Granulocyte-macrophage colony-stimulating factor is a chemoattractant cytokine for human neutrophils: involvement of the ribosomal p70 S6 kinase signaling pathway

GM-CSF stimulates proliferation of myeloid precursors in bone marrow and primes mature leukocytes for enhanced functionality. We demonstrate that GM-CSF is a powerful chemotactic and chemokinetic agent for human neutrophils. GM-CSF-induced chemotaxis is time dependent and is specifically neutralized with Abs directed to either the ligand itself or its receptor. Maximal chemotactic response was achieved at approximately 7 nM GM-CSF, and the EC(50) was approximately 0.9 nM. Both concentrations are similar to the effective concentrations of IL-8 and less than the effective concentrations of other neutrophil chemoattractants such as neutrophil-activating peptide-78, granulocyte chemotactic protein-2, leukotriene B(4), and FMLP. GM-CSF also acts as a chemoattractant for native cells bearing the GM-CSF receptor, such as monocytes, as well as for GM-CSF receptor-bearing myeloid cell lines, HL60 (promyelomonocyte leukemic cell line) and MPD (myeloproliferative disorder cell line), following differentiation induction. GM-CSF induced a rapid, transient increase in F-actin polymerization and the formation of focal contact rings in neutrophils, which are prerequisites for cell migration. The mechanism of GM-CSF-induced chemotaxis appears to involve the cell signaling molecule, ribosomal p70 S6 kinase (p70S6K). Both p70S6K enzymatic activity and T(421)/S(424) and T(389) phosphorylation are markedly increased with GM-CSF. In addition, the p70S6K inhibitor hamartin transduced into cells as active protein, interfered with GM-CSF-dependent migration, and attenuated p70S6K phosphorylation. These data indicate that GM-CSF exhibits chemotactic functionality and suggest new avenues for the investigation of the molecular basis of chemotaxis as it relates to inflammation and tissue injury.     

Aligned electrospun fibers for neural patterning

Objectives

To test a 3D approach for neural network formation, alignment, and patterning that is reproducible and sufficiently stable to allow for easy manipulation.

Results

A novel cell culture system was designed by engineering a method for the directional growth of neurons. This uses NG108-15 neuroblastoma x glioma hybrid cells cultured on suspended and aligned electrospun fibers. These fiber networks improved cellular directionality, with alignment angle standard deviations significantly lower on fibers than on regular culture surfaces. Morphological studies found nuclear aspect ratios and cell projection lengths to be unchanged, indicating that cells maintained neural morphology while growing on fibers and forming a 3D network. Furthermore, fibronectin-coated fibers enhanced neurite extensions for all investigated time points. Differentiated neurons exhibited significant increases in average neurite lengths 96 h post plating, and formed neurite extensions parallel to suspended fibers, as visualized through scanning electron microscopy.

Conclusions

The developed model has the potential to serve as the basis for advanced 3D studies, providing an original approach to neural network patterning and setting the groundwork for further investigations into functionality.

     

MetaboLights: towards a new COSMOS of metabolomics data management

Exciting funding initiatives are emerging in Europe and the US for metabolomics data production, storage, dissemination and analysis. This is based on a rich ecosystem of resources around the world, which has been build during the past ten years, including but not limited to resources such as MassBank in Japan and the Human Metabolome Database in Canada. Now, the European Bioinformatics Institute has launched MetaboLights, a database for metabolomics experiments and the associated metadata (http://www.ebi.ac.uk/metabolights). It is the first comprehensive, cross-species, cross-platform metabolomics database maintained by one of the major open access data providers in molecular biology. In October, the European COSMOS consortium will start its work on Metabolomics data standardization, publication and dissemination workflows. The NIH in the US is establishing 6?C8 metabolomics services cores as well as a national metabolomics repository. This communication reports about MetaboLights as a new resource for Metabolomics research, summarises the related developments and outlines how they may consolidate the knowledge management in this third large omics field next to proteomics and genomics.     

Mechanisms of food selection in Daphnia

A conceptual behavioural and mechanistic Holling-type model of food selection in Daphnia pulicaria is derived from SEM observations with animals feeding on mixtures of spherical-cylindrical diatoms, oblongate green algae, and filamentous cyanobacteria, as well as ultrafine particles. The algae used were Stephanodiscus hantzschii (<- 6 µm length), Monoraphidium setiforme ( 20 µm), and Oscillatoria aghardii (strands, >- 80 µm). Cell (strand) selection can occur at any or all of three stages: (i) interception from the feeding currents, (ii) collection and channeling to the food groove, and (iii) compaction and transport to the mouth. During each stage, given equal initial cell densities, elongate cells are more likely to escape collection than spherical cells and are more likely to be rejected. In addition, filaments require increased handling time at stages (ii) and (iii) and promote entanglement with limb 5 and the postabdominal claw. Food is collected primarily with the aid of limbs 3 (and 4), but limbs 1 and 2 also intervene. Neither the leaky sieve hypothesis alone nor any other single-process hypothesis explains the observations on examined in corpore positions, morphology, and derived movements of the feeding limbs. Attachment and mucus appear to be important for the ingestion of bacteria and ultrafine particles.The model is consistent with many experimental results of differential feeding by Daphnia pulicaria on mixtures of variously shaped algae and other observations on Daphnia feeding behaviour. The paradigm of invariate, nonselective feeding by Daphnia is rejected.     

Myocardial calcium cycling defect in furazolidone cardiomyopathy.

