Zebrafish SPI-1 marks a site of myeloid development independent of primitive erythropoiesis: implications for axial patterning.

The mammalian transcription factor SPI-1 (synonyms: SPI1, PU.1, or Sfpi1) plays a critical role in myeloid development. To examine early myeloid commitment in the zebrafish embryo, we isolated a gene from zebrafish that is a SPI-1 orthologue on the basis of homology and phylogenetic considerations. The zebrafish spi1 (pu1) gene was first expressed at 12 h postfertilization in rostral lateral plate mesoderm (LPM), anatomically isolated from erythroid development in caudal lateral plate mesoderm. Fate-mapping traced rostral LPM cells from the region of initial spi1 expression to a myeloid fate. spi1 expression was lost in the bloodless mutant cloche, but rostral spi1 expression and myeloid development were preserved in the mutant spadetail, despite its complete erythropoietic failure. This dissociation of myeloid and erythroid development was further explored in studies of embryos overexpressing BMP-4, or chordin, in bmp-deficient swirl and snailhouse mutants, and chordin-deficient chordino mutants. These studies demonstrate that, in zebrafish, spi1 marks a rostral population of LPM cells committed to a myeloid fate anatomically separated from and developmentally independent of erythroid commitment in the caudal LPM. Such complete anatomical and developmental dissociation of two hematopoietic lineages adds an interesting complexity to the understanding of vertebrate hematopoietic development and presents significant implications for the mechanisms regulating axial patterning.

10 mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin

Objective
Glucosamine has been previously shown to suppress cartilage aggrecan catabolism in explant cultures. We determined the effect of glucosamine on ADAMTS5 (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type-1 motifs 5), a major aggrecanase in osteoarthritis, and investigated a potential mechanism underlying the observed effects.

Design
HEK293F and CHO-K1 cells transiently transfected with ADAMTS5 cDNA were treated with glucosamine or the related hexosamine mannosamine. Glucosamine effects on FURIN transcription were determined by quantitative RT-PCR. Effects on furin-mediated processing of ADAMTS5 zymogen, and aggrecan processing by glucosamine-treated cells, were determined by western blotting. Post-translational modification of furin and N-glycan deficient furin mutants generated by site-directed mutagenesis was analyzed by western blotting, and the mutants were evaluated for their ADAMTS5 processing ability in furin-deficient CHO-RPE.40 cells.

Results
Ten mM glucosamine and 5–10 mM mannosamine reduced excision of the ADAMTS5 propeptide, indicating interference with the propeptide excision mechanism, although mannosamine compromised cell viability at these doses. Although glucosamine had no effect on furin mRNA levels, western blot of furin from glucosamine-treated cells suggested altered post-translational modification. Glucosamine treatment led to decreased glycosylation of cellular furin, with reduced furin autoactivation as the consequence. Recombinant furin treated with peptide N-glycanase F had reduced activity against a synthetic peptide substrate. Indeed, site-directed mutagenesis of two furin N-glycosylation sites, Asn387 and Asn440, abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells.

Conclusions
Ten mM glucosamine reduces excision of the ADAMTS5 propeptide via interference with post-translational modification of furin and leads to reduced aggrecanase activity of ADAMTS5.

Metabolic consequences of knocking out UGT85B1, the gene encoding the glucosyltransferase required for synthesis of dhurrin in Sorghum bicolor (L. Moench)

Many important food crops produce cyanogenic glucosides as natural defense compounds to protect against herbivory or pathogen attack. It has also been suggested that these nitrogen-based secondary metabolites act as storage reserves of nitrogen. In sorghum, three key genes, CYP79A1, CYP71E1 and UGT85B1, encode two Cytochrome P450s and a glycosyltransferase, respectively, the enzymes essential for synthesis of the cyanogenic glucoside dhurrin. Here, we report the use of targeted induced local lesions in genomes (TILLING) to identify a line with a mutation resulting in a premature stop codon in the N-terminal region of UGT85B1. Plants homozygous for this mutation do not produce dhurrin and are designated tcd2 (totally cyanide deficient 2) mutants. They have reduced vigor, being dwarfed, with poor root development and low fertility. Analysis using liquid chromatography-mass spectrometry (LC-MS) shows that tcd2 mutants accumulate numerous dhurrin pathway-derived metabolites, some of which are similar to those observed in transgenic Arabidopsis expressing the CYP79A1 and CYP71E1 genes. Our results demonstrate that UGT85B1 is essential for formation of dhurrin in sorghum with no co-expressed endogenous UDP-glucosyltransferases able to replace it. The tcd2 mutant suffers from self-intoxication because sorghum does not have a feedback mechanism to inhibit the initial steps of dhurrin biosynthesis when the glucosyltransferase activity required to complete the synthesis of dhurrin is lacking. The LC-MS analyses also revealed the presence of metabolites in the tcd2 mutant which have been suggested to be derived from dhurrin via endogenous pathways for nitrogen recovery, thus indicating which enzymes may be involved in such pathways.

