Purification and mass spectrometry identification of leukotriene D4 synthesized by human alveolar macrophages

Human AM obtained by BAL from normal subjects and asthmatic patients converted [1-14C]-AA into a polar labeled metabolite. The structure of this metabolite, after two successive purifications on TLC (silicagel plates then reversed phase plates) and mass spectrometric analysis was shown to be identical to an authentic sample of LTD4. The amount of LTD4 recovered in the culture medium of AM was attempted to be related to pathological lung profile. In our experimental conditions AM from allergic asthmatics synthetized more LTD4 than cells from healthy subjects and from aspirin sensitive asthmatic patients.     

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.     

Stimulation of leukemia inhibitory factor receptor degradation by extracellular signal-regulated kinase

Leukemia inhibitory factor (LIF) signals via the heterodimeric receptor complex comprising the LIF receptor alpha subunit (LIFRalpha) and the common signal transducing subunit for interleukin-6 cytokine receptors, gp130. This study demonstrates that in different cell types, the level of LIFRalpha decreases during treatment with LIF or the closely related cytokine oncostatin M (OSM). Moreover, insulin and epidermal growth factor induce a similar LIFRalpha down-regulation. The regulated loss of LIFRalpha is specific since neither gp130 nor OSM receptor beta shows a comparable change in turnover. LIFRalpha down-regulation correlates with reduced cell responsiveness to LIF. Using protein kinase inhibitors and point mutations in LIFRalpha, we demonstrate that LIFRalpha down-regulation depends on activation of extracellular signal-regulated kinase 1/2 and phosphorylation of the cytoplasmic domain of LIFRalpha at serine 185. This modification appears to promote the endosomal/lysosomal pathway of the LIFRalpha. These results suggest that extracellular signal-regulated kinase-activating factors like OSM and growth factors have the potential to lower specifically LIF responsiveness in vivo by regulating LIFRalpha half-life.     

hxc2, an Arabidopsis mutant with an altered hypersensitive response to Xanthomonas campestris pv. campestris

A chemical mutagenized population of Arabidopsis Col-0-gl plants was screened for an altered hypersensitive response (HR) after spray inoculation with an HR-inducing isolate of Xanthomonas campestris pv. campestris (strain 147). Three classes of mutant were identified: those exhibiting an HR- phenotype or partial loss of HR; hyper-responsive mutants showing necrotic lesions rapidly leading to the collapse of leaves; and susceptible mutants. One mutant belonging to the susceptible class, hxc-2, was extensively characterized. The compatible phenotype observed several days after initiation of the interaction was confirmed by measurement of in planta bacterial growth and use of bacterial strains constitutively expressing the GUS reporter gene. In the same way, accumulation of autofluorescent compounds, salicylic acid production and defence gene expression in the mutant were found to be similar to that displayed by the susceptible ecotype. Inoculation of hxc-2 with different avirulent bacteria suggests that the mutation is specific for the interaction with the Xcc 147 strain, although the mutation has been shown to affect a single dominant locus, different from the resistance locus defined by genetic analysis of resistance to Xcc 147. Genetic mapping of the mutation indicated that it is located on chromosome III, defining a previously unknown resistance function in response to X. c. campestris.     

DNA damage and DNA adduct formation in rat tissues following oral administration of acrylamide

