Copper stress causes an in vivo requirement for the Escherichia coli disulfide isomerase DsbC

In Escherichia coli, the periplasmic disulfide oxidoreductase DsbA is thought to be a powerful but nonspecific oxidant, joining cysteines together the moment they enter the periplasm. DsbC, the primary disulfide isomerase, likely resolves incorrect disulfides. Given the reliance of protein function on correct disulfide bonds, it is surprising that no phenotype has been established for null mutations in dsbC. Here we demonstrate that mutations in the entire DsbC disulfide isomerization pathway cause an increased sensitivity to the redox-active metal copper. We find that copper catalyzes periplasmic disulfide bond formation under aerobic conditions and that copper catalyzes the formation of disulfide-bonded oligomers in vitro, which DsbC can resolve. Our data suggest that the copper sensitivity of dsbC- strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfides in the absence of functional DsbC. Absence of functional DsbA augments the deleterious effects of copper on a dsbC- strain, even though the dsbA- single mutant is unaffected by copper. This may indicate that DsbA successfully competes with copper and forms disulfide bonds more accurately than copper does. These findings lead us to a model in which DsbA may be significantly more accurate in disulfide oxidation than previously thought, and in which the primary role of DsbC may be to rearrange incorrect disulfide bonds that are formed during certain oxidative stresses.     

Roscovitine targets, protein kinases and pyridoxal kinase

(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.     

V gamma 9V delta 2 T cell response to colon carcinoma cells

During analysis of CD8 T cells derived from ascites of a colon cancer patient, we isolated a Vgamma9Vdelta2 T cell clone showing strong reactivity against autologous tumor cell lines. This clone killed a large fraction of allogeneic colon carcinoma and melanoma cell lines, but did not affect a normal colon cell line, colon fibroblasts, or melanocytes. Tumor cell recognition was TCR and NKG2D dependent and induced TNF-alpha and IFN-gamma secretion by the clone; accordingly, tumor targets expressed several NKG2D ligands, such as MHC class I chain-related gene A and UL16-binding protein molecules. Colon tumor recognition by Vgamma9Vdelta2 T cells was highly dependent on isopentenyl pyrophosphate production and ICAM-1 expression by target cells. Finally, similar reactivity patterns against colon carcinoma cell lines were observed using polyclonal Vgamma9Vdelta2 T cells of various origins, and Vgamma9Vdelta2 lymphocytes were present in the majority of colon tumor samples studied. Together, these results suggest that Vgamma9Vdelta2 T cells contribute to the natural immune surveillance against colon cancers. Therefore, this study provides a strong rationale for the use of Vgamma9Vdelta2 T cell agonists in immunotherapies targeting colon tumors.     

Plasmodium falciparum AMA1 binds a rhoptry neck protein homologous to TgRON4, a component of the moving junction in Toxoplasma gondii

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) coimmunoprecipitates with the Plasmodium homologue of TgRON4, a secreted rhoptry neck protein of Toxoplasma gondii that migrates at the moving junction in association with TgAMA1 during invasion. PfRON4 also originates in the rhoptry necks, suggesting that this unusual collaboration of micronemes and rhoptries is a conserved feature of Apicomplexa.     

Development of enzymatic probes of oxidative and nitrosative DNA damage caused by reactive nitrogen species

Chronic inflammation is associated with a variety of human diseases, including cancer, with one possible mechanistic link involving over-production of nitric oxide (NO*) by activated macrophages. Subsequent reaction of NO* with superoxide in the presence of carbon dioxide yields nitrosoperoxycarbonate (ONOOCO2-), a strong oxidant that reacts with guanine in DNA to form a variety of oxidation and nitration products, such 2'-deoxy-8-oxoguanosine. Alternatively, the reaction of NO and O2 leads to the formation of N2O3, a nitrosating agent that causes nucleobase deamination to form 2'-deoxyxanthosine (dX) and 2'-deoxyoxanosine (dO) from dG; 2'-deoxyinosine (dI) from dA; and 2'-deoxyuridine (dU) from dC, in addition to abasic sites and dG-dG cross-links. The presence of both ONOOCO2- and N2O3 at sites of inflammation necessitates definition of the relative roles of oxidative and nitrosative DNA damage in the genetic toxicology of inflammation. To this end, we sought to develop enzymatic probes for oxidative and nitrosative DNA lesions as a means to quantify the two types of DNA damage in in vitro DNA damage assays, such as the comet assay and as a means to differentially map the lesions in genomic DNA by the technique of ligation-mediated PCR. On the basis of fragmentary reports in the literature, we first systematically assessed the recognition of dX and dI by a battery of DNA repair enzymes. Members of the alkylpurine DNA glycosylase family (E. coli AlkA, murine Aag, and human MPG) all showed repair activity with dX (k(cat)/Km 29 x 10(-6), 21 x 10(-6), and 7.8 x 10(-6) nM(-1) min(-1), respectively), though the activity was considerably lower than that of EndoV (8 x 10(-3) nM(-1) min(-1)). Based on these results and other published studies, we focused the development of enzymatic probes on two groups of enzymes, one with activity against oxidative damage (formamidopyrimidine-DNA glycosylase (Fpg); endonuclease III (EndoIII)) and the other with activity against nucleobase deamination products (uracil DNA glycosylase (Udg); AlkA). These combinations were assessed for recognition of DNA damage caused by N2O3 (generated with a NO*/O2 delivery system) or ONOOCO2- using a plasmid nicking assay and by LC-MS analysis. Collectively, the results indicate that a combination of AlkA and Udg react selectively with DNA containing only nitrosative damage, while Fpg and EndoIII react selectively with DNA containing oxidative base lesions caused by ONOOCO2-. The results suggest that these enzyme combinations can be used as probes to define the location and quantity of the oxidative and nitrosative DNA lesions produced by chemical mediators of inflammation in systems, such as the comet assay, ligation-mediated polymerase chain reaction, and other assays of DNA damage and repair.     