Decreased fatty acit Â-oxidation in riboflavin-responsive, multiple acylocoenzyme a dehydrogenase-deficient patients is associated with an increase in uncoupling protein-3

Riboflavin-responsive, multiple acylcoenzyme A dehydrogenase deficiency (RR-MAD), a lipid storage myopathy, is characterized by, among others, a decrease in fatty acid (FA) ß-oxidation capacity. Muscle uncoupling protein 3 (UCP3) is up-regulated under conditions that either increase the levels of circulating free FA and/or decrease FA ß-oxidation. Using a relatively large cohort of seven RR-MAD patients, we aimed to better characterize the metabolic disturbances of this disease and to explore the possibility that it might increase UCP3 expression. A battery of biochemical and molecular tests were performed, which demonstrated decreases in FA ß-oxidation and in the activities of respiratory chain complexes I and II. These metabolic alterations were associated with increases of 3.1- and 1.7-fold in UCP3 mRNA and protein expression, respectively. All parameters were restored to control values after riboflavin treatment. We postulate that the up-regulation of UCP3 in RR-MAD is due to the accumulation of muscle FA/acylCoA. RR-MAD is an optimal model to support the hypothesis that UCP3 is involved in the outward translocation of an excess of FA from the mitochondria and to show that, in humans, the effects of FA on UCP3 expression are direct and independent of fatty acid ß-oxidation.

Age-related proteostasis and metabolic alterations in Caspase-2-deficient mice.

Ageing is a complex biological process for which underlying biochemical changes are still largely unknown. We performed comparative profiling of the cellular proteome and metabolome to understand the molecular basis of ageing in Caspase-2-deficient (Casp2(-/-)) mice that are a model of premature ageing in the absence of overt disease. Age-related changes were determined in the liver and serum of young (6-9 week) and aged (18-24 month) wild-type and Casp2(-/-) mice. We identified perturbed metabolic pathways, decreased levels of ribosomal and respiratory complex proteins and altered mitochondrial function that contribute to premature ageing in the Casp2(-/-) mice. We show that the metabolic profile changes in the young Casp2(-/-) mice resemble those found in aged wild-type mice. Intriguingly, aged Casp2(-/-) mice were found to have reduced blood glucose and improved glucose tolerance. These results demonstrate an important role for caspase-2 in regulating proteome and metabolome remodelling during ageing.

Copper-induced trafficking of the Cu-ATPase: a key mechanism for copper homeostasis

The Menkes protein (MNK) and Wilson protein (WND) are transmembrane, CPX-type Cu-ATPases with six metal binding sites (MBSs) in the N-terminal region containing the motif GMXCXXC. In cells cultured in low copper concentration MNK and WND localize to the transGolgi network but in high copper relocalize either to the plasma membrane (MNK) or a vesicular compartment (WND). In this paper we investigate the role of the MBSs in Cu-transport and trafficking. The copper transport activity of MBS mutants of MNK was determined by their ability to complement a strain of Saccharomyces cerevisiae deficient in CCC2 (Deltaccc2), the yeast MNK/WND homologue. Mutants (CXXC to SXXS) of MBS1, MBS6, and MBSs1-3 were able to complement Deltaccc2 while mutants of MBS4-6, MBS5-6 and all six MBS inactivated the protein. Each of the inactive mutants also failed to display Cu-induced trafficking suggesting a correlation between trafficking and transport activity. A similar correlation was found with mutants of MNK in which various MBSs were deleted, but two constructs with deletion of MBS5-6 were unable to traffic despite retaining 25% of copper transport activity. Chimeras in which the N-terminal MBSs of MNK were replaced with the corresponding MBSs of WND were used to investigate the region of the molecules that is responsible for the difference in Cu-trafficking of MNK and WND. The chimera which included the complete WND N-terminus localized to a vesicular compartment, similar to WND in elevated copper. Deletions of various MBSs of the WND N-terminus in the chimera indicate that a targeting signal in the region of MBS6 directs either WND/MNK or WND to a vesicular compartment of the cell.

