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BBA - General Subjects (v.1810, #12)

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Endogenous Bmp4 in myoblasts is required for myotube formation in C2C12 cells by Takenao Umemoto; Yuuma Furutani; Masaru Murakami; Tohru Matsui; Masayuki Funaba (pp. 1127-1135).

Our previous study revealed the indispensable activity of endogenous bone morphogenetic protein (Bmp) prior to differentiation induction of C2C12 myoblasts for myogenesis. Here we investigated the Bmp isoform responsible for endogenous Bmp activity during differentiation and its role in myogenesis.Gene expression of Bmp4 during myogenesis was evaluated in C2C12 cells. Effects of inhibition of the Bmp pathway on myogenesis were examined. Cells expressing Bmp4 and regulation of Bmp4 expression in myoblasts were explored.The expression of Bmp4 increased with the progression of myogenesis, although the extent of the increase after differentiation induction was smaller than that before the induction. Down-regulation of Bmp signal components including Bmp4, Bmpr2, and Alk2/3 inhibited the emergence of positive cells for myosin heavy chain II. The treatments also decreased the Myogenin expression. Treatment with cytosine arabinoside decreased the expression of Bmp4. Also, Bmp4 expression was also lower in isolated myotubes than in residual cells. Expression of Rgm c was higher in the myotube fraction. Transcription of Bmp4 was repressed by the conditioned medium of mixed cells consisting of myoblasts and myotubes.Bmp4 expressed in myoblasts has a positive role in myotube formation/maturation through myogenin expression. The presence of myotubes inhibits Bmp4 expression in proliferating myoblasts through transcriptional regulation, although the expression is intrinsically increased with time of culture.Taken previous results on involvement of Bmp in the commitment of osteoblasts and adipocytes with the present results together, Bmp may act as a general promoter of mesenchymal cell differentiation.► Expression of Bmp4 is increased with progression of myogenesis. ► Proliferating myoblasts express Bmp4. ► Rgm c, a co-receptor to enhance Bmp signaling, is predominantly expressed in myotubes. ► Down-regulation of Bmp signal components inhibits myotube differentiation/maturation. ► Transcription of Bmp4 is repressed by the presence of myotubes.

Keywords: Bmp4; Myotube; Myogenesis; Myogenin

Spectrofluorimetric analysis of the interaction of amyloid peptides with neuronal nitric oxide synthase: Implications in Alzheimer's disease by Eden R. Padayachee; Chris G. Whiteley (pp. 1136-1140).

The deposition of aggregated β-amyloid peptide senile plaques and the accumulation of arginine within the astrocytes in the brain of an Alzheimer's patient are classic observations in the neuropathology of the disease. It would be logical, in the aetiology and pathogenesis, to investigate arginine-metabolising enzymes and their intimate association with amyloid peptides.Neuronal nitric oxide synthase (nNOS) was isolated, purified and shown, through fluorescence quenching spectroscopy and fluorescence resonance energy transfer (FRET), to interact with structural fragments of Aβ_1–40 and be catalytic towards amyloid fibril formation.Only one binding site on the enzyme was available for binding. Two amyloid peptide fragments of Aβ_1–40 (Aβ_17–28 and Aβ_25–35) had Stern–Volmer values (K_SV) of 0.111μM⁻¹ and 0.135μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. The polarity of this active site precludes binding of the predominantly hydrophobic amyloid peptide fragments contained within Aβ_17–28 and within two glycine zipper motifs [G-X-X-X-G-X-X-X-G] [Aβ_29–37] and bind to the enzyme at a site remote to the active region.The interaction and binding of Aβ_17–28 and Aβ_25–35 to nNOS causes the movement of two critical tryptophan residues of 0.77nm and 0.57nm respectively towards the surface of the enzyme.The binding of Aβ-peptide fragments with nNOS has been studied by spectrofluorimetry. The information and data presented should contribute towards understanding the mechanism for deposition of aggregated Aβ-peptides and fibrillogenesis in senile plaques in an AD brain.► Interaction of Aβ_17–28, Aβ_25–25, Aβ_32–35 with nitric oxide synthase by fluorescence. ► FRET showed 2 Trp residues moved 0.77 and 0.57nm as Aβ_17–28, Aβ_25–25 became bound. ► Hydrophobic amyloid peptides bind at a site remote to the active region.

Keywords: Neuronal nitric oxide synthase; Alzheimer's disease; Amyloid peptide; Fluorescence resonance energy transfer; Fluorescence quenching

Down-regulation of FUT3 and FUT5 by shRNA alters Lewis antigens expression and reduces the adhesion capacities of gastric cancer cells by Padro Mercè Padró; Lara Cobler; Marta Garrido; de Bolos Carme de Bolós (pp. 1141-1149).

Lewis antigens are fucosylated glycoconjugates involved in the development of several pathologies. The adhesion of sialyl-Lewis antigens to E-selectin is a key step in the development of metastasis and the glycosidic component of CD44 plays a key role in the binding to hyaluronic acid, a component of the extracellular matrix associated to tumor development and invasion. Fucosyltransferases are enzymes that add fucose to precursor glycan structures: FUT3 and FUT5 catalyze the addition of fucose to the α1-3,4 position and are detected in epithelial cells. In this study, we have analyzed the effects of silencing FUT3, FUT5 or FUT3/FUT5, in two gastric cancer cell lines, in the expression of Lewis antigens and in the adhesive and migratory capacities of the cells.FUT3, FUT5 and FUT3/FUT5 were down-regulated using lentiviral delivery of shRNAs in MKN45 and GP220 gastric cancer cells.In the infected cells, decreased levels of FUT3 and FUT5 mRNA detected by quantitative RT-PCR; and lower levels of sialyl-Lewis antigens, evaluated by flow cytometry, were observed. The adhesion to endothelial cells trough the binding to E-selectin, and the binding to hyaluronic acid were reduced in the shFUT3, shFUT5 and shFUT3/FUT5, whereas the levels of CD44, analyzed by western blot, did not change.The down-regulation of FUT3, FUT5 and FUT3/FUT5 reduces the expression of sialyl-Lewis antigens and the adhesion and binding capacities of gastric cancer cells; and allows to identify the specific α1-3,4 fucosyltransferases implicated in the Lewis antigens synthesis in this cellular model.►FUT3 and FUT5 silencing using shRNAs reduces the expression of sialyl-Lewis antigens. ►Decrease of sialyl-Lewis x levels reduces the binding to E-selectin and hyaluronic. ►Also diminishes the migratory abilities of the gastric cancer cells. ►FUT3 and FUT5 overlap activities but FUT3 acts preferably on type 1 and FUT5 on type 2.

Keywords: Fucosyltransferase 3; Fucosyltransferase 5; Lewis antigen; shRNA; Gastric cancer cell; Cell binding

Molecular cloning and functional analysis of scavenger receptor zebrafish CL-P1 by Mitsuko Fukuda; Katsuki Ohtani; Seong-Jae Jang; Takayuki Yoshizaki; Ken-ichiro Mori; Wataru Motomura; Itsuro Yoshida; Yasuhiko Suzuki; Yutaka Kohgo; Nobutaka Wakamiya (pp. 1150-1159).

Scavenger receptors are generally expressed in macrophages and vascular endothelial cells and some scavenger receptors are thought to contribute to the development of atherosclerosis.We cloned the cDNA of a zebrafish CL-P1 (collectin placenta 1) and performed a knockdown study using its antisense morpholino oligonucleotides (MO).Zebrafish CL-P1 (zCL-P1) is 51% identical to human CL-P1 in its amino acid sequence. Microbes and OxLDL bound to zCL-P1 cDNA transfected cells. zCL-P1 mRNA expression gradually increased after 6hours post-fertilization (hpf), reached its highest level at 24hpf, and then decreased, which is similar to the gene expression pattern of Tie-2. The knockdown of zCL-P1 led to an increase in the number of zebrafish embryos with severe morphological abnormalities such as short body lengths and defects in the dorsal aorta at 48hpf. Simultaneous injection of both MO and synthetic zCL-P1 or zVEGF mRNA rescued the abnormal phenotype. In vivo knockdown study shows that zCL-P1 is implicated in vasculogenesis and those of our in vitro study support its role as a scavenger receptor.These results suggest that zCL-P1 might be essential for vasculogenesis during the early embryonic phase in bone fish.► We cloned zebrafish CL-P1 gene. ► Zebrafish CL-P1 is 51% identical to human CL-P1 in its amino acid sequence. ► CL-P1 knockdown induced vascular and developmental defects in the zebrafish. ► The scavenger receptor and collectin was involved in embryonic development in fish.

