Functional characterisation of an intron retaining K(+) transporter of barley reveals intron-mediated alternate splicing.

Intron retention in transcripts and the presence of 5' and 3' splice sites within these introns mediate alternate splicing, which is widely observed in animals and plants. Here, functional characterisation of the K(+) transporter, HvHKT2;1, with stably retained introns from barley (Hordeum vulgare) in yeast (Saccharomyces cerevisiae), and transcript profiling in yeast and transgenic tobacco (Nicotiana tabacum) is presented. Expression of intron-retaining HvHKT2;1 cDNA (HvHKT2;1-i) in trk1, trk2 yeast strain defective in K(+) uptake restored growth in medium containing hygromycin in the presence of different concentrations of K(+) and mediated hypersensitivity to Na(+) . HvHKT2;1-i produces multiple transcripts via alternate splicing of two regular introns and three exons in different compositions. HKT isoforms with retained introns and exon skipping variants were detected in relative expression analysis of (i) HvHKT2;1-i in barley under native conditions, (ii) in transgenic tobacco plants constitutively expressing HvHKT2;1-i, and (iii) in trk1, trk2 yeast expressing HvHKT2;1-i under control of an inducible promoter. Mixed proportions of three HKT transcripts: HvHKT2;1-e (first exon region), HvHKT2;1-i1 (first intron) and HvHKT2;1-i2 (second intron) were observed. The variation in transcript accumulation in response to changing K(+) and Na(+) concentrations was observed in both heterologous and plant systems. These findings suggest a link between intron-retaining transcripts and different splice variants to ion homeostasis, and their possible role in salt stress.

Cardiac myocyte exosomes: stability, HSP60, and proteomics.

Exosomes, which are 50- to 100-nm-diameter lipid vesicles, have been implicated in intercellular communication, including transmitting malignancy, and as a way for viral particles to evade detection while spreading to new cells. Previously, we demonstrated that adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. Thus, release of HSP60 from exosomes would be damaging to the surrounding cardiac myocytes. We hypothesized that 1) pathological changes in the environment, such as fever, change in pH, or ethanol consumption, would increase exosome permeability; 2) different exosome inducers would result in different exosomal protein content; 3) ethanol at "physiological" concentrations would cause exosome release; and 4) ROS production is an underlying mechanism of increased exosome production. We found the following: first, exosomes retained their protein cargo under different physiological/pathological conditions, based on Western blot analyses. Second, mass spectrometry demonstrated that the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins. Third, ethanol did not affect exosome stability but greatly increased the production of exosomes by cardiac myocytes. Fourth, ethanol- and hypoxia/reoxygenation-derived exosomes had different protein content. Finally, ROS inhibition reduced exosome production but did not completely inhibit it. In conclusion, exosomal protein content is influenced by the cell source and stimulus for exosome formation. ROS stimulate exosome production. The functions of exosomes remain to be fully elucidated.

Mycobacterium tuberculosis phagosomes exhibit altered calmodulin-dependent signal transduction: contribution to inhibition of phagosome-lysosome fusion and intracellular survival in human macrophages.

Mycobacterium tuberculosis successfully parasitizes macrophages by disrupting the maturation of its phagosome, creating an intracellular compartment with endosomal rather than lysosomal characteristics. We have recently demonstrated that live M. tuberculosis infect human macrophages in the absence of an increase in cytosolic Ca(2+) ([Ca(2+)](c)), which correlates with inhibition of phagosome-lysosome fusion and intracellular viability. In contrast, killed M. tuberculosis induces an elevation in [Ca(2+)](c) that is coupled to phagosome-lysosome fusion. We tested the hypothesis that defective activation of the Ca(2+)-dependent effector proteins calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) contributes to the intracellular pathogenesis of tuberculosis. Phagosomes containing live M. tuberculosis exhibited decreased levels of CaM and the activated form of CaMKII compared with phagosomes encompassing killed tubercle bacilli. Furthermore, ionophore-induced elevations in [Ca(2+)](c) resulted in recruitment of CaM and activation of CaMKII on phagosomes containing live M. tuberculosis. Specific inhibitors of CaM or CaMKII blocked Ca(2+) ionophore-induced phagosomal maturation and enhanced the bacilli's intracellular viability. These results demonstrate a novel role for CaM and CaMKII in the regulation of phagosome-lysosome fusion and suggest that defective activation of these Ca(2+)-activated signaling components contributes to the successful parasitism of human macrophages by M. tuberculosis.

In vitro and in vivo macrophage function can occur independently of SLP-76.

