Synthesis of phytochelatins in vetiver grass upon lead exposure in the presence of phosphorus

In a hydroponic setting, we investigated the possible role of phytochelatins (metal-binding peptides) in the lead (Pb) tolerance of vetiver grass (Vetiveria zizanioides L.). Pb was added to the nutrient medium at concentrations ranging from 0 to 1,200 mg L^?1. Furthermore, we simulated the effect of soil phosphorus (P) on potentially plant available Pb by culturing vetiver grass in P-rich nutrient media. After 7 days of exposure to Pb, we evaluated the Pb uptake by vetiver grass. Results indicate that vetiver can accumulate Pb up to 3,000 mg kg^?1 dry weight in roots with no toxicity. Formation of lead phosphate inhibited Pb uptake by vetiver, suggesting the need for an environmentally safe chelating agent in conjunction with phytoremediation to clean up soils contaminated with lead-based paint. Unambiguous characterization of phytochelatins (PC_n) was possible using high pressure liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESMS). Vetiver shows qualitative and quantitative differences in PC_n synthesis between root and shoot. In root tissue from vetiver exposed to 1,200 mg Pb L^-1, phytochelatins ranged from PC₁ to PC₃. Collision-induced dissociation of the parent ion allowed confirmation of each PC_n based on the amino acid sequence. Possible Pb-PC₁ and Pb₂-PC₁ complexes were reported in vetiver root at the highest Pb concentration. The data from these experiments show that the most probable mechanism for Pb detoxification in vetiver is by synthesizing PC_n and forming Pb–PC_n complexes.     

Sustained phosphorylation of tyrosine hydroxylase at serine 40: a novel mechanism for maintenance of catecholamine synthesis

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine synthesis. Its activity is known to be controlled acutely (minutes) by phosphorylation and chronically (days) by protein synthesis. Using bovine adrenal chromaffin cells we found that nicotine, acting via nicotinic receptors, sustained the phosphorylation of TH at Ser40 for up to 48 h. Nicotine also induced sustained activation of TH, which for the first 24 h was completely independent of TH protein synthesis, and the phosphorylation of TH at Ser31. Imipramine did not inhibit the acute phosphorylation of TH at Ser40 or TH activation induced by nicotine, but did inhibit the sustained responses to nicotine seen at 24 h. The protein kinase(s) responsible for TH phosphorylation at Ser40 switched from being protein kinase C (PKC) independent in the acute phase to PKC dependent in the sustained phase. Sustained phosphorylation and activation of TH were also observed with histamine and angiotensin II. Sustained phosphorylation of TH at Ser40 provides a novel mechanism for increasing TH activity and this leads to increased catecholamine synthesis. Sustained phosphorylation of TH may be a selective target for drugs or pathology in neurons that contain TH and synthesize dopamine, noradrenaline or adrenaline.     

IrAE: an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus

Ticks are ectoparasitic blood-feeders and important vectors for pathogens including arboviruses, rickettsiae, spirochetes and protozoa. As obligate blood-feeders, one possible strategy to retard disease transmission is disruption of the parasite's ability to digest host proteins. However, the constituent peptidases in the parasite gut and their potential interplay in the digestion of the blood meal are poorly understood. We have characterised a novel asparaginyl endopeptidase (legumain) from the hard tick Ixodes ricinus (termed IrAE), which we believe is the first such characterisation of a clan CD family C13 cysteine peptidase (protease) in arthropods. By RT-PCR of different tissues, IrAE mRNA was only expressed in the tick gut. Indirect immunofluorescence and EM localised IrAE in the digestive vesicles of gut cells and within the peritrophic matrix. IrAE was functionally expressed in Pichia pastoris and reacted with a specific peptidyl fluorogenic substrate, and acyloxymethyl ketone and aza-asparagine Michael acceptor inhibitors. IrAE activity was unstable at pH > or = 6.0 and was shown to have a strict specificity for asparagine at P1 using a positional scanning synthetic combinatorial library. The enzyme hydrolyzed protein substrates with a pH optimum of 4.5, consistent with the pH of gut cell digestive vesicles. Thus, IrAE cleaved the major protein of the blood meal, hemoglobin, to a predominant peptide of 4kDa. Also, IrAE trans-processed and activated the zymogen form of Schistosoma mansoni cathepsin B1 -- an enzyme contributing to hemoglobin digestion in the gut of that bloodfluke. The possible functions of IrAE in the gut digestive processes of I. ricinus are compared with those suggested for other hematophagous parasites.     

