Glutathione is a target in tellurite toxicity and is protected by tellurite resistance determinants inEscherichia coli

2001 ◽  
Vol 47 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Raymond J Turner ◽  
Yair Aharonowitz ◽  
Joel H Weiner ◽  
Diane E Taylor
Keyword(s):  
Reduced Glutathione ◽  
Resistance Mechanism ◽  
Thiol Oxidation ◽  
Wild Type ◽  
Response Curves ◽  
Tellurite Resistance ◽  
Thiol Compounds ◽  
Mechanisms Of Toxicity ◽  
Resistance Determinants ◽  
Glutathione Oxidation

Tellurite (TeO32-) is highly toxic to most microorganisms. The mechanisms of toxicity or resistance are poorly understood. It has been shown that tellurite rapidly depletes the reduced thiol content within wild-type Escherichia coli. We have shown that the presence of plasmid-borne tellurite-resistance determinants protects against general thiol oxidation by tellurite. In the present study we observe that the tellurite-dependent depletion of cellular thiols in mutants of the glutathione and thioredoxin thiol:redox system was less than in wild-type cells. To identify the type of low-molecular-weight thiol compounds affected by tellurite exposure, the thiol-containing molecules were analyzed by reverse phase HPLC as their monobromobimane derivatives. Results indicated that reduced glutathione is a major initial target of tellurite reactivity within the cell. Other thiol species are also targeted by tellurite, including reduced coenzyme A. The presence of the tellurite resistance determinants kilA and ter protect against the loss of reduced glutathione by as much as 60% over a 2 h exposure. This protection of glutathione oxidation is likely key to the resistance mechanism of these determinants. Additionally, the thiol oxidation response curves were compared between selenite and tellurite. The loss of thiol compounds within the cell recovered from selenite but not to tellurite.Key words: tellurite, resistance, thiol oxidation, heavy metal toxicity, selenite, glutathione.

Neither reduced uptake nor increased efflux is encoded by tellurite resistance determinants expressed inEscherichia coli

1995 ◽  
Vol 41 (1) ◽  
pp. 92-98 ◽  
Author(s):  
Raymond J. Turner ◽  
Joel H. Weiner ◽  
Diane E. Taylor
Keyword(s):  
Resistance Mechanism ◽  
Metal Resistance ◽  
Resistance Determinant ◽  
Inescherichia Coli ◽  
Efflux Transporter ◽  
Tellurite Resistance ◽  
E Coli ◽  
Resistance Determinants ◽  
Escherichia Coli Cells ◽  
Resistance Plasmids

Rates of uptake of the TeO32−oxyanion were investigated in Escherichia coli cells containing tellurite resistance determinants from both plasmid (RK2Ter, R478, pMER610, MIP233, pHH1508a, pMUR) and chromosomal (tehAB) sources. The uptake was investigated to determine whether or not reduced uptake or increased efflux is involved in the tellurite resistance mechanism. Reduced TeO32−uptake generated by cultures harboring arsABC from the plasmid R773, which has been previously shown to be an oxyanion efflux transporter, was used as the standard. Uptake curves were found to be essentially identical among E. coli cultures harboring the tellurite resistance plasmids RK2Ter, pMER610, pHH1508a, and pMUR and cultures harboring tellurite-sensitive control plasmids. Cultures harboring clones of the tehAB operon from E. coli showed no change in the TeO32−accumulation. Cultures harboring R478 demonstrated reduced uptake. However, a subclone containing only the tellurite resistance determinant displayed no reduced uptake. This suggests that there may be another determinant on R478 other than the primary tellurite resistance determinant that gives rise to TeO32−efflux. These results demonstrate that neither reduced uptake nor increased efflux is responsible for the tellurite resistance in the resistance determinants investigated here.Key words: tellurite resistance, uptake, metal resistance, resistance.

