Body length rather than routine metabolic rate and body condition correlates with activity and risk-taking in juvenile zebrafish Danio rerio.

In this study, the following hypotheses were explored using zebrafish Danio rerio: (1) individuals from the same cohort differ consistently in activity and risk-taking and (2) variation in activity and risk-taking is linked to individual differences in metabolic rate, body length and body condition. To examine these hypotheses, juvenile D. rerio were tested for routine metabolic rate and subsequently exposed to an open field test. Strong evidence was found for consistent among-individual differences in activity and risk-taking, which were overall negatively correlated with body length, i.e. larger D. rerio were found to be less active in a potentially dangerous open field and a similar trend was found with respect to a more direct measure of their risk-taking tendency. In contrast, routine metabolic rate and body condition were uncorrelated with both activity and risk-taking of juvenile D. rerio. These findings suggest that body length is associated with risk-related behaviours in juvenile D. rerio for which larger, rather than smaller, individuals may have a higher risk of predation, while the role for routine metabolic rate is relatively limited or non-existent, at least under the conditions of the present study.

Behaviour in a standardized assay, but not metabolic or growth rate, predicts behavioural variation in an adult aquatic top predator Esox lucius in the wild.

This study tested for links among behaviour, state and life-history variables as predicted by the pace-of-life hypothesis in adult pike Esox lucius. First, a standardized open-field behavioural assay was developed to assess individual behaviour of wild-captured adult E. lucius. Behaviour within the standardized assay predicted swimming behaviour in the lake, providing an ecological validation of the assay. There was no relationship between standardized behaviour and any of the life-history and state variables, including metabolism, body condition, juvenile growth rate and adult growth rate in contrast to predictions from the pace-of-life hypothesis. This study demonstrates that it is possible to assess ecologically relevant behavioural variation in a large-bodied top predator using a standard open-field assay, but it is noteworthy that this standardized behaviour is not systematically related to standard metabolism or growth.

Species-specific preferences of German recreational anglers for freshwater fishing experiences, with emphasis on the intrinsic utilities of fish stocking and wild fishes.

To answer the question, whether anglers have an intrinsic preference for stocking or a preference for catch outcomes (e.g. catch rates) believed to be maintained by stocking, a discrete choice experiment was conducted among a sample of anglers (n = 1335) in Lower Saxony, Germany. After controlling for catch aspects of the fishing experience, no significant influence of two stocking attributes (stocking frequency and composition of the catch in terms of wild v. hatchery fishes) on the utility gained from fishing was found for any of the freshwater species that were studied. It was concluded that the previously documented large appreciation of fish stocking by anglers may be indicative of an underlying preference for sufficiently high catches rather than reflect an intrinsic preference for stocking or the catching of wild fishes per se.

Experimental assessment of the probabilistic maturation reaction norm: condition matters.

The probabilistic maturation reaction norm (PMRN) describes an individual's probability of maturing at a given age as a function of size and other relevant phenotypic traits. Population-level shifts in the PMRN are often interpreted to indicate genetic as opposed to phenotypic changes in maturation in fish. Inferences derived from trends in the PMRN have been challenged, warranting an experimental assessment of the method. This was accomplished in a laboratory experiment using zebrafish (Danio rerio). Fish were reared under different food levels to induce variation in growth and maturation. Plasticity in maturation was not entirely captured by the demographic age- and length-based PMRN. Adding condition to the PMRN captured a greater amount of environmental variation in maturation probability. Nevertheless, significant differences in the PMRNs among the food levels remained after accounting for the influences of age, size and condition on maturation probability indicating plasticity of the PMRN. This was particularly pronounced between fish held on low food levels as compared with fish experiencing abundant resources, with the latter experiencing higher size-specific maturation probabilities. Our analysis emphasizes the need for incorporating salient physiological traits influencing maturation, such as condition, to make accurate inferences about documented shifts observed in the position of PMRNs on maturation trends in wild fish stocks.

p120 GAP requirement in normal and malignant human hematopoiesis.