Acrylamide is present as a contaminant in the human diet in heated food products. It has been found to be carcinogenic in laboratory rats and has been classified as probably carcinogenic in humans. In order to clarify the possible involvement of a primary genotoxic mechanism in acrylamide-induced carcinogenicity, both the presence of DNA damage, measured by the comet assay, and the formation of N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade), derived from reaction of the active metabolite glycidamide (GA) with the DNA, analyzed by LC/MS/MS, were assessed in selected rat tissues. Rats were administered with single oral doses of acrylamide (18, 36 or 54 mg/kg body weight (b.w.) and the organs (blood leukocytes, brain, bone marrow, liver, testes and adrenals) were sampled at different times after treatment. Results from GA-induced DNA adduct measurements indicated a relatively even organ distribution of the adducts in brain, testes and liver. Organ-specificity in acrylamide carcinogenesis can therefore not be explained by a selective accumulation of GA-DNA adducts in the target organs, at least not after a single dose exposure. The DNA adduct profiles and half-lives were similar in the different organs; except that the N3-GA-Ade adduct was more rapidly removed from tissues than the N7-GA-Gua adduct. Increased extent of DNA migration, as measured by the in vivo rat comet assay, was found in brain and testes, and these specific results seem to be in accordance with the known organ-specificity in acrylamide carcinogenesis in rat. Only weak and transient DNA damage was recorded in the liver, bone marrow and adrenals. The DNA-damaging effect of the compound observed in the blood leukocytes could be a simple biomarker of acrylamide exposure and genotoxicity.     

Extensive mannose phosphorylation on leukemia inhibitory factor (LIF) controls its extracellular levels by multiple mechanisms

In addition to soluble acid hydrolases, many nonlysosomal proteins have been shown to bear mannose 6-phosphate (Man-6-P) residues. Quantification of the extent of mannose phosphorylation and the relevance to physiological function, however, remain poorly defined. In this study, we investigated the mannose phosphorylation status of leukemia inhibitory factor (LIF), a previously identified high affinity ligand for the cation-independent mannose 6-phosphate receptor (CI-MPR), and we analyzed the effects of this modification on its secretion and uptake in cultured cells. When media from LIF-overexpressing cells were fractionated using a CI-MPR affinity column, 35-45% of the total LIF molecules were bound and specifically eluted with free Man-6-P thus confirming LIF as a bona fide Man-6-P-modified protein. Surprisingly, mass spectrometric analysis of LIF glycopeptides enriched on the CI-MPR column revealed that all six N-glycan sites could be Man-6-P-modified. The relative utilization of these sites, however, was not uniform. Analysis of glycan-deleted LIF mutants demonstrated that loss of glycans bearing the majority of Man-6-P residues leads to higher steady-state levels of secreted LIF. Using mouse embryonic stem cells, we showed that the mannose phosphorylation of LIF mediates its internalization thereby reducing extracellular levels and stimulating embryonic stem cell differentiation. Finally, immunofluorescence experiments indicate that LIF is targeted directly to lysosomes following its biosynthesis, providing another mechanism whereby mannose phosphorylation serves to control extracellular levels of LIF. Failure to modify LIF in the context of mucolipidosis II and its subsequent accumulation in the extracellular space may have important implications for disease pathogenesis.     

Factors associated with an individual's decision to withdraw from genetic testing for breast and ovarian cancer susceptibility: implications for counseling

Our study aimed to examine why individuals withdraw from genetic testing for breast and ovarian cancer susceptibility. We explored the characteristics of 334 individuals from high-risk breast and ovarian cancer families who declined genetic testing for BRCA1/2 mutations, when, and why they did so. Individuals who declined genetic testing were older, and a greater proportion had never developed breast or ovarian cancer. Fifty one per cent (51.1%) of individuals withdrew after the first genetic counseling session. Most of those who declined were afraid of the psychological effects of genetic testing (36.3%). The next most-cited explanations concerned logistic problems such as a limited ability to travel, lack of time, personal issues, advanced age, or health problems (21.7%). The third category included individuals who did not see any advantage in being tested (14.5%). Insurability was a concern (5.9%), mainly for men. Surprisingly, confidentiality was not a frequently reported issue (1.3%). Sixty eight per cent (68%) of individuals belonging to a family in which at least one individual has been tested withdrew after the presence of a deleterious BRCA1/2 mutation in a relative was disclosed, compared to 42% after the disclosure of a nonconclusive test result in at least one relative. Concern about the psychological effects of the result was still one of the major reasons. Several factors may influence an individual's decision to decline genetic testing; a greater understanding of these issues may help health professionals to better meet the needs and concerns of individuals from high-risk families, thus possibly improving their health outcomes.