BBA - General Subjects (v.1800, #12)

Effects of exposure to a time-varying 1.5 T magnetic field on the neurotransmitter-activated increase in intracellular Ca2+ in relation to actin fiber and mitochondrial functions in bovine adrenal chromaffin cells by Toshitaka Ikehara; Hirotaka Nishisako; Yuki Minami; Hiromi Ichinose(Sasaki); Tairo Shiraishi; Mitsuo Kitamura; Masayuki Shono; Hitoshi Houchi; Kazuyoshi Kawazoe; Kazuo Minakuchi; Kazuo Yoshizaki; Yohsuke Kinouchi; Hiroshi Miyamoto (1221-1230).
It has been reported that exposure to electromagnetic fields influences intracellular signal transduction. We studied the effects of exposure to a time-varying 1.5 T magnetic field on membrane properties, membrane cation transport and intracellular Ca2+ mobilization in relation to signals. We also studied the mechanism of the effect of exposure to the magnetic field on intracellular Ca2+ release from Ca2+ stores in adrenal chromaffin cells.We measured the physiological functions of ER, actin protein, and mitochondria with respect to a neurotransmitter-induced increase in Ca2+ in chromaffin cells exposed to the time-varying 1.5 T magnetic field for 2 h.Exposure to the magnetic field significantly reduced the increase in [Ca2+]i. The exposure depolarized the mitochondria membrane and lowered oxygen uptake, but did not reduce the intracellular ATP content. Magnetic field-exposure caused a morphological change in intracellular F-actin. F-actin in exposed cells seemed to be less dense than in control cells, but the decrease was smaller than that in cytochalasin D-treated cells. The increase in G-actin (i.e., the decrease in F-actin) due to exposure was recovered by jasplakinolide, but inhibition of Ca2+ release by the exposure was unaffected.These results suggest that the magnetic field-exposure influenced both the ER and mitochondria, but the inhibition of Ca2+ release from ER was not due to mitochondria inhibition. The effect of eddy currents induced in the culture medium may indirectly influence intracellular actin and suppress the transient increase in [Ca2+]i.
Keywords: Electromagnetic field; Ca2+ release; F-actin; mitochondria; endoplasmic reticulum; Eddy currents;

p53 and PPP1R13L (alias iASPP or RAI) form a feedback loop to regulate genotoxic stress responses by Magdalena J. Laska; Ulla B. Vogel; Uffe B. Jensen; Bjørn A. Nexø (1231-1240).
PPP1R13L gene has been found to be over-expressed in variety of cancers and its expression in p53 wild-type background is sufficient to promote tumor growth in vivo. However, in the non-transformed cells it acts as a tumor suppressor which suggests that the role of PPP1R13L is multifaceted.We have used siRNA optimized for inhibition of p53, PPP1R13L, BAX and GADD45 alpha expression and investigated the role of those gene products for PPP1R13L expression and induction in a variety of mouse and human cells with different p53 status. In addition we have applied Western Blot, Q-PCR and proteasome inhibition analysis to further ascertain the link between PPP1R13L induction and p53 status.We show that the pattern and extent of the PPP1R13L expression depend on the presence of active p53. Downregulation of p53 target genes BAX and/or GADD45 alpha led to decreased in PPP1R13L activation after adriamycin and/or etoposide treatments. Treatment of the cells with the proteasome inhibitor MG-132 resulted in the accumulation of both p53 and PPP1R13L proteins.We have provided evidence that endogenous PPP1R13L acts as a negative regulator of p53 function, presumably by direct binding. p53 accumulation and activity after DNA damage is compromised by PPP1R13L expression. We suggest that PPP1R13L and p53 form a negative feedback loop which regulates their amount and activity.The profound modulatory effect of the PPP1R13L protein on the ability of p53 to cause cellular apoptosis has important implications in cancer and presents new therapeutic possibilities.
Keywords: DNA damage; p53 tumor suppressor; PPP1R13L; Apoptosis; Proteasome inhibitor;

Mutational analysis of NHAoc/NHA2 in Saccharomyces cerevisiae by Xiaobin Huang; Leslie R. Morse; Yan Xu; Jaromir Zahradka; Hana Sychrová; Phil Stashenko; Feiyue Fan; Ricardo A. Battaglino (1241-1247).
NHAoc/NHA2 is highly and selectively expressed in osteoclasts and plays a role(s) in normal osteoclast differentiation, apoptosis and bone resorptive function in vitro. Extensive mutational analysis of a bacterial homologue, NhaA, has revealed a number of amino acid residues essential for its activity. Some of these residues are evolutionarily conserved and have been shown to be essential not only for activity of NhaA in bacteria, but also of NHAoc/NHA2 in eukaryotes.The salt-sensitive Saccharomyces cerevisiae strain BW31a was used for heterologous expression of mutants of NHAoc/NHA2. Membrane expression of NHAoc/NHA2 was confirmed by confocal microscopy. Intracellular concentration of Na+ (a measure of Na+ antiporter activity) was estimated by atomic absorption spectroscopy. The growth phenotypes of cells expressing NHAoc/NHA2 mutants were studied on YNB agar supplemented with NaCl and by growth curves in YNB broth.Mutations in amino acid residues V161 and F357 reduced the ability of transfected BW31a cells to remove intracellular sodium and to grow in NaCl-containing medium. Yeast expressing the double mutant F357 F437 cannot grow in 0.4 M NaCl, suggesting that these residues are also essential for antiporter activity.Evolutionarily conserved amino acids are required for full antiporter function.Mutations in these amino acid residues may impact NHAoc activity and therefore osteoclast function in vitro and in vivo.
Keywords: Osteclast; Antiporter; Bone resorption; Yeast; Salt tolerance;