We have previously demonstrated that in furazolidone-induced congestive heart failure in turkeys the specific Ca(2+)-ATPase activity of myocardial sarcoplasmic reticulum (SR) is 60% increased in compensation for a 50% depression in net Ca(2+)-sequestration activity. This study tested the hypothesis that SR Ca(2+)-uptake and Ca(2+)-ATPase activities were uncoupled in this cardiomyopathy because of increased Ca(2+)-release channel activity. A novel microassay was used to monitor Ca2+ transport by myocardial homogenates using the fluorescent Ca2+ dye indo 1 to indicate extravesicular ionized Ca2+. The method is applied to cyropreserved biopsy specimens of myocardium and requires only 50 mg tissue. Both SR Ca(2+)-pump and SR Ca(2+)-channel activity were estimated using the channel-inhibitor ruthenium red (RR) and the mitochondrial inhibitor sodium azide. The specificity of the RR inhibition was confirmed using ryanodine. Cardiomyopathy was induced in 2-week-old turkey poults by the addition of 0.07% furazolidone to their feed for 4 weeks. Compared with controls, myocardial maximal Ca(2+)-channel activity relative to maximal Ca(2+)-pump activity was 22% greater and duration of Ca(2+)-channel activity was 100% increased. However, the heart failure birds had 43 and 53% decreases in absolute maximal Ca(2+)-pumping and Ca(2+)-channel activities, respectively. The abnormal Ca(2+)-channel activity resulted in 200% greater time before initiation of net Ca2+ sequestration and 700% greater final myocardial Ca2+ concentrations. For all birds, the Ca(2+)-accumulating activity was highly correlated with Ca(2+)-release activity (all p less than 0.05). These data indicate that in this animal model of congestive heart failure there is defective SR Ca(2+)-channel function resulting in abnormal Ca2+ homeostasis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accelerated degradation of adenine nucleotide in erythrocytes of patients with chronic renal failure

Recently, we have shown that erythrocytes obtained from patients with chronic renal failure (CRF) exhibited an increased rate of ATP formation from adenine as a substrate. Thus, we concluded that this process was in part responsible for the increase of adenine nucleotide concentration in uremic erythrocytes. There cannot be excluded however, that a decreased rate of adenylate degradation is an additional mechanism responsible for the elevated ATP concentration. To test this hypothesis, in this paper we compared the rate of adenine nucleotide breakdown in the erythrocytes obtained from patients with CRF and from healthy subjects.Using HPLC technique, we evaluated: (1) hypoxanthine production by uremic RBC incubated in incubation medium: (a) pH 7.4 containing 1.2 mM phosphate (which mimics physiological conditions) and (b) pH 7.1 containing 2.4 mM phosphate (which mimics uremic conditions); (2) adenine nucleotide degradation (IMP, inosine, adenosine, hypoxanthine production) by uremic RBC incubated in the presence of iodoacetate (glycolysis inhibitor) and EHNA (adenosine deaminase inhibitor). The erythrocytes of healthy volunteers served as control.The obtained results indicate that adenine nucleotide catabolism measured as a hypoxanthine formation was much faster in erythrocytes of patients with CRF than in the cells of healthy subjects. This phenomenon was observed both in the erythrocytes incubated at pH 7.4 in the medium containing 1.2 mM inorganic phosphate and in the medium which mimics hyperphosphatemia (2.4 mM) and metabolic acidosis (pH 7.1). The experiments with EHNA indicated that adenine nucleotide degradation proceeded via AMP-IMP-Inosine-Hypoxanthine pathway in erythrocytes of both patients with CRF and healthy subjects. Iodoacetate caused a several fold stimulation of adenylate breakdown. Under these conditions: (a) the rate of AMP catabolites (IMP + inosine + adenosine + hypoxanthine) formation was substantially higher in the erythrocytes from patients with CRF; (b) in erythrocytes of healthy subjects degradation of AMP proceeded via IMP and via adenosine essentially at the same rate; (c) in erythrocytes of patients with CRF the rate of AMP degradation via IMP was about 2 fold greater than via adenosine.The results presented in this paper suggest that adenine nucleotide degradation is markedly accelerated in erythrocytes of patients with CRF.     

Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells

Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells.     

Spatially modelling native vegetation condition

Summary   The assessment of vegetation condition is seen as an increasingly important requirement for effective biodiversity conservation in Australia. Condition assessments that operate at the scale of the site are well established. However, there is a need for mapped representations of vegetation condition at regional scales to: (i) assist with regional planning and target setting; (ii) provide regional context for site-based assessment; and (iii) monitor the change in vegetation condition at multiple scales. This paper describes a methodology for converting site condition data collected in plots into maps of vegetation condition across entire regions using a predictive statistical modelling framework (Generalized Additive Modelling) combined with a GIS. The research demonstrates how explanatory variables including topographic position, terrain roughness, landscape connectivity and remote sensing derived indices can be used to map the condition of native vegetation at the scale of a subcatchment. The inclusion of indices derived from remotely sensed imagery (SPOT4) as explanatory variables in the modelling is a novel component of this research. Although the methodology generates statistically and ecologically plausible models of vegetation condition, there are nevertheless limitations associated with the way plot data were collected and some of the explanatory variables, which impacts upon model utility. We discuss how these problems can be minimized when embarking upon studies of this type. We demonstrate how maps produced from exercises such as this could be used for conservation planning and discuss the limitations of these data for monitoring.