The Effect of Mismatch Repair and Heteroduplex Formation on Sexual Isolation in Bacillus

Genetics ◽  
1998 ◽  
Vol 148 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Jacek Majewski ◽  
Frederick M Cohan
Keyword(s):  
Mismatch Repair ◽  
Wild Type Strain ◽  
Resistance Mechanism ◽  
Sequence Divergence ◽  
Sexual Isolation ◽  
Wild Type ◽  
Exponential Relationship ◽  
Dna Strands ◽  
The Relationship ◽  
Heteroduplex Formation

AbstractIn Bacillus transformation, sexual isolation is known to be an exponential function of the sequence divergence between donor and recipient. Here, we have investigated the mechanism under which sequence divergence results in sexual isolation. We tested the effect of mismatch repair by comparing a wild-type strain and an isogenic mismatch-repair mutant for the relationship between sexual isolation and sequence divergence. Mismatch repair was shown to contribute to sexual isolation but was responsible for only a small fraction of the sexual isolation observed. Another possible mechanism of sexual isolation is that more divergent recipient and donor DNA strands have greater difficulty forming a heteroduplex because a region of perfect identity between donor and recipient is required for initiation of the heteroduplex. A mathematical model showed that this heteroduplex-resistance mechanism yields an exponential relationship between sexual isolation and sequence divergence. Moreover, this model yields an estimate of the size of the region of perfect identity that is comparable to independent estimates for Escherichia coli. For these reasons, and because all other mechanisms of sexual isolation may be ruled out, we conclude that resistance to heteroduplex formation is predominantly responsible for the exponential relationship between sexual isolation and sequence divergence in Bacillus transformation.

scholarly journals Forming active recombinant enterokinase expressed in Escherichia coli

2020 ◽  
Vol 18 (3) ◽  
pp. 553-560
Author(s):  
Le Thi Thu Hong ◽  
Luong Kim Phuong ◽  
Trinh Thi Thu Hien ◽  
Nguyen Thi Mai Phuong ◽  
Truong Nam Hai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Light Chain ◽  
Reduced Glutathione ◽  
Recombinant Protein Expression ◽  
Initial Assessment ◽  
Oxidized Glutathione ◽  
Expression Systems ◽  
Insoluble Form ◽  
Glutathione Oxidation

Enterokinase is a serine protease commonly used in some biotechnology researches. For these purposes, the light chain containing enterokinase activity has usually been expressed as recombinant protein in different expression systems because natural enterokinase extraction is often ineffective. In this study, we examined the formation of recombinant enterokinase expressed in Escherichia coli with biological activity. The thioredoxin-enterokinase (trx-ent) fusion protein was autocleavaged into thioredoxin and enterokinase when expressed under insoluble form, denatured with guanidine and then refolded with suitable oxidation and reduction steps. Meanwhile, soluble expression as well as insoluble form denatured by urea had not enzymatic activity. Denaturant solution of 6 M guanidine along with the re-folding conditions in oxidized glutathione oxidation buffers followed by the reduced glutathione buffer with arginine was applied to produce trx-ent protein capable of self-cleavage. The recombinant light-chain enterokinase protein had a size of about 35 kDa on the Tris-glycine gel. Initial assessment on substance had shown that enterokinase was capable of cleaving thioredoxin-sumoprotease into thioredoxin and sumoprotease. This result provides the base for the production of active recombinant enterokinase to be used in recombinant protein expression technology.

scholarly journals Differential Potential of Phytophthora capsici Resistance Mechanisms to the Fungicide Metalaxyl in Peppers

2020 ◽  
Vol 8 (2) ◽  
pp. 278 ◽  
Author(s):  
Weiyan Wang ◽  
Xiao Liu ◽  
Tao Han ◽  
Kunyuan Li ◽  
Yang Qu ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Differentially Expressed Genes ◽  
Phytophthora Capsici ◽  
Resistance Mechanism ◽  
Resistant Mutant ◽  
Resistance Mechanisms ◽  
Differentially Expressed ◽  
Pathogenicity Test ◽  
Wild Type ◽  
Metalaxyl Resistance

