Constitutively Active Mutant D816VKit Induces Megakayocyte and Mast Cell Differentiation of Early Haemopoietic Cells from Murine Foetal Liver

Mutations of Kit at position D816 have been implicated in mastocytosis, acute myeloid leukaemia and germ cell tumours. Expression of this mutant Kit in cell lines results in factor-independent growth, differentiation and increased survival in vitro and tumourigenicity in vivo. Mutant D816VKit and wild-type Kit were expressed in murine primary haemopoietic cells and grown in stem cell factor (SCF) or the absence of factors. Expression of D816VKit did not lead to transformation as assessed by a colony assay, but resulted in enhanced differentiation of cells when compared to control cells. D816VKit induced an increase in the number of cells differentiating along the megakaryocyte lineage in the absence of factors. SCF had an added effect with an increase in differentiation of mast cells. Expression of wild-type Kit in the presence of SCF also failed to cause transformation and induced differentiation of mast cells and megakaryocytes. We conclude that constitutive expression of D816VKit in primary haemopoietic cells is not a sufficient transforming stimulus but leads to the survival and maturation of cells whose phenotype is influenced by the presence of SCF. (C) 2003 Elsevier Science Ltd. All rights reserved.

Cooperation of cytokine signaling with chimeric transcription factors in leukemogenesis: PML-retinoic acid receptor alpha blocks growth factor-mediated differentiation

We utilized a mouse model of acute promyelocytic leukemia (APL) to investigate how aberrant activation of cytokine signaling pathways interacts with chimeric transcription factors to generate acute myeloid leukemia. Expression in mice of the APL-associated fusion, PML-RARA, initially has only modest effects on myelopoiesis. Whereas treatment of control animals with interleukin-3 (IL-3) resulted in expanded myelopoiesis without a block in differentiation, PML-RARA abrogated differentiation that normally characterizes the response to IL-3. Retroviral transduction of bone marrow with an IL-3-expressing retrovirus revealed that IL-3 and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) combined to generate a lethal leukemia-like syndrome in

Can metal complexes serve as hypoxia activated prodrugs? Investigations of a Co(III) complex of the MMP inhibitor marimastat

For many years proof that the hypoxic nature of malignant tumours can be used to selectively target anticancer drugs has been sought. Several classes of potential redox activated anticancer drugs have been developed to take advantage of the reducing environment resulting from the hypoxia. Drug complexes with redox active metal centres as carriers have been investigated, but have largely been employed with cytotoxic drugs that require release of the drug intracellularly, complicating the design of such complexes. MMP inhibitors, a new class of anticancer drug, conversely act in the extracellular environment and we have investigated inhibitor complexes with several redox active transition metals. Marimastat is an MMP inhibitor with potent in-vitro antimetastatic activity and was recently in Phase III clinical trials for a variety of cancer types. We have synthesised a Co(II1) complex of marimastat incorporating the tetradentate ligand tpa (tris(2-methylpyridyl)amine) as a carrier ligand. The complex was structurally characterised in the solid state by single crystal X-ray diffraction, the first example of a crystal structure containing marimastat. 2D COSY and NOESY NMR spectra showed that the complex exists in two isomeric forms in solution, corresponding to the cis and trans isomers yet only crystallises in one of these forms. Biological testing of the complex in mice with 4T1.2 tumours showed interesting and unexpected outcomes. Initial results of the tumour growth inhibition study showed that a significant inhibition of growth was exhibited by the complex over the free inhibitor and the control. However, the metastatic potential of both free marimastat and the complex were higher than the control indicating likely problems with the experimental protocol. Further experiments are needed to determine the potential of such complexes as hypoxia activated prodrugs but there appears at least to be some promise.

Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors

Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip((R)) microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response.

Quantum control and quantum entanglement

We discuss quantum error correction for errors that occur at random times as described by, a conditional Poisson process. We shoo, how a class of such errors, detected spontaneous emission, can be corrected by continuous closed loop, feedback.

Visual biology of Hawaiian coral reef fishes. II. Colors of Hawaiian coral reef fish

The colors of 51 species of Hawaiian reef fish have been measured using a spectrometer and therefore can be described in objective terms that are not influenced by the human visual experience. In common with other known reef fish populations, the colors of Hawaiian reef fish occupy spectral positions from 300-800nm; yellow or orange with blue, yellow with black, and black with white are the most frequently combined colors; and there is no link between possession of ultraviolet (UV) reflectance and UV visual sensitivity or the potential for UV visual sensitivity. In contrast to other reef systems, blue, yellow, and orange appear more frequently in Hawaiian reef fish. Based on spectral quality of reflections from fish skin, trends in fish colors can be seen that are indicative of both visually driven selective pressures and chemical or physical constraints on the design of colors. UV-reflecting colors can function as semiprivate communication signals. White or yellow with black form highly contrasting patterns that transmit well through clear water. Labroid fishes display uniquely complex colors but lack the ability to see the UV component that is common in their pigments. Step-shaped spectral curves are usually long-wavelength colors such as yellow or red, and colors with a peak-shaped spectral curves are green, blue, violet, and UV.