The role of nurses in preventing adverse events related to respiratory dysfunction: literature review

Aims. This paper reports a literature review examining the relationship between specific clinical indicators of respiratory dysfunction and adverse events, and exploring the role of nurses in preventing adverse events related to respiratory dysfunction.

Background. Adverse events in hospital are associated with poor patient outcomes such as increased mortality and permanent disability. Many of these adverse events are preventable and are preceded by a period during which the patient exhibits clearly abnormal physiological signs. The role of nurses in preserving physiological safety by early recognition and correction of physiological abnormality is a key factor in preventing adverse events.

Methods. A search of the Medline and CINAHL databases was conducted using the following terms: predictors of poor outcome, adverse events, mortality, cardiac arrest, emergency, oxygen, supplemental oxygen, oxygen therapy, oxygen saturation, oxygen delivery, assessment, patient assessment, physical assessment, dyspnoea, hypoxia, hypoxaemia, respiratory assessment, respiratory dysfunction, shortness of breath and pulse oximetry. The papers reviewed were research papers that demonstrated a relationship between adverse events and various clinical indicators of respiratory dysfunction.

Results. Respiratory dysfunction is a known clinical antecedent of adverse events such as cardiac arrest, need for medical emergency team activation and unplanned intensive care unit admission. The presence of respiratory dysfunction prior to an adverse event is associated with increased mortality. The specific clinical indicators involved are alterations in respiratory rate, and the presence of dyspnoea, hypoxaemia and acidosis.

Conclusions. The way in which nurses assess, document and use clinical indicators of respiratory dysfunction is influential in identifying patients at risk of an adverse event and preventing adverse events related to respiratory dysfunction. If such adverse events are to be prevented, nurses must not only be able to recognise and interpret signs of respiratory dysfunction, but must also take responsibility for initiating and evaluating interventions aimed at correcting respiratory dysfunction.

Apoptosis may underlie the pathology of zinc-deficient skin

The trace element zinc is essential for the survival and function of all cells. Zinc deficiency, whether nutritional or genetic, is fatal if left untreated. The effects of zinc deficiency are particularly obvious in the skin, seen as an erythematous rash, scaly plaques, and ulcers. Electron microscopy reveals degenerative changes within keratinocytes. Despite the well-documented association between zinc deficiency and skin pathology, it is not clear which cellular processes are most sensitive to zinc deficiency and could account for the typical pathological features. We used the cultured HaCaT keratinocyte line to obtain insight into the cellular effects of zinc deficiency, as these cells show many characteristics of normal skin keratinocytes. Zinc deficiency was induced by growing cells in the presence of the zinc chelator, TPEN, or by growth in zinc-deficient medium. Growth of cells in zinc-deficient medium resulted in a 44% reduction of intracellular zinc levels and a 75% reduction in the activity of the zinc-dependent enzyme, 5'-nucleotidase, relative to the control cells. Over a period of 7 days of exposure to zinc-deficient conditions, no changes in cell viability and growth, or in the cytoskeletal and cell adhesion systems, were found in HaCaT cells. At 7 days, however, induction of apoptosis was indicated by the presence of DNA fragmentation and expression of active caspase-3 in cells. These results demonstrate that apoptosis is the earliest detectable cellular change induced by zinc deficiency in HaCaT keratinocytes. Our observations account for many of the features of zinc deficiency, including the presence of degenerate nuclei, chromatin aggregates and abnormal organization of keratin, that may represent the later stages of apoptosis. In summary, a major causal role for apoptosis in the pathology of zinc deficiency in the skin is proposed. This role is consistent with the previously unexplained diverse range of degenerative cellular changes seen at the ultrastructural level in zinc-deficient keratinocytes.