Keywords: Collectin; Scavenger receptor; Innate immunity; Zebrafish; Embryogenesis

MAPK phosphatase-1 contributes to trichostatin A inhibition of cyclooxygenase-2 expression in human umbilical vascular endothelial cells exposed to lipopolysaccharide by Ya-Fen Hsu; Joen-Rong Sheu; Chien-Huang Lin; Wei-Chuan Chen; George Hsiao; George Ou; Pei-Ting Chiu; Ming-Jen Hsu (pp. 1160-1169).

Histone deacetylase (HDAC) inhibitors have emerged as a new class of antitumor agents because they were demonstrated to induce cell cycle arrest, promote cell apoptosis, and inhibit metastasis. Recently, HDAC inhibitors were also shown to exhibit pronounced anti-inflammatory properties. However, the underlying mechanism contributing to the suppression of inflammatory responses by HDAC inhibitors remains to be fully defined. In the present study, we explored the actions of trichostatin A (TSA), a potent HDAC inhibitor, on lipopolysaccharide (LPS)-induced cyclooxygenase (COX)-2 expression in human umbilical vascular endothelial cells (HUVECs).HUVECs were exposed to LPS in the absence or presence of TSA. COX-2 expression and signaling molecules (JNK, p38MAPK and c-jun) activated by LPS were assessed.The LPS-induced cox-2 messenger RNA and protein were markedly suppressed by TSA. TSA inhibited JNK and p38MAPK phosphorylation in cells exposed to LPS. Treatment of cells with a JNK signaling inhibitor (JNK inhibitor II) or a p38MAPK inhibitor (p38MAPK inhibitor III) markedly inhibited LPS-induced COX-2 expression. TSA suppression of JNK and p38MAPK phosphorylation and subsequent COX-2 expression were restored by selective inhibition of MKP-1 using MKP-1 siRNA. In addition, TSA caused an increase in MKP-1 phosphatase activity in HUVECs. In conclusion, TSA may cause MKP-1 activation to dephosphorylate JNK and p38MAPK, leading to the downregulation of COX-2 in HUVECs stimulated by LPS, a proinflammatory stimulus.MKP-1 contributes to TSA's protective actions in HUVECs exposed to LPS. The present study also supports the therapeutic value of TSA in treating inflammatory vascular diseases.Display Omitted► Trichostatin A attenuated LPS-induced COX-2 expression and PGE₂ release in HUVEC. ► MKP-1 contributes to trichostatin A-induced JNK and p38MAPK dephosphorylation. ► MKP-1 contributes to TSA's protective actions in HUVECs exposed to LPS.

Keywords: MKP-1; Trichostatin A; Human umbilical vascular endothelial cell; Cyclooxygenase-2; Lipopolysaccharide

EGF and angiotensin II modulate lysophosphatidic acid LPA₁ receptor function and phosphorylation state by Colin-Santana Christian C. Colín-Santana; Avendano-Vazquez S. Eréndira Avendaño-Vázquez; Alcantara-Hernandez Rocío Alcántara-Hernández; Garcia-Sainz J. Adolfo García-Sáinz (pp. 1170-1177).

Lysophosphatidic acid (LPA) is a local mediator that exerts its actions through G protein coupled receptors. Knowledge on the regulation of such receptors is scarce to date. Here we show that bidirectional cross-talk exits between LPA₁ and EGF receptors.C9 cells expressing LPA₁ receptor fussed to the enhanced green fluorescent protein were used. We studied intracellular calcium concentration, Akt/PKB phosphorylation, LPA₁ and EGF receptor phosphorylation.EGF diminished LPA-mediated intracellular calcium response and induced LPA₁ receptor phosphorylation, which was sensitive to protein kinase C inhibitors. Angiotensin II and LPA induced EGF receptor transactivation as evidenced by Akt/PKB phosphorylation through metalloproteinase-catalyzed membrane shedding of heparin-binding EGF and autocrine/paracrine activation of EGF receptors. This process was found to be of major importance in angiotensin II-induced LPA₁ receptor phosphorylation. Attempts to define a role for EGF receptor transactivation in homologous LPA₁ receptor desensitization and phosphorylation suggested that G protein-coupled receptor kinases are the major players in this process, overshadowing other events.EGF receptors and LPA₁ receptors are engaged in an intense liaison, in that EGF receptors are capable of modulating LPA₁ receptor function through phosphorylation cascades. EGF transactivation plays a dual role: it mediates some LPA actions, and it modulates LPA₁ receptor function in inhibitory fashion.EGF and LPA receptors coexist in many cell types and play key roles in maintaining the delicate equilibrium that we call health and in the pathogenesis of many diseases. The intense cross-talk described here has important physiological and pathophysiological implications.Model for the role(s) of EGF receptors in the actions of LPA and angiotensin IIDisplay Omitted► Regulation of lysophosphatidic acid receptors is poorly known. ► Here we show that intense crosstalk exits between LPA1 and EGF receptors. ► EGF induced LPA1 receptor desensitization and phosphorylation. ► Angiotensin II and LPA induced EGF receptor transactivation. ► Angiotensin II-induced LPA1 phosphorylation involved EGF receptor transactivation.

Keywords: Lysophospatidic acid receptor; Epidermal growth factor receptor; Transactivation; Desensitization; Phosphorylation

Interaction of the retinoic acid signaling pathway with spicule formation in the marine sponge Suberites domuncula through activation of bone morphogenetic protein-1 by Muller Werner E.G. Müller; Michael Binder; Johannes von Lintig; Yue-Wei Guo; Xiaohong Wang; Jaap A. Kaandorp; Matthias Wiens; Schroder Heinz C. Schröder (pp. 1178-1194).

The formation of the spicules in siliceous sponges involves the formation of cylinder-like structures in the extraspicular space, composed of the enzyme silicatein and the calcium-dependent lectin.Molecular cloning of the cDNAs ( carotene dioxygenase, retinal dehydrogenase, and BMB-1 [bone morphogenic protein-1]) from the demosponge Suberites domuncula was performed. These tools were used to understand the retinoid metabolism in the animal by qRT-PCR, immunoblotting and TEM.We demonstrate that silintaphin-2, a silicatein-interacting protein, is processed from a longer-sized 15-kDa precursor to a truncated, shorter-sized 13kDa calcium-binding protein via proteolytic cleavage at the dipeptide Ala↓Asp, mediated by BMP-1. The expression of this protease as well as the expression of two key enzymes of the carotinoid metabolism, the β,β-carotene-15,15′-dioxygenase and the retinal dehydrogenase/reductase, were found to be strongly up-regulated by retinoic acid. Hence retinoic acid turned out to be a key factor in skeletogenesis in the most ancient still existing metazoans, the sponges.It is shown that retinoic acid regulates the formation of the organic cylinder that surrounds the axis of the spicules and enables, as a scaffold, the radial apposition of new silica layers and hence the growth of the spicules.► Sponges as a model system for molecular skeleton formation. ► Sponges as a model system for studies on the morphogens retinol/retinoic acid. ► Shaping the morphology of siliceous spicules. ► Key enzymes involved in the metabolism of retinol in sponges have been identified. ► Data indicate that retinoids regulate the appositional growth of the siliceous spicules.

Keywords: Retinoic acid; BMP-1; β-Carotene dioxygenase; Retinal dehydrogenase; Silicatein; Silintaphin-2

Enzymatic synthesis of mono and dinucleoside polyphosphates by Hugo Fraga; Rui Fontes (pp. 1195-1204).