Metalaxyl is one of the main fungicides used to control pepper blight caused by Phytophthora capsici. Metalaxyl resistance of P. capsici, caused by the long-term intense use of this fungicide, has become one of the most serious challenges facing pest management. To reveal the potential resistance mechanism of P. capsici to fungicide metalaxyl, a metalaxyl-resistant mutant strain SD1-9 was obtained under laboratory conditions. The pathogenicity test showed that mutant strain SD1-9 had different pathogenicity to different host plants with or without the treatment of metalaxyl compared with that of the wild type SD1. Comparative transcriptome sequencing of mutant strain SD1-9 and wild type SD1 led to the identification of 3845 differentially expressed genes, among them, 517 genes were upregulated, while 3328 genes were down-regulated in SD1-9 compared to that in the SD1. The expression levels of 10 genes were further verified by real-time RT-PCR. KEGG analysis showed that the differentially expressed genes were enriched in the peroxisome, endocytosis, alanine and tyrosine metabolism. The expression of the candidate gene XLOC_020226 during 10 life history stages was further studied, the results showed that expression level reached a maximum at the zoospores stage and basically showed a gradually increasing trend with increasing infection time in pepper leaves in SD1-9 strain, while its expression gradually increased in the SD1 strain throughout the 10 stages, indicated that XLOC_020226 may be related to the growth and pathogenicity of P. capsici. In summary, transcriptome analysis of plant pathogen P. capsici strains with different metalaxyl resistance not only provided database of the genes involved in the metalaxyl resistance of P. capsici, but also allowed us to gain novel insights into the potential resistance mechanism of P. capsici to metalaxyl in peppers.

scholarly journals Low Fitness Cost of the Multidrug Resistance Genecfr

2011 ◽  
Vol 55 (8) ◽  
pp. 3714-3719 ◽  
Author(s):  
Jacqueline M. LaMarre ◽  
Jeffrey B. Locke ◽  
Karen J. Shaw ◽  
Alexander S. Mankin
Keyword(s):  
Resistance Mechanism ◽  
Negative Control ◽  
Growth Characteristics ◽  
Fitness Cost ◽  
23S Rrna ◽  
Control Strain ◽  
Wild Type ◽  
Rapid Spread ◽  
Similar Growth ◽  
Appreciable Reduction

ABSTRACTThe recently described rRNA methyltransferase Cfr that methylates the conserved 23S rRNA residue A2503, located in a functionally critical region of the ribosome, confers resistance to an array of ribosomal antibiotics, including linezolid. A number of reports of linezolid-resistantcfr-positive clinical strains indicate the possible rapid spread of this resistance mechanism. Since the rate of dissemination and the efficiency of maintenance of a resistance gene depend on the fitness cost associated with its acquisition, we investigated the fitness cost ofcfrexpression in a laboratoryStaphylococcus aureusstrain. We found that acquisition of thecfrgene does not produce any appreciable reduction in the cell growth rate. Only in a cogrowth competition experiment was some loss of fitness observed because Cfr-expressing cells slowly lose to thecfr-negative control strain. Interestingly, cells expressing wild-type and catalytically inactive Cfr had very similar growth characteristics, indicating that the slight fitness cost associated withcfracquisition stems from expression of the Cfr polypeptide rather than from the modification of the conserved rRNA residue. In some clinical isolates,cfris coexpressed with theermgene, which encodes a methyltransferase targeting another 23S rRNA residue, A2058. Dimethylation of A2058 by Erm notably increases the fitness cost associated with the Cfr-mediated methylation of A2503. The generally low fitness cost ofcfracquisition observed in our experiments with the laboratoryS. aureusstrain offers a microbiological explanation for the apparent spread of thecfrgene among pathogens.

scholarly journals Bacillus thuringiensis Spores and Vegetative Bacteria: Infection Capacity and Role of the Virulence Regulon PlcR Following Intrahaemocoel Injection of Galleria mellonella