There is evidence to suggest that the p120 GAP (GAP), originally described as an inhibitor of p21ras, may also serve as a downstream effector of ras-regulated signal transduction. To determine whether GAP expression is required for the growth of human normal and leukemic hematopoietic cells, we used GAP antisense oligodeoxynucleotides to inhibit it and analyzed the effects of this inhibition on the colony-forming ability of nonadherent, T lymphocyte-depleted mononuclear cells and of highly purified progenitors (CD34+ MNC) obtained from the bone marrow and peripheral blood of healthy volunteers or chronic myeloid leukemia (CML, bcr-abl-positive) patients. The acute myelogenous leukemia cell line MO7, the Philadelphia BV173 cell line, and the acute promyelocytic leukemia NB4 and HL-60 cell lines were similarly examined. GAP antisense treatment inhibited colony formation from normal myelo-, erythro-, and megakaryopoietic progenitor cells as well as from CML progenitor cells. Proliferation of MO7 (growth factor-dependent) and BV173 (bcr-abl-dependent) cells, but not that of NB4 and HL-60 (growth factor-independent) cells, was also inhibited, even though a specific downregulation of GAP was observed in each cell line, as analyzed by either or both mRNA and protein expression. Stimulation of MO7 cells with hematopoietic growth factors increased the expression of GAP as well as the levels of active GTP-bound p21ras. Stimulation of GAP expression was inhibited upon GAP antisense treatment. These data indicate that p120 GAP is involved in human normal and leukemic hemopoiesis and strongly suggest that GAP is not only a p21ras inhibitor (signal terminator), but also a positive signal transducer.

Harmonizing recreational fisheries and conservation objectives for aquatic biodiversity in inland waters.

The importance of recreational fisheries to local and national economies, and as a generator of immense social welfare throughout the developed world, is well established. Development in the sector and its interaction with non-fishery-related nature conservation objectives for aquatic biodiversity, however, have the potential to generate conflict. This article reviews the intersection between recreational fisheries and nature conservation goals for aquatic biodiversity with specific reference to inland waters in industrialized countries, and the principal management activities and constraints that can lead to conflicts. A SWOT (strengths, weaknesses, opportunities and threats) analysis was used to review the issues facing sectoral development and identify options for future advancement of recreational fisheries to ameliorate potential conflicts with nature conservation goals. It is concluded that reconciliation of recreational fisheries and modern conservation perspectives is both possible and desirable, because many conservation problems also benefit fisheries quality. Angler buy-in to conservation is probable if (1) management scales are small, (2) threats to conservation originate from outside the fisheries sectors and (3) ecological awareness for the conservation problem is high. If these aspects are not present, reconciliation of recreational fisheries and nature conservation goals is less likely, risking both the aquatic biodiversity and the future of angling. To address these issues, enforcement of legislation and continued communication with angler communities is necessary, as well as development of integrated management policies that build on the instrumental values of aquatic biodiversity for recreational fisheries, while curtailing the more insidious threats to such biodiversity that originate directly from the recreational fisheries sector.

Size-dependent reproductive success of wild zebrafish Danio rerio in the laboratory.

Size-dependent reproductive success of wild zebrafish Danio rerio was studied under controlled conditions in the laboratory to further understand the influence of spawner body size on reproductive output and egg and larval traits. Three different spawner size categories attained by size-selective harvesting of the F(1)-offspring of wild D. rerio were established and their reproductive performance compared during a 5 day period. As to be expected, large females spawned more frequently and had significantly greater clutch sizes than small females. Contrary to expectations, small females produced larger eggs when measured as egg diameter with similar amounts of yolk compared to eggs spawned by large spawners. Eggs from small fish, however, suffered from higher egg mortality than the eggs of large individuals. Embryos from small-sized spawners also hatched later than offspring from eggs laid by large females. Larval standard length (L(S))-at-hatch did not differ between the size categories, but the offspring of the large fish had significantly larger area-at-hatch and greater yolk-sac volume indicating better condition. Offspring growth rates were generally similar between offspring from all size categories, but they were significantly higher for offspring spawned by small females in terms of L(S) between days 60 and 90 post-fertilization. Despite temporarily higher growth rates among the small fish offspring, the smaller energy reserves at hatching translated into lower condition later in ontogeny. It appeared that the influence of spawner body size on egg and larval traits was relatively pronounced early in development and seemed to remain in terms of condition, but not in growth, after the onset of exogenous feeding. Further studies are needed to explore the mechanisms behind the differences in offspring quality between large- and small-sized spawners by disentangling size-dependent maternal and paternal effects on reproductive variables in D. rerio.