Chronic hypoxia- and cold-induced changes in cardiac enzyme and gene expression in CD-1 mice by Nicole M. Templeman; Jacqueline L. Beaudry; Christophe M.R. Le Moine; Grant B. McClelland (1248-1255).
In mammals, environmental challenges often result in physical and metabolic cardiac remodeling (i.e., hypertrophy and a shift from lipid to carbohydrate oxidation). While chronic hypoxia and cold are both known to elicit cardiac changes, little is known about their combined effects.To investigate the cumulated effects of these two stressors on cardiac physiology, CD-1 mice were exposed for 4 weeks to normoxia/normothermia, hypoxia, cold, or combined hypoxic–cold. We assessed physical characteristics, left ventricular activities of fatty acid catabolic enzymes short-chain β-hydroxyacyl-CoA dehydrogenase (SCHAD) and medium-chain acyl-CoA dehydrogenase, and mRNA levels of Acadm, muscle- and liver-type carnitine palmitoyltransferase (Cpt-1β, Cpt-1α), and the transcriptional regulator PPARα.1) Chronic hypoxia reduced SCHAD activity without physical remodeling or mRNA changes; 2) chronic cold lead to reduced SCHAD activity in hypertrophied left ventricles and lowered right ventricular Cpt-1α mRNA (compared to chronic hypoxia); and 3) despite causing hypertrophy of both ventricles, chronic exposure to combined hypoxic–cold did not induce significant metabolic remodeling.In response to environmental challenges, cardiac muscles 1) show distinct physical and metabolic remodeling, 2) respond to two stressors simultaneously but not additively, and 3) maintain an adult metabolic phenotype with long-term exposure to environmentally realistic hypoxic–cold.
Keywords: Cardiac hypertrophy; Metabolic remodeling; Peroxisome proliferator-activated receptor α; Medium-chain acyl-CoA dehydrogenase; β-Hydroxyacyl-CoA dehydrogenase; Carnitine palmitoyltransferase I;

Whole blood, flow-chamber studies in real-time indicate a biphasic role for thymosin β-4 in platelet adhesion by Harmanpreet Kaur; Rebecca Heeney; Rupp Carriveau; Gabriel Sosne; Bulent Mutus (1256-1261).
Thymosin beta 4 (Tβ4) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing.In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins–fibrinogen and collagen–were immobilized onto the middle ~ 25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tβ4, on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers.The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026 ± 0.0015 s− 1, corresponding to a half-life of 26.7 s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tβ4 (0.2 μM to 0.5 μM) increased both the rate constant of platelet deposition by ~ 1.5-fold (i.e. half-life decreased from 26.7 s to 17.6 s) and the total number of deposited platelets by ~ 3-fold. However at higher concentrations (> 1 μM) the Tβ4-potentiating effect was diminished to near control levels. Tβ4 did interact with fibrinogen with an estimated K D of ~ 126 ± 18 nM or 66 ± 20 nM under equilibrium or flow, respectively.These results suggest that Tβ4 could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tβ4 could also bind to fibrinogen and as its concentration increased would prevent platelet–fibrinogen interactions resulting in the attenuation of platelet deposition.This work suggests that Tβ4 might have a dual role in platelet function.
Keywords: Platelet deposition; Thymosin β-4; Flow-chamber studies; Collagen; Fibrinogen; ADP; Ca2+; Kinetics;

A previous urine proteomic analysis from our laboratory suggested that hepcidin may be a biomarker for lupus nephritis flare. Immunohistochemical staining of kidney biopsies from lupus patients showed that hepcidin was expressed by infiltrating renal leukocytes. Here we investigated whether inflammatory cytokines relevant to the pathogenesis of lupus nephritis and other glomerular diseases regulate hepcidin expression by human monocytes.Human CD14+ monocytes were incubated with interferon alpha (IFNα), interferon gamma (IFNγ), interleukin-6 (IL6), interleukin-1 beta (IL1β), monocyte chemotactic factor-1 (MCP1), or tumor necrosis factor alpha (TNFα). Hepcidin expression was examined by real-time PCR and enzyme immunoassay.Monocyte hepcidin mRNA increased during adherence to the tissue culture wells, reaching a level 150-fold higher than baseline within 12 h of plating. After accounting for the effects of adhesion, monocytes showed time and dose-dependent up-regulation of hepcidin mRNA upon treatment with IFNα or IL6. One hour of incubation with IFNα or IL6 increased hepcidin mRNA 20 and 80-fold, respectively; by 24 h the mRNA remained 5- and 2.4-fold higher than baseline. IL1β, IFNγ, and MCP-1 did not affect monocyte hepcidin expression. TNFα inhibited hepcidin induction by IL6 in monocytes by 44%. After 24 h of treatment with IFNα or IL6, immunoreactive hepcidin production by monocytes increased 3- and 2.6-fold, respectively.Human monocytes produce hepcidin in response to adhesion and the pro-inflammatory cytokines IFNα and IL6.The appearance of hepcidin in the kidneys or urine during glomerular diseases may be from infiltrating monocytes induced to express hepcidin by adherence and exposure to pro-inflammatory cytokines found in the renal milieu.
Keywords: Hepcidin; Interferon alpha; Human monocytes; Nephritis;