Insects ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 129 ◽  
Author(s):  
Christophe Buisson ◽  
Michel Gohar ◽  
Eugénie Huillet ◽  
Christina Nielsen-LeRoux
Keyword(s):  
Bacillus Thuringiensis ◽  
Galleria Mellonella ◽  
Lethal Dose ◽  
Intestinal Barrier ◽  
Oral Route ◽  
Infection Model ◽  
Wild Type ◽  
Response Curves ◽  
Vegetative Cells ◽  
Crystal Toxins

Bacillus thuringiensis is an invertebrate pathogen that produces insecticidal crystal toxins acting on the intestinal barrier. In the Galleria mellonella larvae infection model, toxins from the PlcR virulence regulon contribute to pathogenicity by the oral route. While B. thuringiensis is principally an oral pathogen, bacteria may also reach the insect haemocoel following injury of the cuticle. Here, we address the question of spore virulence as compared to vegetative cells when the wild-type Bt407cry- strain and its isogenic ∆plcR mutant are inoculated directly into G. mellonella haemocoel. Mortality dose-response curves were constructed at 25 and 37 °C using spores or vegetative cell inocula, and the 50% lethal dose (LD50) in all infection conditions was determined after 48 h of infection. Our findings show that (i) the LD50 is lower for spores than for vegetative cells for both strains, while the temperature has no significant influence, and (ii) the ∆plcR mutant is four to six times less virulent than the wild-type strain in all infection conditions. Our results suggest that the environmental resistant spores are the most infecting form in haemocoel and that the PlcR virulence regulon plays an important role in toxicity when reaching the haemocoel from the cuticle and not only following ingestion.

Resistance mechanism development to the topoisomerase-I inhibitor Hoechst 33342 byLeishmania donovani

Parasitology ◽  
2005 ◽  
Vol 131 (2) ◽  
pp. 197-206 ◽  
Author(s):  
J.-F. MARQUIS ◽  
I. HARDY ◽  
M. OLIVIER
Keyword(s):  
Mammalian Cells ◽  
Topoisomerase I ◽  
Resistance Mechanism ◽  
Resistance Mechanisms ◽  
Drug Accumulation ◽  
Wild Type ◽  
Hoechst 33342 ◽  
In The Wild ◽  
Energy Dependent

The bisbenzimidazole compound Hoechst 33342 (Ho342) has been identified as a specific Topoisomerase-I (Topo-I) inhibitor in mammalian cells. More recently, we have reported the ability of Ho342 to targetL. donovaniTopo-I, leading to parasite growth inhibitionin vitroby mechanisms involving DNA breakage and apoptosis-like phenomenon. As the Ho342 lead molecule (2,5′-Bi-1H-benzimidazole) can be used as a starting structure for derivative compounds more effective againstLeishmania, defining the Ho342 resistance mechanism(s) inLeishmaniarepresents an important strategic tool. In the present study, we selected resistant parasites to Ho342 (LdRHo.300). While we observed an increase of the Topo-I gene expression correlated by a higher Topo-I DNA relaxation activity, the Topo-I genes (LdTOP1AandLdTOP1B) sequencing did not reveal any mutation for the resistant parasites. Moreover, our results on Ho342 cellular accumulation suggested the presence of a potential energy-dependent Ho342 transporter in the wild-type parasite, and that an alteration of this transporter has occurred inLdRHo.300, leading to an altered drug accumulation. Collectively, Ho342 resistance characterization provided results supporting that the resistance developed byLdRHo.300involves complex mechanisms, most likely dominated by an altered drug accumulation, providing new insight in the Ho342 resistance mechanisms.