Detergent-solubilized RNA polymerase from cells infected with foot-and-mouth disease virus.

The foot-and-mouth disease virus RNA polymerase complex was dissociated from cellular membranes with deoxycholate in the presence of dextran sulfate. The soluble polymerase complex was active in the cell-free synthesis of virus-specific RNA; solubilization of the complex permitted direct analysis of the cell-free reaction mixtures without recourse to RNA extraction. A major RNA-containing component found early during cell-free incubation ranged from approximately 140 to 300S. The final major products of the cell-free system were 37S virus RNA, 20S ribonuclease-resistant RNA, and a 50S component containing RNA.

Contrasting pragmatic and suffering-centred approaches to fish welfare in recreational angling.

Two views dealing with fish welfare in recreational fishing are discussed in an effort to stimulate the current discourse on the topic. The pragmatic approach asks whether and how strongly recreational fishing compromises the health and fitness of individual fishes and what can be done to avoid or mitigate such effects. Its implementation rests on accepting recreational fishing as a principally legitimate activity. The second approach to fish welfare focuses on suffering and pain in fishes and is usually morally prescriptive. Its central tenet is that some or all recreational fishing practices may be unacceptable unless sufficient benefits to humans are created, which justify the supposedly cruel treatment of the fishes. The pragmatic approach to fish welfare is preferred because it relies on objectively measurable variables of impaired fish welfare (e.g. physiological, behavioural or fitness indicators) and does not question recreational fishing on moral grounds. Contrary to a suffering-centred approach to fish welfare, a pragmatic perspective emphasizes positive messages and facilitates constructive dialogue among stakeholders. In contrast, a suffering-centred approach to fish welfare tends to promote tension and enduring conflict that cannot be reconciled objectively and thus should be avoided.

Oncogenic activation of c-Abl in non-small cell lung cancer cells lacking FUS1 expression: inhibition of c-Abl by the tumor suppressor gene product Fus1.

In lung cancer, frequent loss of one allele of chromosome 3p is seen in both small cell lung cancer and non-small cell lung cancer (NSCLC), providing evidence of tumor suppressor genes (TSGs) in this chromosomal region. The mechanism of Fus1 tumor suppressor activity is unknown. We have found that a Fus1 peptide inhibits the Abl tyrosine kinase in vitro (IC(50) 35 microM). The inhibitory Fus1 sequence was derived from a region that was deleted in a mutant FUS1 gene (FUS1 (1-80)) detected in some lung cancer cell lines. Importantly, a stearic acid-modified form of this peptide was required for the inhibition, but stearic acid alone was not inhibitory. Two NSCLC cell lines, which lack expression of wild-type Fus1, contain activated c-Abl. Forced expression of an inducible FUS1 cDNA in H1299 NSCLC cells decreased levels of activated c-Abl and inhibited its tyrosine kinase activity. Similarly, treatment of c-Abl immune complexes with the inhibitory Fus1 peptide also reduced the level of c-Abl in these immune complexes. The size and number of colonies of the NSCLC cell line, H1,299, in soft agar was strongly inhibited by the Abl kinase inhibitor imatinib mesylate. Co-expression of FUS1 and c-ABL in COS1 cells blocked activation of c-Abl tyrosine kinase. In contrast, co-expression of mutant FUS1 (1-80) with c-ABL had little inhibitory activity against c-Abl. These findings provide strong evidence that c-Abl is a possible target in NSCLC patients that have reduced expression of Fus1 in their tumor cells.

