The chloramphenicol resistance gene cmlA is disseminated on transferable plasmids that confer multiple-drug resistance in swine Escherichia coli

A recent study of beta-hemolytic Escherichia coli isolated from diarrheic swine found that 53% were resistant to chloramphenicol, a drug that has been prohibited from use in food animals in the US since the mid-1980s. To identify the factors governing the persistence of chloramphenicol resistance in the absence of specific selection pressure, the location of the chloramphenicol resistance gene cmlA and its linkage to other resistance determinants were investigated. Southern blot analysis of plasmid DNA from 46 swine E. coli isolates indicated that cmlA was present on large plasmids greater than 100 kbp. Fifty-two percent of the isolates were able to transfer chloramphenicol resistance to an E. coli recipient at conjugation frequencies ranging from 10(-3) to 10(-8) per recipient. Antimicrobial susceptibility tests on transconjugant strains demonstrated that resistance to sulfamethoxazole, tetracycline, and kanamycin frequently transferred along with chloramphenicol resistance. The transconjugant strains possessed at least two distinct class 1 integrons that linked cmlA to both aminoglycoside resistance genes aadA1 and aadA2 and either to sul1 or to sul3 sulphonamide resistance genes. These results suggest that in the absence of specific chloramphenicol selection pressure, the cmlA gene is maintained by virtue of gene linkage to genes encoding resistance to antimicrobials that are currently approved for use in food animals.     

A reproducible technique combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine for double labelling studies of cell proliferation in paraffin sections of tissues

Abstract. A method is described to combine tritiated thymidine autoradiography with immunoperoxidase detection of bromodeoxyuridine on the same paraffin sections. It overcomes the varied technical artefacts we encountered when first attempting to combine these techniques and results in preparations with extremely low peroxidase and autoradiographic backgrounds. In particular, we find it is important to avoid the use of detergents during immunostaining, otherwise grain counts are reduced and autoradiograph exposures need to be greatly increased, and to avoid excessive peroxidase staining which makes it difficult to visualize silver grains in the overlying emulsion. The advantages of a method to remove emulsion films using acid-alcohol, allowing the same sections to be dipped twice with a long and a short autoradiographic exposure, are presented. The routine combination of high quality tritiated thymidine autoradiography with clean immunoperoxidase staining of bromodeoxyuridine-positive nuclei provides a new and powerful cell kinetic, double-labelling method to augment existing techniques e.g. by labelling the same cells undergoing DNA synthesis in successive cell cycles.     

DNA synthesis in Langerhans'cells of mouse ear epithelium revealed by tritiated thymidine autoradiography and histochemical staining for non-specific esterase and beta-glucuronidase activity

Abstract The proportion of Langerhans'cells in DNA synthesis in normal mouse skin was assessed by combining tritiated thymidine [³H]TdR autoradiography with enzyme histochemistry. After injection of [³H]TdR, ear skin was treated in two ways. Epithelial sheet preparations were stained for the presence of non-specific esterase and cytospin preparations of epithelial cell suspensions were stained for beta-glucuronidase activity. The labelling index (p± SE mean) for cytospins, 40 min after injecting [³H]TdR, was 1.6 ± 0.15%, doubling to 3–4% from 7–17 days after injection. The sheet preparations showed the proportion of label attributable to paired Langerhans'cells rising from 18% at 40 min after injection, to approximately 45%, on days 1–4 after injection. These results suggest that the proliferation of Langerhans'cells in normal mouse skin might be higher than was previously thought to be the case.     

A novel statin-mediated “prenylation block-and-release” assay provides insight into the membrane targeting mechanisms of small GTPases

Ras super-family small GTPases regulate diverse cellular processes such as vesicular transport and signal transduction. Critical to these activities is the ability of these proteins to target to specific intracellular membranes. To allow association with membranes Ras-related GTPases are post-translationally modified by covalent attachment of prenyl groups to conserved cysteine residues at or near their C-terminus. Here we used the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGCR) inhibitor mevastatin to develop a ‘prenylation block-and-release’ assay that allows membrane targeting of prenylated proteins to be visualized in living cells. Using this assay we investigated the cytosol to membrane targeting of several small GTPases to compartments of the secretory and endocytic pathways. We found that all Rabs tested were targeted directly to the membrane on which they reside at steady-state and not via an intermediate location as reported for Ras and Rho proteins. However, we observed that the kinetics of cytosol to membrane targeting differed for each Rab tested. Comparison of the mevastatin sensitivity and kinetics of membrane targeting of Rab23, Rab23 prenylation motif mutants and H-Ras revealed that these parameters are strongly dependent upon the prenyl transferase with Rab geranylgeranyl transferase substrates exhibiting higher sensitivity and requiring greater time to recover from mevastatin inhibition than farnesyl transferase substrates. We propose that this assay is a useful tool to investigate the kinetics, biological functions and the mechanisms of membrane targeting of prenylated proteins.     

The molecular basis for the lack of immunostimulatory activity of vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at approximately 3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 micro g/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.     

Phosphotyrosyl peptides and analogues as substrates and inhibitors of purple acid phosphatases

Purple acid phosphatases are metal-containing hydrolases. While their precise biological role(s) is unknown, the mammalian enzyme has been linked in a variety of biological circumstances (e.g., osteoporosis) with increased bone resorption. Inhibition of the human enzyme is a possible strategy for the treatment of bone-resorptive diseases such as osteoporosis. Previously, we determined the crystal structure of pig purple acid phosphatase to 1.55A and we showed that it is a good model for the human enzyme. Here, a study of the pH dependence of its kinetic parameters showed that the pig enzyme is most efficient at pH values similar to those encountered in the osteoclast resorptive space. Based on the observation that phosphotyrosine-containing peptides are good substrates for pig purple acid phosphatase, peptides containing a range of phosphotyrosine mimetics were synthesized. Kinetic analysis showed that they act as potent inhibitors of mammalian and plant purple acid phosphatases, with the best inhibitors exhibiting low micromolar inhibition constants at pH 3-5. These compounds are thus the most potent organic inhibitors yet reported for the purple acid phosphatases.     

Evidence for L-dopa incorporation into cell proteins in patients treated with levodopa

Levodopa (L-dopa) is the most widely used agent for the symptomatic relief of Parkinson's disease. There is concern that chronic L-dopa treatment may be detrimental, with some studies suggesting that L-dopa may be neurotoxic. A potentially important mechanism whereby L-dopa may exert neurotoxic effects has been overlooked: that of the incorporation of L-dopa into proteins by protein synthesis. L-Dopa competes with tyrosine as a substrate in protein synthesis in vitro. We provide evidence that L-dopa can also be incorporated into proteins in vivo. Blood from L-dopa-treated and -non-treated patients was separated into protein, erythrocyte and lymphocyte fractions and levels of protein-incorporated dopa quantified by HPLC. Levels of protein-incorporated dopa were significantly increased in lymphocyte cell proteins from L-dopa-treated patients. This has not arisen from oxidative pathways as there was no evidence of oxidative damage to proteins. In addition, there was no increase in protein-incorporated dopa in erythrocytes, which are not actively synthesizing proteins. We suggest that protein-incorporated dopa could also be generated in the CNS. The accumulation of protein-incorporated dopa in cells is associated with oxidative stress and impaired function and could contribute to some of the problems associated with long-term L-dopa treatment.