Mono and dinucleoside polyphosphates (p_nNs and Np_nNs) exist in living organisms and induce diverse biological effects through interaction with intracellular and cytoplasmic membrane proteins. The source of these compounds is associated with secondary activities of a diverse group of enzymes.Here we discuss the mechanisms that can promote their synthesis at a molecular level. Although all the enzymes described in this review are able to catalyse the in vitro synthesis of Np_nNs (and/or p_nN), it is not clear which ones are responsible for their in vivo accumulation.Despite the large amount of knowledge already available, important questions remain to be answered and a more complete understanding of p_nNs and Np_nNs synthesis mechanisms is required. With the possible exception of (GTP:GTP guanylyltransferase of Artemia), all enzymes able to catalyse the synthesis of p_nNs and Np_nNs are unspecific and the factors that can promote their synthesis relative to the canonical enzyme activities are unclear.The fact that p_nNs and Np_nNs syntheses are promiscuous activities of housekeeping enzymes does not reduce its physiological or pathological importance. Here we resume the current knowledge regarding their enzymatic synthesis and point the open questions on the field.► The synthesis of mono and dinucleoside polyphosphates (p_nNs and Np_nNs) was reviewed. ► So far only GTP:GTP guanylyltransferase catalyses Np_nNs synthesis in a specific way. ► In all the other enzymes (N)p_nNs synthesis results from promiscuous activities. ► The significance of these promiscuous activities is discussed.

Keywords: Abbreviations; AARS; Aminoacyl-tRNA synthetase; (N)p; _n; Ns; mono- and dinucleoside polyphosphates; (; R; p)Ap; ₄; A[αS]; (; R; p)-adenosine(5′)[α-thio]tetraphospho(5′)adenosine; (; S; p)Ap; ₄; A[αS]; (; S; p)-adenosine(5′)[α-thio]tetraphospho(5′)adenosine; AaaRS; a specific aminoacyl-tRNA synthetase where Aaa is the three letter code of the corresponding amino acid (as in LysRS); Ap; ₂; C(OH)(CH; ₃; )p; etidronate derivative of adenosine 5′triphosphate; Ap; ₂; CCl; ₂; p; adenosine 5′-(β,γ-dichloromethylenetriphosphate); Ap; ₂; CH; ₂; p; adenosine 5′-(β,γ-methylenetriphosphate); Ap; ₂; CH; ₂; p; ₂; A; adenosine(5′)[β,γ-methylene]tetraphospho(5′)adenosine; Ap; ₂; CHClp; ₂; A; adenosine(5′)[β,γ-monochloromethylene]tetraphospho(5′)adenosine; Ap; ₂; Cp; 5′-adenylylated cytidine 5′,3′-biphophonate; Ap; ₂; NHp; ₂; A; adenosine(5′)[β,γ-imido]tetraphospho(5′)adenosine; Ap; ₃; CH; ₂; pA; adenosine(5′)[γ,δ-methylene]tetraphospho(5′)adenosine; Ap; ₃; thia; 5′-adenylyl-triphosphothiamine; Ap; ₄; A; adenosine(5′)tetraphospho(5′)adenosine; Ap; ₄; A[αS]; adenosine(5′)[α-thio]tetraphospho(5′)adenosine; Ap; ₄; A[βS]; adenosine(5′)[β-thio]tetraphospho(5′)adenosine; ApCH; ₂; p; ₃; A; adenosine(5′)[α,β-methylene]tetraphospho(5′)adenosine; arabino-A; adeninearabinofuranoside; AZT; azido-3′-deoxythymidine; d(5-fluoro-U); 5-fluro-2′-deoxyuridine; dim; dimethylallyl; E1; Ubiquitin activating enzyme; far; farnesyl; FHIT; Fragile Histidine Triad protein; ger; geranyl; Gp; ₅; G; guanosine(5′)pentaphospho(5′)guanosine; JAR1; Jasmonate:aminoacid synthetase; Luc; Photinus pyralis; firefly luciferase; LysRS; Lysyl-tRNA synthetase; MITF; Microphthalmia transcription factor; MURD; UDP-MurNAc-L-alanine:D-glutamate ligase; N1p; _n; N2; a specific dinucleoside polyphosphate where N1 and N2 represent the nucleosides and n is the number of phosphates (as in Ap; ₄; A or Gp; ₅; G); Np; _n; N; dinucleoside polyphosphates; NRPS; Non ribosomal peptide synthetase; p; ₄; A; adenosine tetraphosphate; PGK; 3-phosphoglycerate kinase; P; _n; polyphosphate where n is the number of phosphates; p; _n; A; adenosine polyphosphate; p; _n; N; mononucleoside polyphosphate; p; _n; N1; a specific mononucleoside polyphosphate where N1 represent the nucleoside and n is the number of phosphates (as in p; ₄; A); PPi; pyrophosphate; εA; N; ⁶; -ethenoadenosineMononucleoside polyphosphate; Dinucleoside polyphosphate; Enzyme synthesis; Promiscuous activity; Ap; ₄; A; p; ₄; A

Phosphatidylcholine hydroperoxide promotes VEGF-induced angiogenesis in endothelial cells and rat aorta ring cultures by Kiyotaka Nakagawa; Akira Shibata; Tatsuya Saito; Phumon Sookwong; Shunji Kato; Tsuyoshi Tsuduki; Kiminori Matsubara; Teruo Miyazawa (pp. 1205-1211).

Phosphatidylcholine hydroperoxide (PCOOH) is a primary oxidation product of PC, and is markedly accumulated in blood plasma and arterial walls in atherosclerotic animals and humans. The role of PCOOH in the induction of angiogenesis is unknown.In this study, we investigated whether PCOOH stimulated angiogenic responses (e.g., vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, and tube formation, and angiogenesis-related gene/protein expression) in human umbilical vein endothelial cells (HUVEC) and in an ex vivo rat aorta model.VEGF induced proliferation, migration, and tube formation of HUVEC, and these angiogenic responses were all enhanced by PCOOH but not by native (nonoxidized) PC. The angiogenic effects of PCOOH are considered to be mediated via generation of reactive oxygen species and activation of both PI3K/AKT and MAPK pathways. The angiogenic activities of PCOOH were also confirmed by the rat aortic ring assay.These results indicate that PCOOH can elicit several angiogenic responses.The present study implies an important role of PCOOH in atherosclerosis progression and plaque instability.► Angiogenic responses were enhanced by PCOOH but not by native (nonoxidized) PC. ► ROS, PI3K/AKT, and MAPK were important for the PCOOH-driven angiogenesis. ► The angiogenic activities of PCOOH were also confirmed by the rat aortic ring assay.

Keywords: Angiogenesis; Atherosclerosis; Lipid peroxidation; Phosphatidylcholine hydroperoxide

Effect of bitter compounds on amylase secretion in murine submandibular glands: Signaling pathway mechanisms by Maximiliano Dasso; Romina Pagotto; Omar P. Pignataro; Roberto A. Diez; María E. Sales (pp. 1212-1219).

Amylase is synthesized in submandibular glands (SMG) and released into the oral cavity to degrade carbohydrates in the mouth. Bitter taste receptors (T2R) belong to the G-protein coupled receptor (GPCR) family and are expressed in the taste cells and also in the digestive tract.The activity of amylase secreted by murine SMG was measured, detecting maltose by Bernfeld's method. Amylase and T2R6 were detected by imunohistochemistry and Western blot. The expression of Ggustducin, Gi, and phospholipase Cβ2 was also studied by Western blot. cAMP levels were measured by radioimmunoassay and inositol monophosphate production was quantified by ELISA.Theophylline, denatonium and cycloheximide exerted a dose-dependent inhibition on amylase secretion. This effect was reverted by preincubating SMG with an anti-Gαi antibody. cAMP production was increased by the same compounds, an effect that was also abrogated by an anti-Gαi antibody. Bitter compounds reduced inositol monophosphate formation in SMG and H-89, a protein kinase A inhibitor, reverted this action, revealing that this protein kinase down regulates phospholipase C activity.We demonstrated that theophylline, denatonium and cycloheximide inhibit salivary amylase secretion, activating an intracellular signaling pathway that involves cAMP and phospholipase C, that cross talks via protein kinase A.► Bitter agonists inhibit amylase secretion in murine submandibular glands. ► This effect can occur via T2R activation and Gi protein coupling. ► Gαi inhibits fosfodiesterase activity up-regulating cAMP levels. ► This nucleotide activates protein kinase A that down-regulates phospholipase Cβ2 activity. ► The inhibition of phospholipase Cβ2 prevents inositol monophosphate production.