Solution structure and biophysical properties of MqsA, a Zn-containing antitoxin from Escherichia coli

The gene ygiT (mqsA) of Escherichia coli encodes MqsA, the antitoxin of the motility quorum sensing regulator (MqsR). Both proteins are considered to form a DNA binding complex and to be involved in the formation of biofilms and persisters. We have determined the three-dimensional solution structure of MqsA by high-resolution NMR. The protein comprises a well-defined N-terminal domain with a Zn finger motif usually found in eukaryotes, and a defined C-terminal domain with a typical prokaryotic DNA binding helix-turn-helix motif. The two well-defined domains of MqsA have almost identical structure in solution and in the two published crystal structures of dimeric MqsA bound to either MqsR or DNA. However, the connection of the two domains with a flexible linker yields a large variety of possible conformations in solution, which is not reflected in the crystal structures. MqsA binds Zn with all four cysteines, a stoichiometry of 1:1 and a femtomolar affinity (K(a)≥10(17)M(-1) at 23°C, pH 7.0).     

Long time to diagnosis of medulloblastoma in children is not associated with decreased survival or with worse neurological outcome

Background

The long time to diagnosis of medulloblastoma, one of the most frequent brain tumors in children, is the source of painful remorse and sometimes lawsuits. We analyzed its consequences for tumor stage, survival, and sequelae.

Patients and Methods

This retrospective population-based cohort study included all cases of pediatric medulloblastoma from a region of France between 1990 and 2005. We collected the demographic, clinical, and tumor data and analyzed the relations between the interval from symptom onset until diagnosis, initial disease stage, survival, and neuropsychological and neurological outcome.

Results

The median interval from symptom onset until diagnosis for the 166 cases was 65 days (interquartile range 31–121, range 3–457). A long interval (defined as longer than the median) was associated with a lower frequency of metastasis in the univariate and multivariate analyses and with a larger tumor volume, desmoplastic histology, and longer survival in the univariate analysis, but not after adjustment for confounding factors. The time to diagnosis was significantly associated with IQ score among survivors. No significant relation was found between the time to diagnosis and neurological disability. In the 62 patients with metastases, a long prediagnosis interval was associated with a higher T stage, infiltration of the fourth ventricle floor, and incomplete surgical resection; it nonetheless did not influence survival significantly in this subgroup.

Conclusions

We found complex and often inverse relations between time to diagnosis of medulloblastoma in children and initial severity factors, survival, and neuropsychological and neurological outcome. This interval appears due more to the nature of the tumor and its progression than to parental or medical factors. These conclusions should be taken into account in the information provided to parents and in expert assessments produced for malpractice claims.     

A Novel Insight into the Oxidoreductase Activity of Helicobacter pylori HP0231 Protein

Background

The formation of a disulfide bond between two cysteine residues stabilizes protein structure. Although we now have a good understanding of the Escherichia coli disulfide formation system, the machineries at work in other bacteria, including pathogens, are poorly characterized. Thus, the objective of this work was to improve our understanding of the disulfide formation machinery of Helicobacter pylori, a leading cause of ulcers and a risk factor for stomach cancer worldwide.

Methods and Results

The protein HP0231 from H. pylori, a structural counterpart of E. coli DsbG, is the focus of this research. Its function was clarified by using a combination of biochemical, microbiological and genetic approaches. In particular, we determined the biochemical properties of HP0231 as well as its redox state in H. pylori cells.

Conclusion

Altogether our results show that HP0231 is an oxidoreductase that catalyzes disulfide bond formation in the periplasm. We propose to call it HpDsbA.     

An international reference consensus genetic map with 897 marker loci based on 11 mapping populations for tetraploid groundnut (Arachis hypogaea L.)

Only a few genetic maps based on recombinant inbred line (RIL) and backcross (BC) populations have been developed for tetraploid groundnut. The marker density, however, is not very satisfactory especially in the context of large genome size (2800 Mb/1C) and 20 linkage groups (LGs). Therefore, using marker segregation data for 10 RILs and one BC population from the international groundnut community, with the help of common markers across different populations, a reference consensus genetic map has been developed. This map is comprised of 897 marker loci including 895 simple sequence repeat (SSR) and 2 cleaved amplified polymorphic sequence (CAPS) loci distributed on 20 LGs (a01-a10 and b01-b10) spanning a map distance of 3, 863.6 cM with an average map density of 4.4 cM. The highest numbers of markers (70) were integrated on a01 and the least number of markers (21) on b09. The marker density, however, was lowest (6.4 cM) on a08 and highest (2.5 cM) on a01. The reference consensus map has been divided into 20 cM long 203 BINs. These BINs carry 1 (a10_02, a10_08 and a10_09) to 20 (a10_04) loci with an average of 4 marker loci per BIN. Although the polymorphism information content (PIC) value was available for 526 markers in 190 BINs, 36 and 111 BINs have at least one marker with >0.70 and >0.50 PIC values, respectively. This information will be useful for selecting highly informative and uniformly distributed markers for developing new genetic maps, background selection and diversity analysis. Most importantly, this reference consensus map will serve as a reliable reference for aligning new genetic and physical maps, performing QTL analysis in a multi-populations design, evaluating the genetic background effect on QTL expression, and serving other genetic and molecular breeding activities in groundnut.