Copper uptake and translocation in chicory (Cichorium intybus L. cv Grasslands Puna) and tomato (Lycopersicon esculentum Mill. cv Rondy) plants grown in NFT system. II. The role of nicotianamine and histidine in xylem sap copper transport

The effect of rooting media Cu concentration (0.05–20 mg Cu L^-1) on amino acid concentrations and copper speciation in the xylem sap of chicory and tomato plants was measured using 6 week old plants grown in a nutrient film technique system (NFT). Irrespective of the Cu concentration in the nutrient solutions, more than 99.68% and 99.74% of total Cu in tomato and chicory xylem sap was in a bound form. When exposed to high Cu concentrations in the rooting media, amino acid concentrations in the sap increased. Relative to other amino acids, the concentrations of glutamine (Gln), histidine (His), asparagine (Asn), valine (Val), nicotianamine (NA) and proline (Pro) in tomato xylem saps, and His, γ-aminobutyric acid (Gaba), glutamic acid (Glu), leucine (Leu), NA and phenylalanine (Phe) in chicory xylem saps showed the greatest increases. The data indicate that induced synthesis of some free amino acids as a specific and proportional response to Cu treatment. For a single complexation amino acid, the solution Cu²⁺concentration vs pH titration curve for NA at 0.06–0.07 mM was most similar, closely followed by His at 0.5–0.6 mM, to the solution Cu²⁺concentration behaviour in both tomato and chicory xylem sap. It is concluded that increased Cu concentrations in the rooting media induced selective synthesis of certain amino acid which include NA, His, Asn and Gln which have high stability constants with Cu. NA and His have the highest binding constants for Cu and the concentrations of NA and His in chicory and tomato xylem saps can account for all the bound Cu carried in the sap. This revised version was published online in June 2006 with corrections to the Cover Date.     

Beta1 integrin-mediated T cell adhesion and cell spreading are regulated by calpain

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology. However, affinity-related increases in T cell adhesion induced by the anti-beta1 integrin antibody 8A2 occur in a calpain-independent manner and in the absence of morphological shape changes. Furthermore, calpain undergoes activation in response to either alpha4beta1 or alpha5beta1 integrin binding to fibronectin in appropriately stimulated T cells, and calpain II as well as protein tyrosine phosphatase 1B accumulates at sites of focal contact formation. Inhibition of calpain activity not only inhibits the proteolytic modification of protein tyrosine phosphatase 1B, but also decreases the ability of T cells to adhere to and spread on immobilized fibronectin. Thus, we describe a potential regulatory role for calpain in beta1 integrin-mediated signaling events associated with T cell adhesion and cell spreading on fibronectin.     

Decatenation checkpoint‐defective melanomas are dependent on PI3K for survival

Melanoma cell lines are commonly defective for the G2‐phase cell cycle checkpoint that responds to incomplete catenation of the replicated chromosomes. Here, we demonstrate that melanomas defective for this checkpoint response are less sensitive to genotoxic stress, suggesting that the defective cell lines compensated for the checkpoint loss by increasing their ability to cope with DNA damage. We performed an siRNA kinome screen to identify kinases responsible and identified PI3K pathway components. Checkpoint‐defective cell lines were three‐fold more sensitive to small molecule inhibitors of PI3K. The PI3K inhibitor PF‐05212384 promoted apoptosis in the checkpoint‐defective lines, and the increased sensitivity to PI3K inhibition correlated with increased levels of activated Akt. This work demonstrates that increased PI3K pathway activation is a necessary adaption for the continued viability of melanomas with a defective decatenation checkpoint.     

On the Bayesian estimation of a closed population size in the presence of heterogeneity and model uncertainty

Summary .   We consider the estimation of the size of a closed population, often of interest for wild animal populations, using a capture–recapture study. The estimate of the total population size can be very sensitive to the choice of model used to fit to the data. We consider a Bayesian approach, in which we consider all eight plausible models initially described by Otis et al. (1978, Wildlife Monographs 62, 1–135) within a single framework, including models containing an individual heterogeneity component. We show how we are able to obtain a model-averaged estimate of the total population, incorporating both parameter and model uncertainty. To illustrate the methodology we initially perform a simulation study and analyze two datasets where the population size is known, before considering a real example relating to a population of dolphins off northeast Scotland.     

The oxidative DNA lesion 8,5'-(S)-cyclo-2'-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells

Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cell's capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.     

Functional MRI in awake unrestrained dogs

Because of dogs' prolonged evolution with humans, many of the canine cognitive skills are thought to represent a selection of traits that make dogs particularly sensitive to human cues. But how does the dog mind actually work? To develop a methodology to answer this question, we trained two dogs to remain motionless for the duration required to collect quality fMRI images by using positive reinforcement without sedation or physical restraints. The task was designed to determine which brain circuits differentially respond to human hand signals denoting the presence or absence of a food reward. Head motion within trials was less than 1 mm. Consistent with prior reinforcement learning literature, we observed caudate activation in both dogs in response to the hand signal denoting reward versus no-reward.     

Germline variants in oculocutaneous albinism genes and predisposition to familial cutaneous melanoma

Approximately 1%–2% of cutaneous melanoma (CM) is classified as strongly familial. We sought to investigate unexplained CM predisposition in families negative for the known susceptibility genes using next‐generation sequencing of affected individuals. Segregation of germline variants of interest within families was assessed by Sanger sequencing. Several heterozygous variants in oculocutaneous albinism (OCA) genes: TYR, OCA2, TYRP1 and SLC45A2, were present in our CM cohort. OCA is a group of autosomal recessive genetic disorders, resulting in pigmentation defects of the eyes, hair and skin. Missense variants classified as pathogenic for OCA were present in multiple families and some fully segregated with CM. The functionally compromised TYR p.T373K variant was present in three unrelated families. In OCA2, known pathogenic variants: p.V443I and p.N489D, were present in three families and one family, respectively. We identified a likely pathogenic SLC45A2 frameshift variant that fully segregated with CM in a family of four cases. Another four‐case family harboured cosegregating variants (p.A24T and p.R153C) of uncertain functional significance in TYRP1. We conclude that rare, heterozygous variants in OCA genes confer moderate risk for CM.