Keywords: Abbreviations; CV; circumvallate papillae; GCT; granular convoluted tubule; GPCR; G protein coupled receptors; IP; ₁; inositol monophosphate; IP; ₃; inositol triphosphate; PDE; phosphodiesterase; PKA; protein kinase A; PLCβ2; phospholipase Cβ2; SMG; submandibular glands; T2R; bitter taste receptorsAmylase; Bitter agonist; Gi protein; cAMP; Inositol monophosphate; Protein kinase A

Up-regulation of adenylylcyclases I and II induced by long-term adaptation of rats to morphine fades away 20days after morphine withdrawal by Hana Ujcikova; Katerina Dlouha; Lenka Roubalova; Miroslava Vosahlikova; Dmytro Kagan; Petr Svoboda (pp. 1220-1229).

Activation of adenylyl cyclase (AC) by prolonged exposure of mammalian organism to morphine was demonstrated in previous studies of mechanism of action of this drug. However, expression level of individual AC isoforms was not analyzed in crucial cell structure, plasma membrane (PM).Rats were adapted to morphine for 10days and sacrificed 24h (group+M10) or 20days (+M10/−M20) after the last dose. Control animals were sacrificed in parallel with morphine-treated (groups−M10 and (−M10/−M20)). Percoll®-purified PM were isolated from brain cortex and analyzed by immunoblotting and specific radioligand binding.ACI (ACII) was increased 8× (2.5×) in morphine-adapted rats (+M10) when compared with controls (−M10). Increase of ACI and II by long-term adaptation to increasing doses of morphine represented a specific effect as the amount of ACIII–ACIX, of prototypical PM marker, Na, K-ATPase and of trimeric G protein α and β subunits was unchanged. Increase of ACI and II was not detected in PM isolated from group (+M10/−M20). Thus, the marked increase of ACI and ACII faded away 20days since the last dose of morphine.We assume that the specific increase in expression level of ACI and ACII in brain cortex of morphine-adapted rats proceeds as a compensatory, homeostatic response to prolonged exposure to inhibitory drug, morphine.Our findings demonstrate that the dramatic and specific change of the crucial component of the opioid receptor cascade in brain cortex, manifested as an increase in PM level of ACI and II, is reversible.► Long-term adaptation of rat brain cortex to morphine (10 days). ► Increased amount of ACI (8x) and II (2.5x) in plasma-membrane fraction (PM). ► Unchanged amount of prototypical PM marker Na, K-ATPase. ► 20 days since the last dose density of ACI and II in PM returns to the control level.

Keywords: Abbreviations; AC; adenylyl cyclase; β-AR; β-adrenergic receptor; DADLE; [2-; d; -alanine, 5-; d; -leucine]enkephalin; =; Tyr-; d; -Ala-Gly-Phe-; d; -Leu; DAMGO; [2-; d; -alanine, 4-N-methylphenylalanine, 5-glycinol]enkephalin; =; Tyr-; d; -Ala-Gly-N-methyl-Phe-Gly-ol; DOR; δ-opioid receptor; GPCR; G protein-coupled receptor; G proteins; heterotrimeric guanine nucleotide-binding regulatory proteins; G; _s; α; G protein α subunit stimulating adenylyl cyclase activity; G; _i; /G; _o; α; G protein α subunits inhibiting adenylyl cyclase activity in pertussis-toxin sensitive manner; G; _q; /G; ₁₁; α; G protein α subunits stimulating phoshoplipase C in pertussis-toxin independent manner; [; ³⁵; S]GTPγS; guanosine-5′-[γ-; ³⁵; S] triphosphate; KOR; κ-opioid receptor; PM; plasma (cell) membranes; MOR; μ-opioid receptor; Na,K-ATPase; sodium- plus potassium-activated, ouabain-dependent adenosine triphosphatase (EC 3.6.1.3); P; _i; inorganic phosphate; OR; opioid receptor; PBS; phosphate-buffered saline; PM; plasma membrane; PMSF; phenylmethylsulfonyl fluoride; PTX; pertussis toxin; SLB; sample loading buffer; TBS; Tris-buffered saline; w.w.; wet weightMorphine; Long-term adaptation; Adenylyl cyclase isoforms I–IX; Forebrain cortex; Isolated plasma membranes

Synergistic inhibition of butyrylcholinesterase by galantamine and citalopram by Ryan Walsh; Kenneth Rockwood; Earl Martin; Sultan Darvesh (pp. 1230-1235).

Many persons with Alzheimer's disease (AD) treated with galantamine appear to receive additional cognitive benefit from citalopram. Both drugs inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These enzymes co-regulate acetylcholine catabolism. In AD brain, AChE is diminished while BuChE is not, suggesting BuChE inhibition may be important in raising acetylcholine levels. BuChE is subject to activation at high acetylcholine levels reached at the synaptic cleft. The present study explores one way combining galantamine and citalopram could be beneficial in AD.Spectrophotometric studies of BuChE catalysis in the absence or presence of galantamine or citalopram or both, were performed using the Ellman method. Data analysis involved expansion of our previous equation describing BuChE catalysis.Galantamine almost completely inhibited BuChE at low substrate concentrations ( V_S=43.6μM/min; V_S(gal)=0.34 μM/min) without influencing the substrate-activated form of the enzyme ( V_SS=64.0 μM/min; V_SS(gal)=62.3μM/min). Conversely, citalopram inhibited both un-activated ( V_S=43.6μM/min; V_S(cit)=10.2μM/min) and substrate-activated ( V_SS=64.0μM/min; V_SS(cit)=47.3μM/min) forms of BuChE. Combined galantamine and citalopram increased inhibition of un-activated BuChE ( V_S=43.6μM/min; V_S(gal)(cit)=2.73μM/min) and substrate-activated form ( V_SS=64.0μM/min; V_SS(gal)(cit)=42.2μM/min).Citalopram and galantamine produce a combined inhibition of BuChE that is considered to be synergistic.Clinical benefit from combined galantamine and citalopram may be related to a synergistic inhibition of BuChE, facilitating cholinergic neurotransmission. This emphasizes the importance of further study into use of drug combinations in AD treatment.Display Omitted► Alzheimer patients who take galantamine have additional benefit from citalopram. ► Citalopram and galantamine acted synergistically to inhibit butyrylcholinesterase. ► The synergistic effect is on un-activated and substrate-activated enzyme complexes. ► This synergistic effect may explain the additive beneficial effect in AD patients.

Keywords: Alzheimer's disease; Cholinesterase inhibitor; Selective serotonin reuptake inhibitor; Enzyme kinetics; Dementia

Electromagnetic fields as first messenger in biological signaling: Application to calmodulin-dependent signaling in tissue repair by Arthur Pilla; Robert Fitzsimmons; David Muehsam; June Wu; Christine Rohde; Diana Casper (pp. 1236-1245).

The transduction mechanism for non-thermal electromagnetic field (EMF) bioeffects has not been fully elucidated. This study proposes that an EMF can act as a first messenger in the calmodulin-dependent signaling pathways that orchestrate the release of cytokines and growth factors in normal cellular responses to physical and/or chemical insults.Given knowledge of Ca²⁺ binding kinetics to calmodulin (CaM), an EMF signal having pulse duration or carrier period shorter than bound Ca²⁺ lifetime may be configured to accelerate binding, and be detectable above thermal noise. New EMF signals were configured to modulate calmodulin-dependent signaling and assessed for efficacy in cellular studies.Configured EMF signals modulated CaM-dependent enzyme kinetics, produced several-fold increases in key second messengers to include nitric oxide and cyclic guanosine monophosphate in chondrocyte and endothelial cultures and cyclic adenosine monophosphate in neuronal cultures. Calmodulin antagonists and downstream blockers annihilated these effects, providing strong support for the proposed mechanism.Knowledge of the kinetics of Ca²⁺ binding to CaM, or for any ion binding specific to any signaling cascade, allows the use of an electrochemical model by which the ability of any EMF signal to modulate CaM-dependent signaling can be assessed a priori or a posteriori. Results are consistent with the proposed mechanism, and strongly support the Ca/CaM/NO pathway as a primary EMF transduction pathway.The predictions of the proposed model open a host of significant possibilities for configuration of non-thermal EMF signals for clinical and wellness applications that can reach far beyond fracture repair and wound healing.► EMF can act as first messengers in calmodulin-dependent signaling pathways. ► EMF signals were configured to modulate calmodulin-dependent signaling in cells. ► EMF increased NO and cGMP, blocked by calmodulin antagonists. ► Results support Ca/CaM/NO signaling as a primary EMF transduction pathway. ► Model predictions open a host of clinical applications for non-thermal EMF signals.