Rhizoctonia bataticola lectin (RBL) induces mitogenesis and cytokine production in human PBMC via p38 MAPK and STAT-5 signaling pathways by Radha Pujari; Nagaraja N. Nagre; Vishwanath B. Chachadi; Shashikala R. Inamdar; Bale M. Swamy; Padma Shastry (1268-1275).
Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus Rhizoctonia bataticola is highly mitogenic towards human peripheral blood mononuclear cells (PBMC). The lectin has sugar specificity towards N-glycans and binds to glycoproteins containing complex N-glycans (Nagre et al., Glycoconj J. 2010). In this study, we investigated the role of Mitogen Activated Protein Kinase (MAPK) and Signal Transducers and Activators of Transcription (STAT)-5 signaling in RBL-induced proliferation and production of Th1/Th2 cytokines.Human PBMC were stimulated with RBL and proliferation was determined by tritiated thymidine incorporation assay, cytokine profiles by ELISA and activation of MAPK and STAT-5 by western blotting. RBL binding was monitored by immunofluorescence staining. Expression of IL-2Rα (CD25) was measured by flow cytometry.The binding and mitogenic activities of RBL were inhibited by glycoproteins– mucin, asialofetuin and fetuin. RBL stimulated expression of IL-2Rα and production of Th1/Th2 cytokines– IL-2, IFN-γ, IL-4 and IL-10. RBL-induced phosphorylation of ERK1/2 and p38 MAPK was detected at 1 h and 3 h respectively. Significant phosphorylation of STAT-5 (tyr694) was observed at 12 h. Pharmacological inhibitors of p38 MAPK (SB203580) and JAK/STAT (AG490) but not ERK (PD98059) abrogated proliferation. RBL-induced expression of IL-2Rα and secretion of cytokines were drastically inhibited by SB203580 and AG490.RBL-induced proliferation and production of Th1/Th2 cytokines are mediated via p38 MAPK and STAT-5 signaling.RBL, a lectin with complex sugar specificity, is strongly mitogenic to human PBMC and stimulates the production of Th1 and Th2 cytokines. The results identified the signaling mechanism underlying the immunostimulatory activity of RBL.
Keywords: PBMC; Proliferation; Signaling; MAPK; STAT-5; Cytokine;

Binding and intracellular routing of the plant-toxic lectins, lanceolin and stenodactylin by Maria Giulia Battelli; Vittoria Scicchitano; Letizia Polito; Valentina Farini; Luigi Barbieri; Andrea Bolognesi (1276-1282).
The present research studied the interaction of two ribosome-inactivating proteins (RIPs) from Adenia genus with HeLa cells. Namely, lanceolin and stenodactylin were examined in comparison to volkensin, another toxic two-chain RIP from Adenia genus.The binding, endocytosis, intracellular routing, degradation and exocytosis were investigated by measuring the distribution of radiolabelled RIP and by determining its cytotoxicity.Stenodactylin was the most toxic, resulting in the greater inhibition of protein synthesis and cell death. Lanceolin and stenodactylin bound to cells with comparable affinity and have a similar number of binding sites (105/cell). The uptake of lanceolin and stenodactylin was 13 and 36 times greater, respectively, than that reported for volkensin. The two toxins bound to cell membrane receptors via their lectin B chain, were endocytosed through a clathrin-independent pathway, were internalised in a manner independent from endosomal acidification, and required routing through the Golgi apparatus, as reported for modeccin and volkensin. Stenodactylin showed greater uptake, exocytosis and re-uptake of non-degraded RIP than lanceolin and volkensin, whereas volkensin had the highest residual activity after being released from the cell.The high cytotoxicity of RIPs from the Adenia genus may depend on the following: high affinity binding to the cell and efficient endocytosis, intracellular routing that appears similar to that of other ricin-like toxic RIPs, partial resistance to proteolysis, and, regarding stenodactylin, high accumulation in cell.The data provide a model that could lead to new strategies for anti-cancer therapy and neuroscience studies.
Keywords: Adenia; HeLa cells; Lanceolin; Ribosome-inactivating proteins; Stenodactylin; Volkensin;