Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line

Several organochlorinated pesticides including DDT, PCBs and dieldrin have been reported to cause immune suppression and increase susceptibility to infection in animals. Often this manifestation is accompanied by atrophy of major lymphoid organs. It has been suggested that increased apoptotic cell death leading to altered T-B cell ratios, and loss of regulatory cells in critical numbers leads to perturbations in immune function. The major objective of our study was to define the mechanism by which endosulfan, an organochlorinated pesticide, induces human T-cell death using Jurkat, a human T-cell leukemic cell line, as an in vitro model. We exposed Jurkat cells to varying concentrations of endosulfan for 0-48 h and analyzed biochemical and molecular features characteristic of T-cell apoptosis. Endosulfan lowered cell viability and inhibited cell growth in a dose- and time-dependent manner. DAPI staining was used to enumerate apoptotic cells and we observed that endosulfan at 10-200 M induced a significant percentage of cells to undergo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 M of endosulfan. We confirmed these observations using both DNA fragmentation and annexin-V binding assays. It is now widely being accepted that mitochondria undergo major changes early during the apoptotic process. We examined mitochondrial transmembrane potential (_m) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptosis. Within 30 min of chemical exposure, a significant percentage of cells exhibited a decreased incorporation of DiOC₆(3), a cationic lipophilic dye into mitochondria indicating the disruption of _m. This drop in _m was both dose- and time-dependent and correlated well with other parameters of apoptosis. We also examined whether this occurred by the down regulation of bcl-2 protein expression that is likely to increase the susceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the intracellular expression of bcl-2 protein was elevated in a dose dependent manner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathway. Based on these data, as well as those reported elsewhere, we propose the following sequence of events to account for T-cell apoptosis induced by endosulfan: uncoupling of oxidative phosphorylation excess ROS production GSH depletion oxidative stress disruption of _m release of cytochrome C and other apoptosis related proteins to cytosol apoptosis. This study reports for the first time that endosulfan can induce apoptosis in a human T-cell leukemic cell line which may have direct relevance to loss of T cells and thymocytes in vivo. Furthermore, our data strongly support a role of mitochondrial dysfunction and oxidative stress in endosulfan toxicity.     

Mitochondrial targeting of farnesyl diphosphate synthase is a widespread phenomenon in eukaryotes

Cellular internalization of cell-penetrating peptide HIV-1 Tat basic domain (RKKRRQRRR) was studied in Triticale cv AC Alta mesophyll protoplasts. Fluorescently labeled monomer (Tat) and dimer (Tat(2)) of Tat basic domain efficiently translocated through the plasma membrane of mesophyll protoplast and showed distinct nuclear accumulation within 10 min of incubation. Substitution of first arginine residue with alanine in Tat basic domain (M-Tat) severely reduced cellular uptake of the peptide (3.8 times less than Tat). Tat(2) showed greater cellular internalization than Tat (1.6 times higher). However, characteristics of cellular uptake remained same for Tat and Tat(2). Cellular internalization of Tat and Tat(2) was concentration dependent and non-saturable whereas no significant change in cellular uptake was observed even at higher concentrations of M-Tat. Low temperature (4 degrees C) remarkably increased cellular internalization of Tat as well as Tat(2) but M-Tat showed no enhanced uptake. Viability test showed that peptide treatment had no cytotoxic effect on protoplasts further indicating involvement of a common mechanism of peptide uptake at all the temperatures. Endocytic inhibitors nocodazole (10 muM), chloroquine (100 muM) and sodium azide (5 mM) did not show any significant inhibitory effect on cellular internalization of either Tat or Tat(2). These results along with stimulated cellular uptake at low temperature indicate that Tat peptide is internalized in the plant protoplasts in a non-endocytic and energy-independent manner. Competition experiments showed that non-labeled peptide did not inhibit or alter nuclear accumulation of fluorescent Tat or Tat(2) suggesting active transport to the nucleus was not involved. Studies in mesophyll protoplasts show that internalization pattern of Tat peptide is apparently similar to that observed in mammalian cell lines.     

Molecular Identification of Bacteria by Total Sequence Screening: Determining the Cause of Death in Ancient Human Subjects

Research of ancient pathogens in ancient human skeletons has been mainly carried out on the basis of one essential historical or archaeological observation, permitting specific pathogens to be targeted. Detection of ancient human pathogens without such evidence is more difficult, since the quantity and quality of ancient DNA, as well as the environmental bacteria potentially present in the sample, limit the analyses possible. Using human lung tissue and/or teeth samples from burials in eastern Siberia, dating from the end of 17^th to the 19^th century, we propose a methodology that includes the: 1) amplification of all 16S rDNA gene sequences present in each sample; 2) identification of all bacterial DNA sequences with a degree of identity ≥95%, according to quality criteria; 3) identification and confirmation of bacterial pathogens by the amplification of the rpoB gene; and 4) establishment of authenticity criteria for ancient DNA. This study demonstrates that from teeth samples originating from ancient human subjects, we can realise: 1) the correct identification of bacterial molecular sequence signatures by quality criteria; 2) the separation of environmental and pathogenic bacterial 16S rDNA sequences; 3) the distribution of bacterial species for each subject and for each burial; and 4) the characterisation of bacteria specific to the permafrost. Moreover, we identified three pathogens in different teeth samples by 16S rDNA sequence amplification: Bordetella sp., Streptococcus pneumoniae and Shigella dysenteriae. We tested for the presence of these pathogens by amplifying the rpoB gene. For the first time, we confirmed sequences from Bordetella pertussis in the lungs of an ancient male Siberian subject, whose grave dated from the end of the 17^th century to the early 18^th century.     