CEP701 and PKC412 Predictably and Reliably Inhibit FLT3 Phosphorylation in Primary AML Blasts but Their Induction of a Cytotoxic Response Appears to Be Much More Variable.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 95-95 ◽  
Author(s):  
Steven Knapper ◽  
Alan K. Burnett ◽  
Amanda F. Gilkes ◽  
Kenneth I. Mills ◽  
Val Walsh
Keyword(s):  
Statistical Significance ◽  
Annexin V ◽  
Expression Level ◽  
Wild Type ◽  
Response Curves ◽  
Allele Ratio ◽  
Cytotoxic Response ◽  
Significant Difference ◽  
Flt3 Expression

Abstract Activating mutations of the receptor tyrosine kinase FLT3 are present in approximately one-third of AML cases and are associated with an adverse prognosis. FLT3 is expressed in over 90% of cases of AML and many non-mutants show evidence of FLT3 activation, which may play a significant signalling role in leukaemogenesis, making FLT3 an attractive therapeutic target. CEP701 (Cephalon) and PKC412 (Novartis) are orally-bioavailable indolocarbazole derivatives that potently inhibit FLT3 phosphorylation. We studied the relationship between in vitro inhibition of FLT3 phosphorylation and induction of cytotoxicity in primary AML blasts from 12 patients. 7 of the cases were FLT3 mutants (6 ITDs and 1 D835 point mutant), the amount of mutant RNA varying between 7% and 84% of total FLT3 RNA expressed. The blasts were exposed for 1 hour to a range of concentrations of CEP701 and PKC412, lysed and immunoprecipitated with an anti-FLT3 antibody. After sequential immunoblotting with anti-phosphotyrosine and anti-FLT3 antibodies, inhibition of FLT3 phosphorylation was measured by densitometry. Both drugs inhibited FLT3 phosphorylation in all samples with lower concentrations required in FLT3 mutants. CEP701 inhibited FLT3 phosphorylation with median IC50s of 3.7nM and 11.9nM in mutant and wild type (WT) cases respectively (p=0.0006). IC50s for PKC412 were 7.7nM and 59.8nM in mutant and WT cases (p=0.0268). Induction of cytotoxicity was assessed by MTS assay following 72-hour exposure of blasts to a range of concentrations of CEP701 and PKC412. Cytotoxic responses to both drugs were greater in FLT3 mutants than WT cases at each dose studied and in terms of IC50 dose (median IC50s in mutant and WT cases: 95nM and 231nM with CEP701, 1.24 μM and 1.61μM with PKC412) although these differences did not reach statistical significance. Annexin V binding apoptosis assay produced similar dose response curves. Both agents showed greater inter-case variability in cytotoxic response than in sensitivity to inhibition of FLT3 phosphorylation. A lack of cytotoxic response to FLT3 inhibition with CEP701 was seen in the ITD mutant with the lowest ratio of mutant to WT FLT3 RNA (0.08) and several WT samples displayed resistance to in vitro induction of cytotoxicity despite almost complete inhibition of FLT3. Induction of cytotoxicity with PKC412 in both mutant and WT cases generally required doses well in excess of those required to fully inhibit FLT3 phosphorylation. Cases were further stratified by flow cytometric measurement of surface FLT3 expression, and by immunoblotting to measure STAT5 dephosphorylation in response to both drugs. No significant difference in overall FLT3 expression was seen between mutant and WT cases. Interestingly the highest FLT3 expression level was seen in a wild type case that was highly sensitive to CEP701. Inhibition of STAT5 phosphorylation appeared closely linked to FLT3 inhibition, although in some cases a good cytotoxic response was achieved despite failure to inhibit STAT5, suggesting involvment of other signalling pathways. In summary, although both CEP701 and PKC412 predictably and reliably inhibit FLT3 phosphorylation in primary AML blasts, their induction of cytotoxicity appears to be much more variable. A number of factors may influence this including variations in level of dependency on FLT3 signalling for blast survival, mutant to WT allele ratio and overall FLT3 expression level. Effects on targets other than FLT3 also need to be considered.

Reduced glutathione oxidation ratio and 8 ohdG accumulation by mild ischemic pretreatment

2000 ◽  
Vol 856 (1-2) ◽  
pp. 28-36 ◽  
Author(s):  
Sun-Hee Baek ◽  
Ji-Yeon Kim ◽  
Jin-Hee Choi ◽  
Eun-Mi Park ◽  
Mi-Young Han ◽  
...  
Keyword(s):  
Reduced Glutathione ◽  
Glutathione Oxidation ◽  
Oxidation Ratio
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