Species adaptation in serpentine soils in Lesbos Island (Greece): metal hyperaccumulation and tolerance

Serpentine (ultramafic) soils, containing relatively high nickel and other metal concentrations, present a stressful environment for plant growth but also a preferred substrate for some plants which accumulate nickel in their tissues. In the present study we focused on: (1) the relationships between serpentine soils of Lesbos Island (Greece) and serpentinophilic species in order to test their adaptation to the ‘serpentine syndrome’, and (2) the Ni-hyperaccumulation capacity of Alyssum lesbiacum, a serpentine endemic, Ni-hyperaccumulating species, recorded over all its distribution for the first time. We sampled soil and the most abundant plant species from the four serpentine localities of Lesbos Island. Soil and leaf elemental concentrations were measured across all the sites. Our results confirmed our hypothesis that serpentinophilic species are adapted to elevated heavy metal soil concentrations but restricting heavy metal concentration in their leaves. We demonstrated that different A. lesbiacum populations from Lesbos Island present differences in Ni hyperaccumulation according to soil Ni availability. Our results highlighted the understanding of serpentine ecosystems through an extensive field study in an unexplored area. Alyssum lesbiacum and Thlaspi ochroleucum emerge as two strong Ni hyperaccumulators with the former having a high potential for phytoextraction purposes.     

Global similarity and local divergence in human and mouse gene co-expression networks

Background  

A genome-wide comparative analysis of human and mouse gene expression patterns was performed in order to evaluate the evolutionary divergence of mammalian gene expression. Tissue-specific expression profiles were analyzed for 9,105 human-mouse orthologous gene pairs across 28 tissues. Expression profiles were resolved into species-specific coexpression networks, and the topological properties of the networks were compared between species.     

Urinary excretion of three nucleic acid oxidation adducts and isoprostane F(2)alpha measured by liquid chromatography-mass spectrometry in smokers, ex-smokers, and nonsmokers

To assess novel liquid chromatography/mass spectrometric methods for measuring oxidative damage to nucleic acids and lipids, we compared urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 5-hydroxymethyl-2'-deoxyuridine (5-OHmU), and 8-hydroxyguanosine (8-OxoG), and an isoprostane, 8-iso-prostaglandin F(2)alpha (IsopF(2)alpha) in 234 healthy men (n = 113) and women (n = 121), 80 current smokers, 96 never-smokers), and 58 ex-smokers (no tobacco use for 3 years). The 8-OHdG and 8-OxoG did not differ significantly by group; 5-OHmU was higher in smokers, compared with ex- (p <.003) and never- (p <.0001) smokers and in ex- vs. never-smokers (p =.014) at, respectively, 13.5 +/- 0.7, 11.3 +/- 1.0, and 8.7 +/- 0.3 microg/g creatinine. IsopF(2)alpha was higher in smokers, compared with ex- (p =.007) and never-smokers (p <.0001) and in ex- vs. never- smokers (p =.002) at, respectively, 1.1 +/- 0.10; 0.74 +/- 0.07, and 0.51 +/- 0.04 microg/g creatinine. There were significant correlations among all three nucleic acid adducts and between IsopF(2)alpha and both 5-OHmU and 8-OHdG. Many smokers and ex-smokers had high levels of either 5-OHmU excretion or IsopF(2)alpha excretion, but not both. We conclude that 5-OHmU and IsopF(2)alpha are more discriminating of oxidative stress from tobacco smoke than the other two compounds measured. Whether characteristic patterns of excretion of these indicators forecast differential disease risk should be explored in future research.     