Insulin signaling after exercise in insulin receptor substrate-2-deficient mice

The period immediately after exercise is characterized by enhanced insulin action in skeletal muscle, and on the molecular level, by a marked increase in insulin-stimulated, phosphotyrosine-associated phosphatidylinositol (PI) 3-kinase activity. Because the increase in PI 3-kinase activity cannot be explained by increased insulin receptor substrate (IRS)-1 signaling, the present study examined whether this effect is mediated by enhanced IRS-2 signaling. In wild-type (WT) mice, insulin increased IRS-2 tyrosine phosphorylation (2.5-fold) and IRS-2-associated PI 3-kinase activity (3-fold). Treadmill exercise, per se, had no effect on IRS-2 signaling, but in the period immediately after exercise, there was a further increase in insulin-stimulated IRS-2 tyrosine phosphorylation (3.5-fold) and IRS-2-associated PI 3-kinase activity (5-fold). In IRS-2-deficient (IRS-2-/-) mice, the increase in insulin-stimulated, phosphotyrosine-associated PI 3-kinase activity was attenuated as compared with WT mice. However, in IRS-2-/- mice, the insulin-stimulated, phosphotyrosine-associated PI 3-kinase response after exercise was slightly higher than the insulin-stimulated response alone. In conclusion, IRS-2 tyrosine phosphorylation and associated PI 3-kinase activity are markedly enhanced by insulin in the immediate period after exercise. IRS-2 signaling can partially account for the increase in insulin-stimulated phosphotyrosine-associated PI 3-kinase activity after exercise.

Casitas b-lineage lymphoma-deficient mice are pretected against high-fat diet-induuced obesity and insulin resistance

Casitas b-lineage lymphoma (c-Cbl) is a multiadaptor protein with E3-ubiquitin ligase activity involved in regulating the degradation of receptor tyrosine kinases. We have recently reported that c-Cbl^–/– mice exhibit a lean phenotype and enhanced peripheral insulin action likely due to elevated energy expenditure. In the study reported here, we examined the effect of a high-fat diet on energy homeostasis and glucose metabolism in these animals. When c-Cbl^–/– mice were fed a high-fat diet for 4 weeks, they maintained hyperphagia, higher whole-body oxygen consumption (27%), and greater activity (threefold) compared with wild-type animals fed the same diet. In addition, the activity of several enzymes involved in mitochondrial fat oxidation and the phosphorylation of acetyl CoA carboxylase was significantly increased in muscle of high-fat–fed c-Cbl–deficient mice, indicating a greater capacity for fat oxidation in these animals. As a result of these differences, fat-fed c-Cbl^–/– mice were 30% leaner than wild-type animals and were protected against high-fat diet–induced insulin resistance. These studies are consistent with a role for c-Cbl in regulating nutrient partitioning in skeletal muscle and emphasize the potential of c-Cbl as a therapeutic target in the treatment of obesity and type 2 diabetes.

c-Cbl-deficient mice have reduced adiposity, higher energy expenditure, and improved peripheral insulin action

Casitas b-lineage lymphoma (c-Cbl) is an E3 ubiquitin ligase that has an important role in regulating the degradation of cell surface receptors. In the present study we have examined the role of c-Cbl in whole-body energy homeostasis. c-Cb^-/- mice exhibited a profound increase in whole-body energy expenditure as determined by increased core temperature and whole-body oxygen consumption. As a consequence, these mice displayed a decrease in adiposity, primarily due to a reduction in cell size despite an increase in food intake. These changes were accompanied by a significant
increase in activity (2- to 3-fold). In addition, cc-Cb-/- mice displayed a marked improvement in whole-body insulin action, primarily due to changes in muscle metabolism. We observed increased protein levels of the insulin receptor (4-fold) and uncoupling protein-3 (2-fold) in skeletal muscle and a significant increase in the phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase. These fmdings suggest that c-Cbl plays an integral role in whole-body fuel homeostasis by regulating whole-body energy expenditure and insulin action.

