras. SDS-polyacrylamide gel electrophoresis, under reducing conditions, revealed a highly similar pattern of these venoms, and between them and the venom of the same species from Costa Rica. Similar patterns were also observed in ion-exchange chromatography on CM-Shephadex C-25, in which a highly basic myotoxic fraction was present.

The impact of Haemophilus ducreyi cytolethal distending toxin on cells involved in immune response.

The Haemophilus ducreyi cytolethal distending toxin (HdCDT) induces cell cycle arrest and thereby inhibits cell proliferation of many cultured mammalian cell-lines. We investigated the effect of HdCDT on circulating human hematopoietic cells, including T- and B-cells, monocytes and polymorphonuclear cells (PMN). Lymphocytes were stimulated with T- and B-cell specific mitogens, whereas monocytes and PMN with endotoxin.