Expression of SH2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), a hematopoietic cell-specific adapter protein, is required to couple Syk family tyrosine kinase activation to downstream mediators such as phospholipase C (PLC)-gamma following TCR, platelet collagen receptor and mast cell Fc epsilon R stimulation. In addition to T cells, mast cells and platelets, SLP-76 is expressed in monocytes and macrophages. To determine the role of SLP-76 in Fc gamma R-stimulated signaling pathways in macrophages, we examined cultured bone marrow-derived macrophages (BMM) from SLP-76(-/-) and wild-type mice. In this study, we show that Fc gamma R cross-linking rapidly induces tyrosine phosphorylation of SLP-76 in wild-type BMM. Surprisingly, however, BMM from SLP-76(-/-) mice activate ERK2 and phosphorylate PLC-gamma 2 following Fc gamma R ligation. Furthermore, SLP-76(-/-) BMM display normal Fc gamma R-dependent phagocytic function and reactive oxygen intermediate production. SLP-76(-/-) and SLP-76(+/+) BMM secrete comparable levels of IL-12 in response to lipopolysaccharide and IFN-gamma. To examine macrophage function in vivo, SLP-76(-/-) mice were challenged i.v. with Listeria monocytogenes. SLP-76(-/-) mice survive and efficiently contain the acute phase of infection similar to wild-type mice but exhibit a stable chronic infection attributed to the lack of mature T cells. These data show that, although SLP-76 is required to couple Syk family PTK activity to downstream mediators and effector functions in Fc gamma R-induced pathways in some cell types, activation of Fc gamma R-dependent pathways occurs independently of SLP-76 in BM

Inhibition of Ca(2+) signaling by Mycobacterium tuberculosis is associated with reduced phagosome-lysosome fusion and increased survival within human macrophages.

Complement receptor (CR)-mediated phagocytosis of Mycobacterium tuberculosis by macrophages results in intracellular survival, suggesting that M. tuberculosis interferes with macrophage microbicidal mechanisms. As increases in cytosolic Ca(2+) concentration (¿Ca(2+)(c)) promote phagocyte antimicrobial responses, we hypothesized that CR phagocytosis of M. tuberculosis is accompanied by altered Ca(2+) signaling. Whereas the control complement (C)-opsonized particle zymosan (COZ) induced a 4.6-fold increase in ¿Ca(2+)(c) in human macrophages, no change in ¿Ca(2+)(c) occurred upon addition of live, C-opsonized virulent M. tuberculosis. Viability of M. tuberculosis and ingestion via CRs was required for infection of macrophages in the absence of increased ¿Ca(2+)(c), as killed M. tuberculosis or antibody (Ab)-opsonized, live M. tuberculosis induced elevations in ¿Ca(2+)(c) similar to COZ. Increased ¿Ca(2+)(c) induced by Ab-opsonized bacilli was associated with a 76% reduction in intracellular survival, compared with C-opsonized M. tuberculosis. Similarly, reversible elevation of macrophage ¿Ca(2+)(c) with the ionophore A23187 reduced intracellular viability by 50%. Ionophore-mediated elevation of ¿Ca(2+)(c) promoted the maturation of phagosomes containing live C-opsonized bacilli, as evidenced by acidification and accumulation of lysosomal protein markers. These data demonstrate that M. tuberculosis inhibits CR-mediated Ca(2+) signaling and indicate that this alteration of macrophage activation contributes to inhibition of phagosome-lysosome fusion and promotion of intracellular mycobacterial survival.

Ketamine for bone marrow aspiration and trephine biopsy in children.

To make bone marrow aspiration and trephine biopsy less painful and more acceptable in children a short acting anaesthetic ketamine was used in a dose of 1-2 mg/kg body weight intravenously or 4-5 mg/kg intramuscularly. One hundred cases aged 2 to 13 years were studied. The actual procedure time ranged between 20 seconds and 3 minutes, adequate samples were obtained in 80% of children on first attempt. Vomiting was the only side effect noted in two children. Ketamine is safe and recommended in all children undergoing bone marrow aspiration and trephine biopsies.

Properties of ATPase activity associated with peroxisomes of rat and bovine liver.

1. Peroxisomes were isolated from bovine and rat liver by use of differential and density gradient centrifugations. 2. In the final density gradient (Nycodenz) a distinct peak of ATPase activity codistributed with the peroxisome marker catalase and was well separated from the bulk of the ATPase activity and from markers for other subcellular organelles. 3. The peroxisome-associated ATPase had a pH optimum of 7.5 and was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was unaffected by up to 30 microM n-tributyltin chloride. 4. Prolonged incubation with oligomycin at high concentrations indicated that 50% of peroxisomal ATPase was resistant to this inhibitor. The oligomycin-sensitive ATPase activity required at least a four-fold higher ratio of inhibitor to protein for inhibition than mitochondrial ATPase did. It was concluded that oligomycin-sensitive and oligomycin-resistant ATPase may be associated with liver peroxisomes.