Lipoxygenase activities of human platelets and their subcellular fractions: Comparison between lipoxygenase-deficient platelets and normal platelets

Lipoxygenase activities were estimated in washed platelets (intact platelets) and their subcellular fractions obtained from 7 patients with deficient platelet lipoxygenase activities and 9 normal subjects. From sonicated platelet preparations, 12,000 g supernatant (F-I), cytosol (F-II) and microsomal fractions (F-III) were prepared by differential centrifugation. When 12-hydroxyeicosatetraenoic acid (12-HETE) produced by the incubation of arachidonic acid with intact platelets or each of their subcellular fractions from normal subjects was measured by reversed-phase high-performance liquid chromatography analysis, the lipoxygenase activities of F-I, F-II and F-III were 87%, 31% and 17%, respectively, of the enzyme activity of intact platelets. One of the patients showed no detectable lipoxygenase activity in any preparation, while the other patients showed reduced enzyme activities in all preparations. The addition of CaCl2 significantly increased 12-HETE synthesis solely by F-I from these patients. In most of these patients, contrary to normal subjects, it appeared that the lipoxygenase activity was not fully expressed in intact platelets, since the F-I produced more 12-HETE than the intact platelets.     

Phytosulfokine stimulates somatic embryogenesis in Cryptomeria japonica

Phytosulfokine (PSK), which has been identified as a plant growth factor, had a dramatic stimulatory effect on the formation of somatic embryos of sugi (Cryptomeria japonica) in the presence of polyethylene glycol. The resultant somatic embryos germinated with synchronous sprouting of cotyledons, hypocotyls and roots, and most of the seedlings grew normally. A cDNA clone for the precursor to the PSK peptide of C. japonica was identified in an expressed sequence tags database. Our results support the existence of a PSK signaling pathway in C. japonica.     

A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors.

Cytokines manifest their function through alteration of gene expression. However, target genes for signals from cytokine receptors are largely unknown. We therefore searched for immediate-early cytokine-responsive genes and isolated a novel gene, CIS (cytokine inducible SH2-containing protein) which is induced in hematopoietic cells by a subset of cytokines including interleukin 2 (IL2), IL3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO), but not by stem cell factor, granulocyte colony-stimulating factor and IL6. The CIS message encodes a polypeptide of 257 amino acids that contains an SH2 domain of 96 amino acids in the middle. To clarify the function of CIS in cytokine signal transduction, we expressed CIS in IL3-dependent hematopoietic cell lines under the control of a steroid-inducible promoter. The CIS product stably associated with the tyrosine-phosphorylated beta chain of the IL3 receptor as well as the tyrosine-phosphorylated EPO receptor. Forced expression of CIS by steroid reduced the growth rate of these transformants, suggesting a negative role of CIS in signal transduction. CIS induction requires the membrane-proximal region of the cytoplasmic domain of the EPO receptor as well as that of the common beta chain of the IL3, IL5 and GM-CSF receptor, whereas CIS binds to the receptor that is tyrosine phosphorylated by cytokine stimulation. Thus CIS appears to be a unique regulatory molecule for cytokine signal transduction.     

Limited myogenic response to a single bout of weight-lifting exercise in old rats

The purpose of the present study was to compare themyogenic response of hindlimb muscles in young (14-20 wk of age)and old (>120 wk of age) rats with a single exhaustive bout of heavyresistance weight lifting. [³H]thymidine and[¹⁴C]leucine labeling were monitored for up to2 wk after the exercise bout to estimate serial changes in mitoticactivity and the level of amino acid uptake and myosin synthesis.Histological, histochemical, and immunohistochemical[anti-5-bromo-2'-deoxyuridine and myogenic determinationgenes (MyoD)] analyses of whole muscles and analysis ofmuscle-specific gene expression (MyoD) using Western blotting andRT-PCR were performed. Old rats showed significant muscle atrophy and alower exercise capacity than young rats. Exercise-induced muscledamage, as assessed in histological sections, and increases in serumcreatine kinase activity were evident in both young and old exercisedgroups. Mitotic activity was increased in young, but not old, rats 2 days after exercise. There was a biphasic increase in[¹⁴C]leucine uptake during the 14 dayspostexercise (peaks at 1-4 and 10 days) in young rats: only thefirst peak was observed in old rats. There was a lower uptake of[¹⁴C]leucine in the myosin fraction and animpaired expression of MyoD at the protein (immunohistochemistry andWestern blotting) and mRNA (RT-PCR) levels in old rats throughout thepostexercise period. These results demonstrate a reduced reparativecapability of muscle in response to a single bout of exercise in oldcompared with young rats.

     

Automated monitoring of cell concentration and viability using an image analysis system

In order to automate measurements of cell concentration and viability in a suspended animal cell culture, we have developed anin situ microscopic image analysis system with an effective cell recognition algorithm. With a small amount of sample, this system can measure the cell density rapidly and aseptically. In addition, it can measure a cell size histogram including cell debris small particle distribution. These small particles have been found to be related to the viability of the mouse-mouse hybridoma STK1 cell line. By using cell debris small particle density as an indicator of cell viability, the developed system provides non-destructive viability monitoring without trypan blue staining.     

Galectin-9 Enhances Cytokine Secretion,but Suppresses Survival and Degranulation,in Human Mast Cell Line

Galectin-9 (Gal-9), a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.     

Delayed adenosine A1 receptor preconditioning in rat myocardium is MAPK dependent but iNOS independent

Adenosine A1 receptor delayed preconditioning (PC) against myocardial infarction has been well described; however, there have been limited investigations of the signaling mechanisms that mediate this phenomenon. In addition, there are multiple conflicting reports on the role of inducible nitric oxide synthase (iNOS) in mediating A1 late-phase PC. The purpose of this study was to determine the roles of the p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) in in vivo delayed A1 receptor PC and whether this protection at the myocyte level is due to upregulation of iNOS. Myocardial infarct size was measured in open-chest anesthetized rats 24 h after treatment with vehicle or the adenosine A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 100 microg/kg ip). Additional rats receiving CCPA were pretreated with the p38 inhibitor SB-203580 (1 mg/kg ip) or the MAPK/ERK kinase (MEK) inhibitor PD-098059 (0.5 mg/kg ip). At 24 h after CCPA administration, a group of animals was given the iNOS inhibitor 1400 W 10 min before ischemia. Treatment with CCPA reduced infarct size from 48 +/- 2 to 28 +/- 2% of the area at risk, an effect that was blocked by both SB-203580 and PD-098059 but not 1400 W. Ventricular myocytes isolated 24 h after CCPA injection exhibited significantly reduced oxidative stress during H2O2 exposure compared with myocytes from vehicle-injected animals, and this effect was not blocked by the iNOS inhibitor 1400 W. Western blot analysis of whole heart and cardiac myocyte protein samples revealed no expression of iNOS 6 or 24 h after CCPA treatment. These results indicate that adenosine A1 receptor delayed PC in rats is mediated by MAPK-dependent mechanisms, but this phenomenon is not associated with the early or late expression of iNOS.     

Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible genes in human amniotic epithelial cells

Background  

Exposure to dioxins results in a broad range of pathophysiological disorders in human fetuses. In order to evaluate the effects of dioxins on the feto-placental tissues, we analyzed the gene expression in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated primary cultures of human amniotic epithelial cells.     

Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.     

Systematic phenome analysis of Escherichia coli multiple‐knockout mutants reveals hidden reactions in central carbon metabolism

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple‐knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose‐7‐phosphate to erythrose‐4‐phosphate and dihydroxyacetone phosphate were observed in transaldolase‐deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose‐7‐phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP‐dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.