Phenotypic plasticity, heterochrony and ontogenetic repatterning during juvenile development of divergent Arctic charr (Salvelinus alpinus)

Phenotypic plasticity is a developmental process that plays a role as a source of variation for evolution. Models of adaptive divergence make the prediction that increasing ecological specialization should be associated with lower levels of plasticity. We tested for differences in the magnitude, rate and trajectory of morphological plasticity in two lake populations of Arctic charr (Salvelinus alpinus) that exhibited variation in the degree of resource polymorphism. We reared offspring on diet treatments that mimicked benthic and pelagic prey. Offspring from the more divergent population had lower levels of morphological plasticity. Allometry influenced the rate of shape change over ontogeny, with differences in rate among ecomorphs being minimal when allometric variation was removed. However, plasticity in the spatial trajectory of development was extensive across ecomorphs, both with and without the inclusion of allometric variation, suggesting that different aspects of shape development can evolve independently.     

Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells

New anti-cancer agents are being developed that specifically recognise tumour cells. Recognition is dependent upon the enhanced expression of antigenic determinants on the surface of tumour cells. The tumour exposure and the extracellular accessibility of the mucin MUC-1 make this marker a suitable target for tumour diagnosis and therapy. We isolated and characterised six human scFv antibody fragments that bound to the MUC-1 core protein, by selecting a large naive human phage display library directly on a MUC-1-expressing breast carcinoma cell line. Their binding characteristics have been studied by ELISA, FACS and indirect immunofluorescence. The human scFv antibody fragments were specific for the tandem repeat region of MUC-1 and their binding is inhibited by soluble antigen. Four human scFv antibody fragments (M2, M3, M8, M12) recognised the hydrophilic PDTRP region of the MUC-1 core protein, which is thought to be an immunodominant region. The human scFv antibody fragments were stable in human serum at 37 °C and retained their binding specificity. For imaging or targeting to tumours over-expressing MUC-1, it might be feasible to use these human scFv, or multivalent derivatives, as vehicles to deliver anti-cancer agents. Received: 2 November 2000 / Accepted: 11 January 2001     

Inhibition of p56(lck) tyrosine kinase by isothiazolones

Lck encodes a 56-kDa protein-tyrosine kinase, predominantly expressed in T lymphocytes, crucial for initiating T cell antigen receptor (TCR) signal transduction pathways, culminating in T cell cytokine gene expression and effector functions. As a consequence of a high-throughput screen for selective, novel inhibitors of p56(lck), an isothiazolone compound was identified, methyl-3-(N-isothiazolone)-2-thiophenecarboxylate(A-125800), which inhibits p56(lck) kinase activity with IC50 = 1-7 microM. Under similar assay conditions, the isothiazolone compound was equipotent in blocking the ZAP-70 tyrosine kinase activity but was 50 to 100 times less potent against the catalytic activities of p38 MAP kinase and c-Jun N-terminal kinase 2alpha. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC50 = 35 microM) and blocked T cell proliferation in response to alloantigen (IC50 = 14 microM) and CD3/CD28-induced IL-2 secretion (IC50 = 2.2 microM) in primary T cell cultures. Inhibition of p56(lck )by A-125800 was dose- and time-dependent and was irreversible. A substitution of methylene for the sulfur atom in the isothiazolone ring of the compound completely abrogated the ability to inhibit p56(lck) kinase activity and TCR-dependent signal transduction. Incubation with thiols such as beta-ME or DTT also blocked the ability of the isothiazolone to inhibit p56(lck) kinase activity. LC/MS analysis established the covalent modification of p56(lck) at cysteine residues 378, 465, and 476. Together these data support an inhibitory mechanism, whereby cysteine -SH groups within the p56(lck) catalytic domain react with the isothiazolone ring, leading to ring opening and disulfide bond formation with the p56(lck) enzyme. Loss of p56(lck) activity due to -SH oxidation has been suggested to play a role in the pathology of AIDS. Consequently, a similar mechanism of sulfhydryl oxidation leading to p56(lck) inhibition, described in this report, may occur in the intact T cell and may underlie certain T cell pathologies.     

A toolbox for integrative species delimitation in Machilis jumping bristletails (Microcoryphia: Machilidae)

Accurate species delimitation is fundamental for many fields of biology. Although a wide range of evolutionary-biological patterns are known to promote failure in species delimitation when any single information source is considered, the majority of species characterisations are still based on single disciplines. Using Alpine jumping bristletails we here showcase the establishment of morphological and molecular tools for multi-source species delimitation in a taxonomically problematic arthropod group and stress its advantages over single-discipline approaches. Once sound species delimitations have been achieved, Alpine jumping bristletails will be available as a prime model system for addressing questions related to the emergence of endemism and parthenogenesis.     

A comparative analysis of carbon dioxide displacement rates for euthanasia of the ferret

Though carbon dioxide asphyxiation is a common method of euthanasia for laboratory animals, species-specific guidelines have not been established for this procedure in the domestic ferret (Mustela putorius furo). The authors investigated the efficacy and stress effects of carbon dioxide euthanasia in 24 ferrets that had participated in previous experimental protocols. They euthanized ferrets by placing them in cages that were either prefilled with carbon dioxide or gradually filled at a displacement rate of 10%, 20% or 50% of the cage volume per min. Blinded observers subjectively evaluated ferret distress. Prefilling the cage or filling it at a rate of 50% volume per min resulted in less time to recumbency and to last breath than did filling the cage at a slower displacement rate. Slower carbon dioxide displacement rates also caused an increase in ferret blood glucose concentrations, which may indicate distress. Overall, observers found that prefilling the euthanasia cage caused the least stress to ferrets.