Keywords: EMF Mechanism; Signaling; Electrochemical model; Calmodulin; Nitric oxide

The antimicrobial peptide arenicin-1 promotes generation of reactive oxygen species and induction of apoptosis by Jaeyong Cho; Dong Gun Lee (pp. 1246-1251).

Arenicin-1, a 21-residue antimicrobial peptide, is known to exert significant broad-spectrum antimicrobial activity without cytotoxicity in mammalian cells except at high concentration. However, the mechanism of fungal cell death by arenicin-1 is weakly understood.We confirmed an increase in reactive oxygen species (ROS) in Candida albicans exposed to arenicin-1 and investigated the apoptotic response to ROS accumulation using apoptosis detecting methods.Cells exposed to arenicin-1 showed an increase in the production of ROS and cytotoxic hydroxyl radicals, which are the major factors of apoptosis. The increase in ROS was due to mitochondrial dysfunction caused by arenicin-1. We confirmed that arenicin-1 induced mitochondrial membrane depolarization and also triggered release of activated metacaspases. Further, it initiated an apoptotic mechanism acting on the plasma membrane, including plasma membrane depolarization and exposure of phosphatidylserine on the outer surface. Cells finally died, showing morphological changes in the nucleus and DNA structure. Based on these apoptotic phenomena induced by arenicin-1, we concluded that arenicin-1 exerts antifungal activity by inducing apoptosis.This study suggests that the antimicrobial peptide arenicin-1 induces apoptosis in C. albicans via intracellular ROS accumulation and mitochondrial damage, resulting in fungal cell death.► Arenicin-1 increased ROS generation in C. albicans cells. ► Arenicin-1 may induce formation of hydroxyl radical. ► Arenicin-1 activates metacaspases in yeast. ► Arenicin-1 can induce apoptosis.

Keywords: Abbreviations; DHR-123; dihydrorhodamine-123; HPF; hydroxyphenyl fluorescein; DiOC; ₆; (3); 3,3′-dihexyloxacarbocyanine iodide; DiBAC; ₄; (3); bis-(1,3-dibutylbarbituric acid)trimethine oxonol; DAPI; 4′,6-diamidino-2-phenylindole; TUNEL; terminal deoxynucleotidyl transferase dUTP nick end labeling; ROS; reactive oxygen speciesArenicin-1; Apoptosis; Reactive oxygen species; Mitochondrial damage; Candida albicans

Interscapular brown adipose tissue metabolic reprogramming during cold acclimation: Interplay of HIF-1α and AMPKα by Milica Vucetic; Vesna Otasevic; Aleksandra Korac; Ana Stancic; Aleksandra Jankovic; Milica Markelic; Igor Golic; Ksenija Velickovic; Biljana Buzadzic; Bato Korac (pp. 1252-1261).

Brown adipose tissue thermogenic program includes complex molecular and structural changes. However, energetic aspects of this process are poorly depicted.^{We investigated time-dependent reprogramming of interscapular brown adipose tissue (IBAT) energy metabolism during cold-acclimation, as well as the effects of nitric oxide (}NO) on those changes. Rats were exposed to cold (4±1°C) for periods of 1, 3, 7, 12, 21, and 45days, and divided into three groups: control, treated with L-arginine, and treated with N^ω-nitro-L-arginine methyl ester (L-NAME).In the early phase of cold-acclimation (up to 7days), the protein levels of all metabolic parameters and oxidative phosphorylation components were below the control. However, metabolic parameters and respiratory chain components entered a new homeostatic level in the late phase of cold-acclimation. These changes were accompanied with increased protein levels of phospho-AMP-dependent protein kinase-α (phospho-AMPKα) on the first day of cold-acclimation, and hypoxia-inducible factor-1α (HIF-1α) throughout early cold-acclimation. L-arginine positively affected protein expression of enzymes involved in glucose metabolism and β-oxidation of fatty acids in the early phase of cold-acclimation, and oxidative phosphorylation components throughout cold-acclimation. In contrast, L-NAME had the opposite effects.^{Results suggest that IBAT structural remodeling is followed by energy metabolism reprogramming, which control might be orchestrated by the action of AMPKα and HIF-1α. Data also indicated the involvement of L-arginine-}NO in the regulation of IBAT metabolism.Results obtained in this study might be of great importance for elucidating regulatory pathways governing energy metabolism in both physiological and pathophysiological states.► Protein level of metabolic enzymes decreased during acute cold-exposure. ► Protein level of respiratory chain components decreased during 7-day cold-exposure. ► Levels of all abovementioned enzymes raised in the late phase of cold acclimation. ► Orchestrated protein expressions of AMPKα and HIF-1α during 45^{days of cold exposure. ►}NO stimulates expression of metabolic and enzymes of oxidative phosphorylation.

Keywords: Abbreviations; BAT; brown adipose tissue; IBAT; interscapular BAT; L-NAME; N; ^ω; -nitro-L-arginine methyl ester; phospho-AMPKα; phosphorylated AMP-activated protein kinase α; HIF-1α; hypoxia-inducible factor-1α; COX IV; subunit IV of cytochrome; c; oxidase; PDH; pyruvate dehydrogenase; SCAS; succinyl-CoA synthetase; ACADM; medium chain fatty acids acyl-CoA dehydrogenase; FAS; fatty acids synthase; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; LDH; lactate dehydrogenase; PGC-1α; peroxisome proliferation-activated receptor-γ coactovator-1α; NOS; nitric oxide synthase; eNOS; endothelial NOS; iNOS; inducible NOS; PAS reagent; Period-Acid-Shiff reagent; TEM; transmission electron microscopyBrown adipose tissue; Metabolism reprogramming; Nitric oxide; phospho-AMPKα; HIF-1α

Thioredoxin glutathione reductase: Its role in redox biology and potential as a target for drugs against neglected diseases by Stefanie Prast-Nielsen; Hsin-Hung Huang; David L. Williams (pp. 1262-1271).

There are two, largely autonomous antioxidant pathways in many organisms, one based on thioredoxin and one based on glutathione, with each pathway having a unique flavoprotein oxidoreductase to maintain them in a reduced state. A recently discovered protein, thioredoxin glutathione reductase (TGR) potentially connects these two pathways. In a large group of parasitic worms, responsible for hundreds of millions of infections in humans and animals, untold morbidity and significant mortality, TGR is the sole enzyme present to maintain redox balance.In this review, the current understanding of the biochemical properties of TGR enzymes is compared to the related enzymes thioredoxin reductase and glutathione reductase. The role of the rare amino acid selenocysteine is discussed. An overview of the potential to target TGR for drug development against a range of parasitic worms and preliminary results to identify TGR inhibitors for schistosomiasis treatment is presented.TGR has properties that are both unique and common to other flavoprotein oxidoreductases. TGR plays a fundamentally different and essential role in the redox biology of parasitic flatworms. Therefore, TGR is a promising target for drug development for schistosomiasis and other trematode and cestode infections.TGR may have differing functions in host organisms, but through analyses to understand its ability to reduce both glutathione and thioredoxin we can better understand the reaction mechanisms of an important class of enzymes. The unique properties of TGR in parasitic flatworms provide promising routes to develop new treatments for diseases.► Redox biology of schistosoma ► Selenoproteins in schistosoma ► The structure and catalytic mechanisms of thioredoxin–glutathione reductase enzymes ► Drug development based on thioredoxin–glutathione reductase for neglected tropical diseases

Keywords: Abbreviations; FAD; flavin adenine dinucleotide; Grx; glutaredoxin; GSH; glutathione; GSSG; glutathione disulfide; GR; glutathione disulfide reductase; PZQ; praziquantel; Sec; selenocysteine; SECIS; Sec insertion sequence; Trx; thioredoxin; TrxR; thioredoxin reductase; TGR; thioredoxin–glutathione reductaseDrug development; Flavoprotein oxidoreductase; Glutathione; Schistosomiasis; Selenocysteine; Thioredoxin

Functional properties of the newly observed^Gγ-chain fetal hemoglobin variant Hb F-Monserrato-Sassari (HBG2:c.280T>C) or [^Gγ93 (F9) Cys→Arg] by Mariagiuseppina Pellegrini; Barbara Manconi; Alessandra Olianas; Maria Teresa Sanna; Claudia Meloni; Monica Pirastru; Paolo Mereu; Giovanni Leoni; Bruno Masala; Laura Manca (pp. 1272-1277).