Improved glusoce homeostasis and enhanced insulin signalling in Grb 14-deficient mice

Gene targeting was used to characterize the physiological role of growth factor receptor-bound (Grb)14, an adapter-type signalling protein that associates with the insulin receptor (IR). Adult male Grb14^-/- mice displayed improved glucose tolerance, lower circulating insulin levels, and increased incorporation of glucose into glycogen in the liver and skeletal muscle. In ex vivo studies, insulin-induced 2-deoxyglucose uptake was enhanced in soleus muscle, but not in epididymal adipose tissue. These metabolic effects correlated with tissue-specific alterations in insulin signalling. In the liver, despite lower IR autophosphorylation, enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and activation of protein kinase B (PKB) was observed. In skeletal muscle, IR tyrosine phosphorylation was normal, but signalling via IRS-1 and PKB was increased. Finally, no effect of Grb14 ablation was observed on insulin signalling in white adipose tissue. These findings demonstrate that Grb14 functions in vivo as a tissue-specific modulator of insulin action, most likely via repression of IR-mediated IRS-1 tyrosine phosphorylation, and highlight this protein as a potential target for therapeutic intervention.

Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Y704, Y729, Y744, Y764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation and cell survival. However, it is unclear whether these tyrosines are equally important under more physiological conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated GCSF- R deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (mO), single tyrosine "add back" mutants or wild type (WT) receptors. G-CSFinduced responses were determined in primary colony assays, serial replatings and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Y764 strongly enhanced proliferativeresponses, which was reverted by inhibition of ERK activitity. Y729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing mO gradually dropped compared to WT. The presence of Y729, but also Y704 and Y744, both involved in activation of STAT3, further reduced replating
efficiencies. Conversely, Y764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a >104–fold increase of colony forming cells over mO after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.

Retinal sensitivity loss in third-generation n-3 PUFA-deficient rats

A previous study conducted in guinea pigs suggested that ingestion of diets high in EPA and DHA may result in suboptimal retinal function. The aim of the present study was to evaluate retinal function in pigmented (Long-Evans) rats, raised to a third generation on diets that were either deficient in n-3 PUFA or adequate (with the addition of DHA). Electroretinographic assessment employed full-field white flash stimulation. Photoreceptor responses were evaluated in terms of peak amplitudes and implicit times (a-wave, b-wave), intensity-response functions (Naka-Rushton), and the parameters of a model of transduction (P3). Retinal phospholipid FA composition was measured by capillary GLC. DHA levels were reduced by 55% in n-3-deficient animals compared with the n-3-adequate group, whereas the levels of docosapentaenoic acid n-6 were 44 times higher in n-3-deficient animals. The level of arachidonic acid was marginally higher (12.8%) in n-6-adequate animals. The n-3-deficient animals exhibited significantly reduced retinal sensitivity (σ and S values were both affected by 0.29 log units) and increased b-wave implicit times compared with those fed the n-3-adequate diet. These data suggest that n-3 PUFA are required for development of retinal sensitivity, more so than other indices of retinal function assessed by current methods, such as maximal response amplitude. However, the benefit for retinal function of adding preformed DHA to diets already replete in n-3 PUFA remains unclear.

The cost of seasonal respiratory illnesses in Australian children: the dominance of patient and family costs and implications for vaccine use

Respiratory viral infections are one of the next group of diseases likely to be targeted for prevention in childhood by the use of vaccines. To begin collecting necessary epidemiology and cost information about the illnesses caused by these viruses, we conducted a prospective cohort study in 118 Melbourne children between 12 and 71 months of age during winter and spring 2001. We were interested in calculating an average cost per episode of community-managed acute respiratory disease, in identifying the key cost drivers of such illness, and to identify the proportion of costs borne by the patient and family. There were 202 community-managed influenza-like illnesses identified between July and December 2001, generating 89 general practitioner visits, and 42 antibiotic prescriptions. The average cost of community-managed episodes (without hospitalisation) was $241 (95% CI $191 to $291), with the key cost drivers being carer time away from usual activities caring for the ill child (70% of costs), use of non-prescription medications (5.4%), and general practice visits (5.0%). The patient and family met 87per cent of total costs. The lowest average cost occurred in households from the highest income bracket. Acute respiratory illness managed in the community is common, with the responsibility for meeting the cost of episodes predominantly borne by the patient and family in the form of lost productivity. These findings have implications for preventive strategies in children, such as the individual use of, or implementation of public programs using, currently available vaccines against influenza and vaccines under development against other viral respiratory pathogens.