Isolation and characterization of a novel bladder cancer cell line: Inhibition by epidermal growth factor

Summary A novel continuous cell line, designated BC3c, was established from a surgical biopsy of an invasive solid transitional cell carcinoma of the bladder derived from an 82-yr-old Caucasian female. BC3c cells were near-triploid bearing multiple structural and numerical chromosome anomalies. The epithelial origin of the cancer cells was indicated by the expression of cytokeratins 8 and 19 as well as by the absence of mesenchymal markers. Polymerase chain reaction-restriction-fragment length polymorphisms and single-strand conformation polymorphism mutation detection assays did not reveal any mutations in H-ras codon 12 and K-ras codons 12 and 13. In addition, no mutation in specific hot-spot codons of the p53 gene and no accumulation of the p53 protein were observed. BC3c cells grew rapidly in vitro, even in the absence of exogenous growth factors, because they were found to stimulate their growth in an autocrine manner. BC3c cells were found to express the epidermal growth factor-receptor (EGF-r) abundantly, but in contrast to other established bladder cancer cell lines, human recombinant epidermal growth factor inhibited the cells’ proliferation in vitro. These features render the newly established bladder cancer cell line BC3c a useful tool for further experimentation.     

Comparative analysis of signal transduction by CD40 and the Epstein-Barr virus oncoprotein LMP1 in vivo

There is much evidence, based primarily on in vitro studies, indicating that the Epstein-Barr virus oncoprotein latent membrane protein 1 (LMP1) mimics an activated CD40 receptor. In order to investigate the extent of similarity between LMP1 and CD40 functions in vivo, we analyzed the cytoplasmic signaling properties of LMP1 and CD40 in B cells in a directly comparable manner. For this purpose, we generated transgenic mice expressing either LMP1 or a chimeric LMP1CD40 molecule, which constitutively activates the CD40 pathway, under the control of the CD19 promoter. LMP1 and LMP1CD40 were expressed at similar levels in a B-lymphocyte-specific manner. Similar to LMP1, LMP1CD40 suppressed germinal center (GC) formation and antibody production in response to thymus-dependent antigens, albeit to a greater extent than LMP1. Furthermore, the avidity of the antibodies produced against thymus-dependent antigens was lower for LMP1CD40 transgenic mice than for wild-type and LMP1 transgenic mice. GC suppression was linked to the ability of LMP1CD40 and LMP1 to downregulate mRNA and protein levels of BCL6 and to suppress the activity of the BCL6 promoter. In contrast to LMP1, LMP1CD40 caused an upregulation of CD69, CD80, and CD86 in B cells and a dramatic increase in serum immunoglobulin M. In addition, LMP1CD40 but not LMP1 transgenic mice had elevated numbers of marginal-zone B cells and increased populations of polymorphonuclear cells and/or neutrophils. Consistent with these findings, LMP1CD40 but not LMP1 transgenic mice showed signs of spontaneous inflammatory reactions and the potential for autoimmunity.     

Analysis of glycoprotein hormone receptor extracellular domain interactions using a solid-phase capture assay

The receptors for the glycoprotein hormones are unique in having a large extracellular domain that is responsible for mediating ligand binding. We describe the characterization, validation, and application of a solid-phase radioligand binding assay that can be used to assess the interaction of peptides and small molecules at the extracellular domain (ECD) of the follicle-stimulating hormone receptor (FSHR). The assay utilizes a C-terminal tag on the FSHR-ECD, which is used to capture the ECD and position it in a sterically favorable orientation on a solid-phase platform. Competition experiments with the cognate ligand, FSH, indicated that the interaction at the FSHR-ECD using the solid-phase assay was comparable to the full-length receptor assayed using a standard filtration assay. The utility of the assay was evaluated by competing several peptides and a small molecule for both the full-length FSHR and the FSHR-ECD. The solid-phase capture format allowed for the establishment of an assay to specifically evaluate compounds that interact at the ECD or require the full-length receptor, thereby facilitating structure-activity studies. This assay format should be applicable to the other receptors of this family.     