HbF-Monserrato-Sassari is a newly discovered abnormal fetal hemoglobin observed in an apparently normal newborn baby during a hemoglobinopathies survey at birth in North Sardinian population.Electrophoretic analysis of the cord blood lysate evidenced for an abnormal tetramer due to a mutated fetal globin chain. Electrospray ionisation-mass spectrometry and gene sequencing were used to identify the mutation. Oxygen binding ability of the variant Hb was determined.Sequencing of the γ globin genes revealed the TGT→ CGT transition at codon 93 in one of the two^Gγ genes, which leads to the Arg for Cys amino acid replacement at position 9 of the F α-helix. The amino acid substitution was confirmed by mass spectrometric analysis of the globin chains. Since modifications or substitutions at position β93 are known to affect the arrangement of a salt bridge at the α1β2 sliding contacts that are crucial for subunit cooperativity, the functional properties of the variant were studied to evaluate the effect of the replacement at the same position in the γ globin chain. With respect to normal HbF, the variant showed a significant increase in oxygen affinity and a slight decrease of both Bohr effect and cooperativity.Result indicates a key role of the Cys γ93 residue for subunit cooperativity in the T→R transition of the HbF tetramer. Substitutions at the F9 position of the^Gγ globin may result in stabilization of the high affinity R-state of the Hb tetramer. Because of the loss of Cys γ93 residue, this variant is considered to be potentially compromised in nitric oxide transport.► HbF-Monserrato-Sassari is a newly discovered abnormal human fetal hemoglobin. ► It is characterized by the Arg for Cys replacement at position 93 of the Gγ globin. ► It shows increased oxygen affinity, slightly decreased Bohr effect and cooperativity. ► Cys γ93 has a key role for subunit cooperativity in the T→R transition of the HbF. ► Substitutions at this position may stabilize the high affinity R-state of the Hb and endanger nitric oxide transport.

Keywords: Abbreviations; HbA; adult hemoglobin; HbF; fetal hemoglobin; CE-HPLC; cation exchange high performance liquid chromatography; IEF; isoelectric focusing; AUT-PAGE; acid-urea Triton X-100 electrophoresis; RP-HPLC; reversed phase-HPLC; PCR; polymerase chain reaction; ESI-MS; electrospray ionisation-mass spectrometry; FPLC; fast protein liquid chromatography; BPG; 2,3-bisphosphoglycerate; P; ₅₀; partial pressure of oxygen required to saturate 50% of the hemes; n; ₅₀; slope of the Hill plot at 50% saturation; Mav; mass averageHbF variant; ^G; γ globin gene; Gene sequencing; α1γ2 interface; Oxygen affinity; NO transport

Orai1/CRACM1 overexpression suppresses cell proliferation via attenuation of the store-operated calcium influx-mediated signalling pathway in A549 lung cancer cells by Ming-Feng Hou; Ho-Chang Kuo; Jih-Heng Li; Yu-Shiuan Wang; Chen-Chia Chang; Ku-Chung Chen; Wei-Chiao Chen; Chien-Chih Chiu; Shengyu Yang; Wei-Chiao Chang (pp. 1278-1284).

Orai1/CRACM1 is a principal component of the store-operated calcium channels. Store-operated calcium influx is highly correlated with inflammatory reactions, immunological regulation, and cell proliferation. Epidermal growth factor (EGF), which plays an important role in the regulation of cell proliferation, can activate store-operated calcium channels. However, the consequences of Orai1/CRACM1 overexpression in EGF-mediated lung cancer cells growth are not known.To investigate the role of Orai1/CRACM1 in EGF-mediated lung cancer cell proliferation, Orai1/CRACM1 plasmids were transfected into cells by lipofection. A cell proliferation assay, immunofluorescence staining, flow cytometry, and real-time polymerase chain reaction were employed to monitor cell proliferation. The calcium influx signals were investigated using a fluorescent-based calcium assay.Transfection of Orai1/CRACM1 plasmids resulted in the inhibition of EGF-mediated cell proliferation. ERK1/2 and Akt phosphorylation were inhibited by Orai1/CRACM1 overexpression. Expression of the cell cycle modulator p21 was induced in the Orai1/CRACM1-overexpressing cells, whereas the expression of cyclin D3 was reduced. Flow cytometry revealed that overexpression of Orai1/CRACM1 resulted in G0/G1 cell cycle arrest. Importantly, Orai1/CRACM1 overexpression significantly attenuated EGF-mediated store-operated calcium influx. In addition, application of 2-APB, a store-operated calcium channel inhibitor, resulted in the inhibition of EGF-mediated cancer cell proliferation.We conclude that Orai1/CRACM1 overexpression attenuates store-operated Ca²⁺ influx that in turn blocks EGF-mediated proliferative signaling and drives cell cycle arrest.► Orai1/CRACM1 overexpression resulted in the reduction of cell proliferation ► Phosphorylation of ERK1/2 and Akt is blocked by Orai1/CRACM1 overexpression. ► Orai1/CRACM1 overexpression resulted in a cell cycle G0/G1 phase arrest. ► Store-operated calcium inhibitors influenced cell proliferation.

Keywords: Orai1/CRACM1; Cell proliferation; Lung cancer; p21

Partial restoration of the long QT syndrome associated KCNQ1 A341V mutant by the KCNE1 β-subunit by Ikuomi Mikuni; Carlos G. Torres; Martin W. Bienengraeber; Wai-Meng Kwok (pp. 1285-1293).

The A341V mutation in the pore-forming KCNQ1 subunit of the slowly activating delayed-rectifier potassium current (IKs) underlies a common form of the long QT syndrome, and is associated with an unusually severe phenotype. However, there is controversy regarding the underlying mechanism responsible for the clinically observed phenotype. We investigated the biophysical characteristics of A341V in a cardiac environment by utilizing a cardiac cell line, and in particular the impact of the KCNE1 β-subunit.Whole-cell current were recorded from transiently transfected HL-1 cells, a cardiac cell line. Mutant KCNQ1 and KCNE1 were constructed by site-directed mutagenesis.The A341V mutant resulted in a non-functional channel when expressed alone. When co-expressed with wild type KCNE1, A341V produced a slowly activating current, with a smaller current density, slower rates of activation, and a depolarized shift in its activation curve compared to the wild type KCNQ1+KCNE1. Confocal microscopy confirmed the surface expression of GFP-tagged A341V, suggesting a functionally defective protein. A T58A mutation in KCNE1 abolished functional restoration of A341V. Under heterozygous conditions, the expression of A341V+KCNQ1+KCNE1 reduced but did not abolish the electrophysiological changes observed in A341V+KCNE1. A dominant negative effect of A341V was also observed. Action potential simulations revealed that the A341V mutation is arrhythmogenic.The KCNE1 β-subunit partially rescued the non-functional A341V mutant, with electrophysiological properties distinct from the wild type IKs.The severity of the A341V phenotype may be due to a combination of a significant suppression of the IKs with altered biophysical characteristics.► KCNE1 functionally restored a long QT syndrome-associated A341V mutant. ► The resultant A341V+KCNE1 current had distinct biophysical characteristics. ► A341V had a dominant negative effect on channel current. ► Heterozygous expression of A341V+KCNQ1+KCNE1 altered cardiac excitability in a computational model of ventricular action potentials. ► These changes contribute to the severe clinical phenotype of carriers of A341V.