Schistosoma comparative genomics: integrating genome structure, parasite biology and anthelmintic discovery

Schistosoma genomes provide a comprehensive resource for identifying the molecular processes that shape parasite evolution and for discovering novel chemotherapeutic or immunoprophylactic targets. Here, we demonstrate how intragenus and intergenus comparative genomics can be used to drive these investigations forward, illustrate the advantages and limitations of these approaches and review how post-genomic technologies offer complementary strategies for genome characterisation. Although sequencing and functional characterisation of other schistosome/platyhelminth genomes continues to expedite anthelmintic discovery, we contend that future priorities should equally focus on improving assembly quality, and chromosomal assignment, of existing schistosome/platyhelminth genomes.     

Air trapping on chest CT is associated with worse ventilation distribution in infants with cystic fibrosis diagnosed following newborn screening

Background

In school-aged children with cystic fibrosis (CF) structural lung damage assessed using chest CT is associated with abnormal ventilation distribution. The primary objective of this analysis was to determine the relationships between ventilation distribution outcomes and the presence and extent of structural damage as assessed by chest CT in infants and young children with CF.

Methods

Data of infants and young children with CF diagnosed following newborn screening consecutively reviewed between August 2005 and December 2009 were analysed. Ventilation distribution (lung clearance index and the first and second moment ratios [LCI, M₁/M₀ and M₂/M₀, respectively]), chest CT and airway pathology from bronchoalveolar lavage were determined at diagnosis and then annually. The chest CT scans were evaluated for the presence or absence of bronchiectasis and air trapping.

Results

Matched lung function, chest CT and pathology outcomes were available in 49 infants (31 male) with bronchiectasis and air trapping present in 13 (27%) and 24 (49%) infants, respectively. The presence of bronchiectasis or air trapping was associated with increased M₂/M₀ but not LCI or M₁/M₀. There was a weak, but statistically significant association between the extent of air trapping and all ventilation distribution outcomes.

Conclusion

These findings suggest that in early CF lung disease there are weak associations between ventilation distribution and lung damage from chest CT. These finding are in contrast to those reported in older children. These findings suggest that assessments of LCI could not be used to replace a chest CT scan for the assessment of structural lung disease in the first two years of life. Further research in which both MBW and chest CT outcomes are obtained is required to assess the role of ventilation distribution in tracking the progression of lung damage in infants with CF.     

Proteomic identification of IPSE/alpha-1 as a major hepatotoxin secreted by Schistosoma mansoni eggs

Background

Eggs deposited in the liver of the mammalian host by the blood fluke parasite, Schistosoma mansoni, normally drive a T-helper-2 (Th2)-mediated granulomatous response in immune-competent mice. By contrast, in mice deprived of T-cells and incapable of producing granulomata, egg-secreted proteins (ESP) induce acute hepatic injury and death. Previous work has shown that one such ESP, the T2 ribonuclease known as omega-1, is hepatotoxic in vivo in that specific antisera to omega-1 prevent hepatocyte damage.