Arsonoliposomes: effect of lipid composition on their stability and morphology

The influence of the lipid composition of arsonoliposomes on their membrane integrity was investigated to evaluate whether it is possible to combine their action with drugs that can be encapsulated in their aqueous interior. This was investigated by measuring the retention of vesicle-encapsulated calcein (100 mM) during incubation, in the absence and presence of serum proteins. Liposomes containing various concentrations of arsonolipid (with the palmitoyl side chain) as well as egg-lecithin (phosphatidylcholine, PC) and cholesterol (lipid/chol 2:1 mol:mol) were prepared. In some experiments, PC was replaced by the synthetic phospholipid DSPC. All PC/arsonoliposomes tested are stable after 24 h of incubation in buffer at 37 degrees C. After incubation in the presence of serum proteins, arsonoliposomes that contain low amounts of arsonolipid (up to 5 mol% of the lipid content without cholesterol) are stable, whereas increased release of calcein is observed when vesicle arsonolipid concentration is raised (from 5 to 15 mol%). Further increase of arsonolipid content results in immediate decrease of calcein latency while the remaining calcein is rapidly released during incubation. DSPC/arsonoliposomes are comparably more stable, and membrane integrity is independent of the vesicle arsonolipid content, in the range investigated (15-40 mol% of the lipid content without cholesterol). Thereby, we conclude that more stable arsonoliposomes that incorporate high arsonolipid concentrations may be produced when PC is replaced by DSPC. The latter arsonoliposomes provide a system that may be used for combining arsonolipid activity with the activity of other drugs.     

A Sequence Alignment-Independent Method for Protein Classification

Annotation of the rapidly accumulating body of sequence data relies heavily on the detection of remote homologues and functional motifs in protein families. The most popular methods rely on sequence alignment. These include programs that use a scoring matrix to compare the probability of a potential alignment with random chance and programs that use curated multiple alignments to train profile hidden Markov models (HMMs). Related approaches depend on bootstrapping multiple alignments from a single sequence. However, alignment-based programs have limitations. They make the assumption that contiguity is conserved between homologous segments, which may not be true in genetic recombination or horizontal transfer. Alignments also become ambiguous when sequence similarity drops below 40%. This has kindled interest in classification methods that do not rely on alignment. An approach to classification without alignment based on the distribution of contiguous sequences of four amino acids (4-grams) was developed. Interest in 4-grams stemmed from the observation that almost all theoretically possible 4-grams (20(4)) occur in natural sequences and the majority of 4-grams are uniformly distributed. This implies that the probability of finding identical 4-grams by random chance in unrelated sequences is low. A Bayesian probabilistic model was developed to test this hypothesis. For each protein family in Pfam-A and PIR-PSD, a feature vector called a probe was constructed from the set of 4-grams that best characterised the family. In rigorous jackknife tests, unknown sequences from Pfam-A and PIR-PSD were compared with the probes for each family. A classification result was deemed a true positive if the probe match with the highest probability was in first place in a rank-ordered list. This was achieved in 70% of cases. Analysis of false positives suggested that the precision might approach 85% if selected families were clustered into subsets. Case studies indicated that the 4-grams in common between an unknown and the best matching probe correlated with functional motifs from PRINTS. The results showed that remote homologues and functional motifs could be identified from an analysis of 4-gram patterns.     

Synthesis of arsinolipids. II. A non-isosteric analogue of fully acylated cardiolipin

2-Hydroxypropane-1,3-bis(arsonic acid) after six successive reactions gives the arsinolipid 2-acyloxy-As, As'-bis[2,3-di(acyloxy)propyl]propane-1,3-diylbis(arsinic acid) in 20-40% overall yields. This arsinolipid is a non-isosteric analogue of the fully acylated cardiolipin. The R- and S-glycidol, used to create the backbone of the lipid, give the optically active RR and SS, respectively, arsinolipids, while the rac-glycidol produces a mixture of diastereomers (a racemic pair, RR and SS, and two meso forms with an RS configuration). Some properties of these arsinolipids are described, from which the most interesting are the facile hydrolysis of the middle acyl group and their tendency to absorb environmental water.