Keywords: Long QT syndrome; IKs; A341V; KCNE1; Arrhythmia; Action potentials

The T/NK cell co-stimulatory molecule SECTM1 is an IFN “early response gene” that is negatively regulated by LPS in Human monocytic cells by Trevor Huyton; Gottmann Wiebke Göttmann; Bade-Doding Christina Bade-Döding; Ananta Paine; Rainer Blasczyk (pp. 1294-1301).

SECTM1 is a T/NK cell “co-stimulatory” molecule that is expressed in the peripheral blood by neutrophils and monocytes.We used qRT-PCR to investigate the mRNA expression of SECTM1 in human monocytic cells after stimulation with interferons and LPS and confirmed the protein expression by flow cytometry.The kinetics of interferon induced SECTM1 mRNA expression in MM6 cells are time dependent occurring rapidly within 3h of stimulation and reaching a maximal level at ~6h for IFN-α and ~12h for IFN-β and IFN-γ. Co-treatment of MM6 cells with IFN-γ and cycloheximide caused a superinduction of SECTM1 mRNA expression while cycloheximide alone had no effect, illustrating that de novo protein synthesis is not required for IFN-γ enhanced expression of SECTM1 mRNA, a characteristic of IFN early response genes. The kinetics of IFN induced SECTM1 mRNA expression in primary monocytes is comparable although it occurs much quicker with rapid induction by IFN-α, IFN-β and IFN-γ and maximal levels reached in <6h. Human monocytic cells also displayed a pronounced negative regulation of SECTM1 mRNA expression by LPS, while at the protein level SECTM1 expression was also shown to be regulated by IFN and LPS.Bioinformatic analysis of the SECTM1 promoter region identified STAT1α/GAS, STAT3, ISRE, NFκB and putative p63 binding sites suggesting a complex transcriptional control. This tight regulation of SECTM1 gene expression and rapid upregulation highlights its relevance in the innate immune response.Human monocytes produce SECTM1 in response to interferon stimuli that is negatively regulated by LPS.The level of SECTM1 expression is likely to be a key factor in innate immune responses and in the immune tolerance of cancerous cells.► Sectm1 is an immune system protein expressed in monocytes. ► Sectm1 mRNA and protein expression is regulated by interferons and LPS. ► SECTM1 is likely to play an important role in innate immunity and cancer tolerance.

Keywords: Abbreviations; SECTM1; Secreted and transmembrane 1; IFN; Interferon; LPS; Lipopolysacharide; TNF-α; Tumor necrosis factor alpha; TGF-β; Transforming growth factor beta; IL10; Interleukin 10SECTM1; CD7; RT-PCR; Interferon; LPS tolerance; Monocyte

Biosurfactant mannosyl-erythritol lipid inhibits secretion of inflammatory mediators from RBL-2H3 cells by Yosuke Morita; Satoshi Tadokoro; Masao Sasai; Dai Kitamoto; Naohide Hirashima (pp. 1302-1308).

Biosurfactant mannosyl-erythritol lipids (MELs) are glycolipids produced by microbes that have various biological activities. It has been reported that MELs exhibit excellent surface-activity and also various bioactivities, such as induction of cell differentiation and apoptosis. However, little is known about their action related to drug discovery or drug seeds.We investigated the effects of MELs on the secretion of inflammatory mediators from mast cells that play a central role in allergic responses. Mast cells secrete three kinds of inflammatory mediators and we quantified these secreted mediators by photometer or ELISA. The action mechanisms of MELs were studied by Ca²⁺-sensitive fluorescence dye and Western blotting of phosphorylated proteins.MELs inhibited exocytotic release by antigen stimulation in a dose-dependent manner. We also found that MELs inhibited antigen-induced secretion of leukotriene C₄ and cytokine TNF-α (tumor necrosis factor-α). The inhibitory action of MELs on mediator secretion was mediated by inhibition of Ca²⁺ increase, phosphorylation of MAP kinases and SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) that serve as a molecular machinery for exocytotic membrane fusion.MELs have anti-inflammatory action inhibiting the secretion of inflammatory mediators from mast cells.MELs affects two of major intracellular signaling pathways including Ca²⁺ increase and MAP kinases. MELs also inhibited the phosphorylation of SNARE proteins that is crucial for not only exocytosis but also intracellular vesicular trafficking.► Biosurfactants MELs inhibited exocytotic release from mast cells. ► MELs inhibited secretion of leukotriene C₄ and TNF-α from mast cells. ► MELs inhibited Ca²⁺ increase, phosphorylation of MAP kinases and SNAREs. ► MELs had anti-allergic actions inhibiting secretion of inflammatory mediators.

Keywords: Biosurfactant; Glycolipid; Mast cell; Exocytosis; TNF-α; Leukotriene

Carbamoyl-PROXYL-enhanced MRI detects very small disruptions in brain vascular permeability induced by dietary cholesterol by Atsuyuki Tomizawa; Itsuko Ishii; Zhivko Zhelev; Ichio Aoki; Sayaka Shibata; Mitsukazu Kitada; Rumiana Bakalova (pp. 1309-1316).

Gd-DTPA-enhanced magnetic resonance imaging (MRI) is a conventional method for non-invasive investigation of blood-brain-barrier (BBB) permeability in animal models. It allows the visualization of serious injury to the BBB. We developed a novel approach for detecting very small disruptions in BBB permeability induced by dietary cholesterol by using carbamoyl-PROXYL (CMP) as an MRI contrast probe.Mice were separated into two groups: normal diet (ND-mice) and high cholesterol diet (CD-mice). MRI-signal dynamics, plasma cholesterol, matrix metalloproteinase (MMP-9, MMP-2), and the white blood cell profile were analyzed. For the MRI analysis, two regions-of-interest (ROI) were selected: brain (ROI-1) and surrounding area (ROI-2).In the ROI-2 of ND-mice, CMP- or Gd-enhanced MRI-signal followed typical kinetics with a half-life of signal decay (τ_1/2) ~8 or ~15min, respectively. In CD-mice, the MRI-signal increased continuously without decay.In the ROI-1 of ND- and CD-mice, MRI-signal enhancement was not detected by Gd-DTPA. In the ROI-1 of ND-mice, CMP-induced MRI-signal enhancement was negligible, while in CD-mice, it was significant (τ_1/2>15min).Hypercholesterolemia increased the plasma levels of MMP-9 and neutrophils.Hypercholesterolemia increases vascular permeability, which is mediated by MMP-9 and neutrophils.Even very small disruptions in brain vascular permeability could be detected by CMP-enhanced MRI but not by Gd-DTPA-enhanced MRI.► Hypercholesterolemia-induced disruptions of vascular permeability in the brain. ► New MRI approach for detection of very small disruptions of vascular permeability. ► For this purpose, carbamoyl-PROXYL is most appropriate contrast probe than Gd. ► Dietary cholesterol increases the level of plasma matrix metalloproteinase-9. ► Dietary cholesterol increases the level of plasma neutrophils.

Keywords: Abbreviations; BBB; blood-brain barrier; carbamoyl-PROXYL; 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl; CD; high cholesterol diet; CSF; cerebrospinal fluid; Gd-DTPA; gadolinium-diethylenetriamine pentaacetic acid; MCAO; middle cerebral artery occlusion; MMP; matrix metalloproteinase; MRI; magnetic resonance imaging; ND; normal diet; ROI; regions of interest; TIMP; tissue inhibitor of metalloproteinase; ZO-1; zona occludens-1Blood-brain barrier; 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl; Hypercholesterolemia; Magnetic resonance imaging; Matrix metalloproteinase; Neutrophils

Rice OsERG3 encodes an unusual small C2-domain protein containing a Ca²⁺-binding module but lacking phospholipid-binding properties by Chang Ho Kang; Byeong Cheol Moon; Hyeong Cheol Park; Seong Cheol Koo; Joo Mi Jeon; Yong Hwa Cheong; Woo Sik Chung; Chae Oh Lim; Jae-Yeon Kim; Byung-Dae Yoon; Sang Yeol Lee; Cha Young Kim (pp. 1317-1322).