Methodology/Principal Findings

Using an in vitro culture system employing mouse primary hepatocytes and alanine transaminase (ALT) activity as a marker of heptocyte injury, we demonstrated that S. mansoni eggs, egg-secreted proteins (ESP), soluble-egg antigen (SEA), and omega-1 are directly hepatotoxic and in a dose-dependent manner. Depletion of omega-1 using a monoclonal antibody abolished the toxicity of pure omega-1 and diminished the toxicity in ESP and SEA by 47 and 33%, respectively. Anion exchange chromatography of ESP yielded one predominant hepatotoxic fraction. Proteomics of that fraction identified the presence of IPSE/alpha-1 (IL-4 inducing principle from S. mansoni eggs), a known activator of basophils and inducer of Th2-type responses. Pure recombinant IPSE/alpha-1 also displayed a dose-dependent hepatotoxicity in vitro. Monoclonal antibody depletion of IPSE/alpha-1 abolished the latter''s toxicity and diminished the total toxicity of ESP and SEA by 32 and 35%, respectively. Combined depletion of omega-1 and IPSE/alpha-1 diminished hepatotoxicity of ESP and SEA by 60 and 58% respectively.

Conclusions

We identified IPSE/alpha-1 as a novel hepatotoxin and conclude that both IPSE/alpha-1 and omega-1 account for the majority of the hepatotoxicity secreted by S. mansoni eggs.     

Oxidative damage to DNA related to survivorship and carotenoid-based sexual ornamentation in the common yellowthroat

Carotenoid-based sexual ornaments are hypothesized to be reliable signals of male quality, based on an allocation trade-off between the use of carotenoids as pigments and their use in antioxidant defence against reactive oxygen species. Carotenoids appear to be poor antioxidants in vivo, however, and it is not clear whether variation in ornament expression is correlated with measures of oxidative stress (OXS) under natural conditions. We used single-cell gel electrophoresis to assay oxidative damage to erythrocyte DNA in the common yellowthroat (Geothlypis trichas), a sexually dichromatic warbler in which sexual selection favours components of the males' yellow 'bib'. We found that the level of DNA damage sustained by males predicted their overwinter survivorship and was reflected in the quality of their plumage. Males with brighter yellow bibs showed lower levels of DNA damage, both during the year the plumage was sampled (such that yellow brightness signalled current OXS) and during the previous year (such that yellow brightness signalled past OXS). We suggest that carotenoid-based ornaments can convey information about OXS to prospective mates and that further work exploring the proximate mechanism(s) linking OXS to coloration is warranted.     

Hypertonic Saline Attenuates Colonic Tumor Cell Metastatic Potential by Activating Transmembrane Sodium Conductance

Hypertonic saline (HTS) suppresses tumor cell-endothelial interactions by reducing integrin expression. This translates into reduced adhesion, migration and metastatic potential. This study determined the relative contributions of hyperosmolarity and sodium-specific hypertonicity on the inhibitory effects of HTS, the intracellular pH and sodium responses to HTS and the role of cytoskeletal remodeling in these changes. Human colonic tumor cells (LS174T) were exposed to lipopolysaccharide under isotonic, hypertonic, sodium-free (N-methyl- D-glucamine), hyperosmolar (mannitol or urea), disrupted cytoskeletal (10 μg/ml cytochalasin D) conditions or in the presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA). β₁ integrin expression was measured flow-cytometrically. Intracellular sodium and pH were measured with confocal laser microscopic imaging. Statistical analysis was performed with analysis of variance, and P < 0.05 was considered significant. Data are represented as mean ± SEM. Hypertonic exposure attenuated integrin expression (62.03 ± 4.7% of control, P < 0.04). No discernible effect was observed with sodium-free or hyperosmolar solutions. HTS evoked a cellular alkalinization (by a mean 0.2 pH units) and an increase in cytosolic sodium concentration (by a mean 12.4 mM, P < 0.001) via upregulation of sodium-hydrogen exchange. Disassembly of actin microfilaments by cytochalasin D and antiporter inhibition with EIPA abrogated the effect of hypertonicity on integrin expression and intracellular sodium and pH (P < 0.05). HTS downregulates adhesion molecule expression via a hypertonic, sodium-specific, cytoskeletally mediated mechanism that involves activation of sodium-hydrogen exchange with associated changes in intracellular pH and sodium concentrations.