Engaging recreational fishers in management and conservation: global case studies.

Globally, the number of recreational fishers is sizeable and increasing in many countries. Associated with this trend is the potential for negative impacts on fish stocks through exploitation or management measures such as stocking and introduction of non-native fishes. Nevertheless, recreational fishers can be instrumental in successful fisheries conservation through active involvement in, or initiation of, conservation projects to reduce both direct and external stressors contributing to fishery declines. Understanding fishers' concerns for sustained access to the resource and developing methods for their meaningful participation can have positive impacts on conservation efforts. We examined a suite of case studies that demonstrate successful involvement of recreational fishers in conservation and management activities that span developed and developing countries, temperate and tropical regions, marine and freshwater systems, and open- and closed-access fisheries. To illustrate potential benefits and challenges of involving recreational fishers in fisheries management and conservation, we examined the socioeconomic and ecological contexts of each case study. We devised a conceptual framework for the engagement of recreational fishers that targets particular types of involvement (enforcement, advocacy, conservation, management design [type and location], research, and monitoring) on the basis of degree of stakeholder stewardship, scale of the fishery, and source of impacts (internal or external). These activities can be enhanced by incorporating local knowledge and traditions, taking advantage of leadership and regional networks, and creating collaborations among various stakeholder groups, scientists, and agencies to maximize the probability of recreational fisher involvement and project success.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays: towards new standards for gene expression measurements.

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

Vaccination with leukemia cells expressing cell-surface-associated GM-CSF blocks leukemia induction in immunocompetent mice.

The fundamental basis for immunotherapy of leukemia is that leukemic cells express specific antigens that are not expressed by normal hematopoietic cells. However, the host immune system appears to be tolerant to leukemia cells. To overcome this tolerance, we vaccinated immunocompetent mice with murine leukemia cells (WEHI-3B and BCR-ABL+ 32D cells) transduced with a specifically constructed transmembrane form of granulocyte-macrophage colony-stimulating factor (tmGM-CSF). The transduced cells expressed tmGM-CSF on the cell-surface. To determine whether tmGM-CSF-expressing WEHI-3B leukemia cells would prevent leukemia formation as a vaccine, immunocompetent mice (BALB/c and C3H/HEJ) were immunized with lethally irradiated murine leukemia cells expressing cell-surface tmGM-CSF before challenging mice with murine leukemia cells. Two immunocompetent mouse models were investigated, either WEHI-3B cells in BALB/c mice or BCR-ABL+ 32D cells in C3H/HEJ mouse. The results showed that 100% of WEHI-3B/tmGM-CSF-vaccinated BALB/c mice and about 65% of 32D+ BCR-ABL/tmGM-CSF-vaccinated C3H/HEJ mice were protected from leukemia after leukemia cell challenge, whereas all non-vaccinated mice succumbed to leukemia. Spleen and marrow cell suspensions from vaccinated mice challenged with WEHI-3B cells lacked detectable GFP+ WEHI-3B cells at 82 days post-challenge. A significant delay of death was observed in C3H/HEJ mice challenged with the very aggressive DA-1 cell line expressing BCR-ABL. Vaccination of mice with WEHI-3B/CD40L cells protected 80% of the mice from the WEHI-3B challenge. Notably, 60% of the WEHI-3B/BALB/c mice were also protected from leukemia when WEHI-3B/tmGM-CSF vaccination was carried out after the leukemia challenge. In order to determine whether cellular immunity is involved in this vaccine-mediated protection, either CD4+ or CD8+ T cells were depleted from mice after the WEHI-3B/tmGM-CSF vaccination. The results indicate that CD8+ T-cells mediated the protective immune response provided by the irradiated tmGM-CSF-expressing WEHI-3B cells. In addition, vaccination of nude mice did not provide protection from WEHI-3B leukemia induction. Importantly, 80% of non-vaccinated mice were also protected from a WEHI-3B cell challenge after receiving spleen cells from vaccinated mice 1 day before challenge with leukemia cells. These results indicate that overexpression of tmGM-CSF on the leukemia cell-surface can enhance the recognition of leukemic cells by CD8+ T cells, and can either prevent or significantly delay leukemia induction. These findings suggest that injection of irradiated leukemia cells expressing cell-surface-bound GM-CSF has the potential as an immunological approach to treat leukemia.