Short/branched-chain acyl-CoA dehydrogenase deficiency due to an IVS3+3A>G mutation that causes exon skipping

Short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD) is an autosomal recessive disorder of l-isoleucine catabolism. Little is known about the clinical presentation associated with this enzyme defect, as it has been reported in only a limited number of patients. Because the presence of C5-carnitine in blood may indicate SBCADD, the disorder may be detected by MS/MS-based routine newborn screening. It is, therefore, important to gain more knowledge about the clinical presentation and the mutational spectrum of SBCADD. In the present study, we have studied two unrelated families with SBCADD, both with seizures and psychomotor delay as the main clinical features. One family illustrates the fact that affected individuals may also remain asymptomatic. In addition, the normal level of newborn blood spot C5-acylcarnitine in one patient underscores the fact that newborn screening by MS/MS currently lacks sensitivity in detecting SBCADD. Until now, seven mutations in the SBCAD gene have been reported, but only three have been tested experimentally. Here, we identify and characterize an IVS3+3A>G mutation (c.303+3A>G) in the SBCAD gene, and provide evidence that this mutation is disease-causing in both families. Using a minigene approach, we show that the IVS3+3A>G mutation causes exon 3 skipping, despite the fact that it does not appear to disrupt the consensus sequence of the 5′ splice site. Based on these results and numerous literature examples, we suggest that this type of mutation (IVS+3A>G) induces missplicing only when in the context of non-consensus (weak) 5′ splice sites. Statistical analysis of the sequences shows that the wild-type versions of 5′ splice sites in which +3A>G mutations cause exon skipping and disease are weaker on average than a random set of 5′ splice sites. This finding is relevant to the interpretation of the functional consequences of this type of mutation in other disease genes.     

Reporting randomised clinical trials of analgesics after traumatic or orthopaedic surgery is inadequate: a systematic review

Background  

Several randomised clinical trials (RCTs) of analgesics in postoperative pain after traumatic or orthopaedic surgery (TOS) have been published, but no studies have assessed the quality of these reports. We aimed to examine the quality of reporting RCTs on analgesics for postoperative pain after TOS.     

Ambient Air Pollution and the Progression of Atherosclerosis in Adults

Background

Cross-sectional studies suggest an association between exposure to ambient air pollution and atherosclerosis. We investigated the association between outdoor air quality and progression of subclinical atherosclerosis (common carotid artery intima-media thickness, CIMT).

Methodology/Principal Findings

We examined data from five double-blind randomized trials that assessed effects of various treatments on the change in CIMT. The trials were conducted in the Los Angeles area. Spatial models and land-use data were used to estimate the home outdoor mean concentration of particulate matter up to 2.5 micrometer in diameter (PM2.5), and to classify residence by proximity to traffic-related pollution (within 100 m of highways). PM2.5 and traffic proximity were positively associated with CIMT progression. Adjusted coefficients were larger than crude associations, not sensitive to modelling specifications, and statistically significant for highway proximity while of borderline significance for PM2.5 (P = 0.08). Annual CIMT progression among those living within 100 m of a highway was accelerated (5.5 micrometers/yr [95%CI: 0.13–10.79; p = 0.04]) or more than twice the population mean progression. For PM2.5, coefficients were positive as well, reaching statistical significance in the socially disadvantaged; in subjects reporting lipid lowering treatment at baseline; among participants receiving on-trial treatments; and among the pool of four out of the five trials.

Conclusion

Consistent with cross-sectional findings and animal studies, this is the first study to report an association between exposure to air pollution and the progression of atherosclerosis – indicated with CIMT change – in humans. Ostensibly, our results suggest that air pollution may contribute to the acceleration of cardiovascular disease development – the main causes of morbidity and mortality in many countries. However, the heterogeneity of the volunteering populations across the five trials, the limited sample size within trials and other relevant subgroups, and the fact that some key findings reached statistical significance in subgroups rather than the sample precludes generalizations to the general population.     

A Meta-Analysis of the Relationship between FGFR3 and TP53 Mutations in Bladder Cancer

TP53 and FGFR3 mutations are the most common mutations in bladder cancers. FGFR3 mutations are most frequent in low-grade low-stage tumours, whereas TP53 mutations are most frequent in high-grade high-stage tumours. Several studies have reported FGFR3 and TP53 mutations to be mutually exclusive events, whereas others have reported them to be independent. We carried out a meta-analysis of published findings for FGFR3 and TP53 mutations in bladder cancer (535 tumours, 6 publications) and additional unpublished data for 382 tumours. TP53 and FGFR3 mutations were not independent events for all tumours considered together (OR = 0.25 [0.18–0.37], p = 0.0001) or for pT1 tumours alone (OR = 0.47 [0.28–0.79], p = 0.0009). However, if the analysis was restricted to pTa tumours or to muscle-invasive tumours alone, FGFR3 and TP53 mutations were independent events (OR = 0.56 [0.23–1.36] (p = 0.12) and OR = 0.99 [0.37–2.7] (p = 0.35), respectively). After stratification of the tumours by stage and grade, no dependence was detected in the five tumour groups considered (pTaG1 and pTaG2 together, pTaG3, pT1G2, pT1G3, pT2-4). These differences in findings can be attributed to the putative existence of two different pathways of tumour progression in bladder cancer: the CIS pathway, in which FGFR3 mutations are rare, and the Ta pathway, in which FGFR3 mutations are frequent. TP53 mutations occur at the earliest stage of the CIS pathway, whereas they occur would much later in the Ta pathway, at the T1G3 or muscle-invasive stage.