The protease inhibitor cystatin C is differentially expressed among dendritic cell populations, but does not control antigen presentation

Dendritic cells (DC) undergo complex developmental changes during maturation. The MHC class H (MHC H) molecules of immature DC accumulate in intracellular compartments, but are expressed at high levels on the plasma membrane upon DC maturation. It has been proposed that the cysteine protease inhibitor cystatin C (CyC) plays a pivotal role in the control of this process by regulating the activity of cathepsin S, a protease involved in removal of the MHC H chaperone E, and hence in the formation of MHC H-peptide complexes. We show that CyC is differentially expressed by mouse DC populations. CD8(+) DC, but not CD4(+) or CD4(-)CD8(-) DC, synthesize CyC, which accumulates in MHC II(+)Lamp(+) compartments. However, II processing and MHC H peptide loading proceeded similarly in all three DC populations. We then analyzed MHC H localization and Ag presentation in CD8(+) DC, bone marrow-derived DC, and spleen-derived DC lines, from CyC-deficient mice. The absence of CyC did not affect the expression, the subcellular distribution, or the formation of peptide-loaded MHC II complexes in any of these DC types, nor the efficiency of presentation of exogenous Ags. Therefore, CyC is neither necessary nor sufficient to control MHC II expression and Ag presentation in DC. Our results also show that CyC expression can differ markedly between closely related cell types, suggesting the existence of hitherto unrecognized mechanisms of control of CyC expression.

Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase

in Escherichia coli, the DnaG primase is the RNA polymerase that synthesizes RNA primers at replication forks. It is composed of three domains, a small N-terminal zinc-binding domain, a larger central domain responsible for RNA synthesis, and a C-terminal domain comprising residues 434-581 [DnaG(434-581)] that interact with the hexameric DnaB helicase. Presumably because of this interaction, it had not been possible previously to express the C-terminal domain in a stably transformed E coli strain. This problem was overcome by expression of DnaG(434-581) under control of tandem bacteriophage gimel-promoters, and the protein was purified in yields of 4-6 mg/L of culture and studied by NMR. A TOCSY spectrum of a 2 mM solution of the protein at pH 7.0, indicated that its structured core comprises residues 444-579. This was consistent with sequence conservation among most-closely related primases. Linewidths in a NOESY spectrum of a 0.5 mM sample in 10 mM phosphate, pH 6.05, 0.1 M NaCl, recorded at 36 degreesC, indicated the protein to be monomeric. Crystals of selenomethionine-substituted DnaG(434-581) obtained by the hanging-drop vapor-diffusion method were body-centered tetragonal, space group I4(1)22, with unit cell parameters a = b 142.2 Angstrom, c = 192.1 Angstrom, and diffracted beyond 2.7 Angstrom resolution with synchrotron radiation. (C) 2003 Elsevier Inc. All rights reserved.

Potential triggers for akinete differentiation in an Australian strain of the cyanobacterium Cylindrospermopsis raciborskii (AWT 205/1)

Understanding the triggers for some cyanobacteria of the Nostocales and Stigonematales orders to produce specialised reproductive cells termed akinetes, is very important to gain further insights into their ecology. By improving our understanding of their life cycle, appropriate management options may be devised to control the formation of these cells, and therefore the potential bloom inoculum which they are thought to provide, may be reduced. This study investigated the effect of chemical (phosphorus limitation), and environmental variables (temperature shock) on akinete differentiation in the freshwater cyanobacterium Cylindrospermopsis raciborskii (AWT 205/1). From the preliminary results, it is suggested that the availability of phosphorus and changes in temperature were a necessary requirement for the formation of akinetes in this particular strain of C. raciborskii. In the four phosphorus treatments investigated (0, 3, 38 and 75 mug l(-1) P), only the two higher treatments produced akinetes (approximately 220 ml(-1)). When the first akinetes were observed in the 38 and 75 mug l(-1) P treatments, filterable reactive phosphorus (FRP) concentrations in the medium were approximately 22 and 52 mug l(-1) P, respectively, indicating that there was no phosphorus limitation. In the temperature shock experiment, akinetes were observed in the 15 and 20degreesC treatments. However, akinetes were degraded (pale yellow colour, limited swelling and shrivelled edges) and in much lower concentrations, which was thought to be a result of the daily temperature shock. We suggest that the formation of akinetes in C. raciborskii (AWT 205/1) can be triggered by an initial temperature shock and that phosphorus is a necessary requirement to allow further growth and full development of akinetes.