Inhibition of v-Mos kinase activity by protein kinase A.

We investigated the effect of cyclic AMP-dependent protein kinase (PKA ) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37v-mos in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Combined tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies indicated that Ser-56 is the major in vivo phosphorylation site on v-Mos. In vivo phosphorylation at Ser-56 correlated with slower migration of the v-Mos protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, even though Ser-56 was phosphorylated by PKA, this phosphorylation was not involved in the inhibition of v-Mos kinase. The alanine-for-serine substitution at residue 56 did not affect the ability of v-Mos to autophosphorylate in vitro or, more importantly, to activate MEK1 in transformed NIH 3T3 cells. We identified Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. From our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation in mice could be explained at least in part by its inhibition of Mos kinase.

Inhibition of Bcr serine kinase by tyrosine phosphorylation.

The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired.

Detection of BCR-ABL proteins in blood cells of benign phase chronic myelogenous leukemia patients.

More than 95% of patients with chronic myelogenous leukemia (CML) contain an abnormal chromosome termed the Philadelphia chromosome (Ph1). Ph1 and the resulting BCR-ABL fused genes are markers for this type of leukemia. The product of the fused BCR-ABL genes is a protein of about 2000 amino acids termed P210 BCR-ABL. Although the BCR-ABL protein can be routinely detected in blood cells from blast crisis CML patients by assaying for its activated tyrosine kinase activity, detection of P210 BCR-ABL in early stage CML patients (chronic phase) has not yet been possible (S. A. Maxwell et al., Cancer Res., 47: 1731, 1987). A procedure involving Western blotting with an anti-ABL monoclonal antibody was developed that allows detection of P210 BCR-ABL and P145 ABL in cells from chronic phase and blast crisis CML patients, but as expected only P145 ABL was found in normal white blood cells. Most chronic phase patients also contained one to two ABL proteins with a molecular weight of about 190,000. Interestingly, the ratio of BCR-ABL to ABL proteins increased in four blast crisis patients compared to 18 chronic phase patients. Also, one chronic phase patient analyzed on three separate occasions lacked P210 BCR-ABL and exhibited only the Mr 190,000 form. This assay should also be useful in other leukemias that express altered forms of the ABL protein.

Sequences within the first exon of BCR inhibit the activated tyrosine kinases of c-Abl and the Bcr-Abl oncoprotein.

The Bcr-Abl oncoprotein is the primary causative factor in Philadelphia chromosome-associated leukemias. The activated tyrosine kinase of the Bcr-Abl oncoprotein is the primary driving force behind its oncogenic activity. We report here that a deleted form of Bcr [Bcr(64-413)], encompassing the Abl SH2 binding domains of Bcr, reduced the phosphotyrosine content of c-Abl and Bcr-Abl within cells and inhibited Bcr-Abl autophosphorylation activity in vitro. Similarly, a Bcr peptide phosphorylated on Ser-354 blocked the c-Abl and Bcr-Abl kinases in vitro, whereas the same peptide phosphorylated on Tyr-360 was not inhibitory. Bcr(64-413) was also resistant to tyrosine phosphorylation by either activated c-Abl or Bcr-Abl. Importantly, Bcr(64-413) interfered with the growth of Bcr-Abl-expressing cell lines. Our findings indicate that the Abl SH2 binding domain of Bcr in the phosphoserine form inhibits the Bcr-Abl oncoprotein but that tyrosine phosphorylation of this domain of Bcr reverses its inhibitory effects on Bcr-Abl. These results raise interesting questions about a possible role of Bcr or a Bcr-related molecule in modulating the activity of the Bcr-Abl oncoprotein and c-Abl itself.