Active and passive smoking and the risk of subarachnoid hemorrhage - An international population-based case-control study

Background and Purpose - This study was undertaken to better clarify the risks associated with cigarette smoking and subarachnoid hemorrhage (SAH). Methods - The study included 432 incident cases of SAH frequency matched to 473 community SAH-free controls to determine dose-dependent associations of active and passive smoking ( at home) and smoking cessation with SAH. Results - Compared with never smokers not exposed to passive smoking, the adjusted odds ratio for SAH among current smokers was 5.0 (95% confidence interval [CI], 3.1 to 8.1); for past smokers, 1.2 ( 95% CI, 0.8 to 2.0); and for passive smokers, 0.9 ( 95% CI, 0.6 to 1.5). Current and lifetime exposures showed a clear dose-dependent effect, and risks appeared more prominent in women and for aneurysmal SAH. Approximately 1 in 3 cases of SAH could be attributed to current smoking, but risks decline quickly after smoking cessation, even among heavy smokers. Conclusions - A strong positive association was found between cigarette smoking and SAH, especially for aneurysmal SAH and women, which is virtually eliminated within a few years of smoking cessation. Large opportunities exist for preventing SAH through smoking avoidance and cessation programs.

The Arabidopsis mutant iop1 exhibits induced over-expression of the plant defensin gene PDF1.2 and enhanced pathogen resistance

Jasmonate and ethylene are concomitantly involved in the induction of the Arabidopsis plant defensin gene PDF1.2. To define genes in the signal transduction pathway leading to the induction of PDF1.2, we screened for-mutants with induced over-expression of a beta-glucuronidase reporter, under the control of the PDF1.2 promoter. One mutant, iop1 (induced over-expressor of PDF1.2) produced small plants that showed induced over-expression of the pathogenesis-related genes PR-3, PR-4 and PR-1,2 (PDF1.2), combined with a down-regulated induction of PR-1 upon pathogen inoculation. The iop1 mutant showed enhanced resistance to a number of necrotrophic pathogens.

A comparison of the performance and biological efficiency of new knapsack sprayers and a controlled droplet application (CDA) sprayer for the control of the diamondback moth, Plutella xylostella L. (Lepidoptera : Plutellidae)

Field trials and laboratory bioassays were undertaken to compare the performance and efficacy (mortality of diamondback moth larvae) of insecticides applied to cabbages with three high volume hydraulic knapsack sprayers (NS-16, PB-20 and Selecta 12V) and a controlled droplet application (CDA) sprayer. In field experiments, the high volume knapsack sprayers (application rate 500-600 L ha(-1)) provided better spray coverage on the upper and lower surfaces of inner leaves, the upper surfaces of middle and outer leaves, and greater biological efficacy than the CDA sprayer (application rate 20similar to40 L ha(-1)). The PB-20 provided better spray coverage on the upper surface of middle leaves and both surfaces of outer leaves when compared with the Selecta 12V. However, its biological efficacy in the field was not significantly different from that of the other high volume sprayers. Increasing the application rate from 20 to 40 L ha(-1) for the CDA sprayer significantly increased droplet density but had no impact on test insect mortality. Laboratory evaluations of biological efficacy yielded higher estimates than field evaluations and there was no significant difference between the performance of the PB-20 and the CDA sprayer. Significant positive relationships were detected between insect mortality and droplet density deposited for both the PB-20 and the CDA sprayers.

When the going gets rough: effect of maternal size manipulation on larval quality

Variation in larval size has been shown to be an important factor for the post-metamorphic performance of marine invertebrates but, despite its importance, few sources of this variation have been identified. For a range of taxa, offspring size is positively correlated with maternal size but the reasons for this correlation remain unclear. We halved the size of colonies in the bryozoan Bugula neritina 1 wk prior to reproduction (but during embryogenesis) to determine if larval size is a fixed or plastic trait. We manipulated colonies in such a way that the ratio of feeding zooids to reproductive zooids was constant between treatment and control colonies. We found that manipulating colony size strongly affects larval size; halved colonies produced larvae that were similar to13% smaller than those produced by intact colonies. We entered these data into a simple model based on previous work to estimate the likely post-metamorphic consequences of this reduction in larval size. The model predicted that larvae that came from manipulated colonies would suffer similar to300% higher post-metamorphic mortality and similar to50% lower fecundity as adults. Colonies that are faced with a stress appear to be trading off current offspring fitness to maximize their own long-term fitness and this may explain previous observations of compensatory growth in colonial organisms. This study demonstrates that larval size is a surprisingly dynamic trait and strong links exist between the maternal phenotype and the fitness of the offspring. The performance of settling larvae may be determined not only by their larval experience but also by the experience of their mothers.