The C2 domain is a Ca²⁺/phospholipid-binding motif found in many proteins involved in signal transduction or membrane trafficking. OsERG3 is a homolog of OsERG1, a gene encoding a small C2-domain protein in rice.OsERG3 Ca²⁺-binding and phospholipid-binding assays were carried out using³H-labeled phospholipid liposomes and a⁴⁵Ca²⁺ overlay assay, respectively. Cytosolic expression of OsERG3 was investigated by Western blot analysis and the OsERG3::smGFP transient expression assay. OsERG3 transcript levels were greatly enhanced by treatment with a fungal elicitor and Ca²⁺-ionophore. OsERG3 protein proved unable to interact with phospholipids regardless of the presence or absence of Ca²⁺ ions. Nonetheless, OsERG3 displayed calcium-binding activity in an in vitro⁴⁵Ca²⁺-binding assay, a property not observed with OsERG1. The cytosolic location of OsERG3 was not altered by the presence of fungal elicitor or Ca²⁺-ionophore. OsERG3 encodes a small C2-domain protein consisting of a single C2 domain. OsERG3 binds Ca²⁺ ions but not phospholipids. OsERG3 is a cytosolic soluble protein. The OsERG3 gene may play a role in signaling pathway involving Ca²⁺ ions.The data demonstrate that OsERG3 is an unusual small C2-domain protein containing a Ca²⁺-binding module but lacking phospholipid-binding properties.► We examine rice small C2-domain proteins, which are responsive to elicitor signals. ► We show that OsERG3 binds Ca²⁺ ions but not phospholipids. ► OsERG3 keeps its cytosolic location even in the presence of elicitor signals.

Keywords: Abbreviations; GST; glutathione; S; -transferase; smGFP; soluble modified green fluorescent protein; PC; phosphatidylcholine; PI; phosphatidylinositol; PS; phosphatidylserine; PE; phosphatidylethanolamineRice; Signal transduction; Small C2-domain protein; Ca; ²⁺; /phospholipid binding

Interaction of fosfomycin with the Glycerol 3-phosphate Transporter of Escherichia coli by Antonella Santoro; Anna Rita Cappello; Marianna Madeo; Emanuela Martello; Domenico Iacopetta; Vincenza Dolce (pp. 1323-1329).

Fosfomycin is widely used to treat urinary tract and pediatric gastrointestinal infections of bacteria. It is supposed that this antibiotic enters cells via two transport systems, including the bacterial Glycerol-3-phosphate Transporter (GlpT). Impaired function of GlpT is one mechanism for fosfomycin resistance.The interaction of fosfomycin with the recombinant and purified GlpT of Escherichia coli reconstituted in liposomes has been studied. IC₅₀ and the half-saturation constant of the transporter for external fosfomycin (K_i) were determined by transport assay of [¹⁴C]glycerol-3-phosphate catalyzed by recombinant GlpT. Efficacy of fosfomycin on growth rates of GlpT defective bacteria strains transformed with recombinant GlpT was measured.Fosfomycin, externally added to the proteoliposomes, poorly inhibited the glycerol-3-phosphate/glycerol-3-phosphate antiport catalyzed by the reconstituted transporter with an IC5₀ of 6.4mM. A kinetic analysis revealed that the inhibition was completely competitive, that is, fosfomycin interacted with the substrate-binding site and the K_i measured was 1.65mM. Transport assays performed with proteoliposomes containing internal fosfomycin indicate that it was not very well transported by GlpT. Complementation study, performed with GlpT defective bacteria strains, indicated that the fosfomycin resistance, beside deficiency in antibiotic transporter, could be due to other gene defects.The poor transport observed in a reconstituted system together with the high value of K_i and the results of complementation study well explain the usual high dosage of this drug for the treatment of the urinary tract infections.This is the first report regarding functional analysis of interaction between fosfomycin and GlpT.► The interaction of fosfomycin with the purified GlpT of E. coli has been studied. ► Fosfomycin poorly inhibited the G3P/G3P antiport catalyzed by the transporter. ► Fosfomycin is not very well transported by the GlpT. ► Fosfomycin resistance could involve several gene defects, besides GlpT deficiency.

Keywords: Abbreviations; G3P; Glycerol-3-phosphate; GlpT; Glycerol-3-phosphate Transporter; HEPES; (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); LB; Luria-Bertani broth; MES; 2-[N-morpholino]ethanesulfonic acid; SDS-PAGE; polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; Ugp; Uptake of glycerol phosphateGlycerol-3-phosphate Transporter; GlpT; Transport; Fosfomycin; Escherichia coli

The interrelationship between ligand binding and self-association of the folate binding protein. The role of detergent–tryptophan interaction by Jan Holm; Christian Schou; Linnea N. Babol; Anders J. Lawaetz; Susanne W. Bruun; Morten Z. Hansen; Steen I. Hansen (pp. 1330-1339).

The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of folate binding.Self-association behavior of apo- and holo-FBP was addressed through size exclusion chromatography, SDS-PAGE, mass spectrometry, surface plasmon resonance and fluorescence spectroscopy.Especially holo-FBP exhibits concentration-dependent self-association at pH 7.4 (pI), and is more prone to associate into stable complexes than apo-FBP. Even more pronounced was the tendency to complexation between apo-FBP and holo-FBP in accord with a model predicting association between apo and holo monomers . This will lead to removal of apo monomers from the reaction scheme resulting in a weak incomplete ligand binding similar to that observed at FBP concentrations <10nM. The presence of synthetic and natural detergents normalized folate binding kinetics and resulted in appearance of monomeric holo-FBP. Fluorescence spectroscopy indicated molecular interactions between detergent and tryptophan residues located in hydrophobic structures of apo-FBP which may participate in protein associations.Self-association into multimers may protect binding sites, and in case of holo-FBP even folate from biological degradation. High-affinity folate binding in body secretions, typically containing 1–10nM FBP, requires the presence of natural detergents, i.e. cholesterol and phospholipids, to avoid complexation between apo- and holo-FBP.► Folate binding protein shows concentration-dependent self-association at pH 7.4–pI. ► Holo-forms are more prone to self-association into stable complexes than apo-forms. ► Apo- and holo-forms also tend to self-associate into asymmetric and stable complexes. ► Asymmetric complexes are formed at <10nM protein and lead to a weak folate binding. ► Detergents may inhibit asymmetric complexation by interaction with tryptophan groups.

Keywords: Abbreviations; AC; analytical ultracentrifugation; CMC; critical micelle concentration; FBP; folate binding protein; FR; folate receptor; GPI; glycosyl phosphatidyl inositol; MALDI-TOF; matrix-assisted laser desorption ionization time-of-flight; PARAFAC; parallel factor analysis; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEC; size exclusion chromatography; SPR; surface plasmon resonanceFolate binding protein; Ligand mediated self-association; Asymmetric apo–holo complexes; Surface plasmon resonance; Detergent–tryptophan interaction; Fluorescence spectroscopy

Streptonigrin inhibits β-Catenin/Tcf signaling and shows cytotoxicity in β-catenin-activated cells by Seyeon Park; Sohyun Chun (pp. 1340-1345).

Activation of β-catenin/T-cell factor (Tcf) signaling plays a role in human carcinogenesis. This suggests a possibility that the β-catenin/Tcf signaling activated by the accumulation of β-catenin in the nucleus is related to some type of human carcinogenesis. Therefore, if β-catenin's transcriptional activity can be markedly down-regulated, tumor growth will be suppressed in β-catenin activated types of cancer.To investigate the activation or suppression of β-catenin/Tcf transcription, we established a transiently transfected cell line with a constitutively active β-catenin mutant gene whose product is not degraded. This cell line was also co-transfected with luciferase reporter gene constructs containing either an optimized (TOPflash) or mutant (FOPflash) Tcf-binding element.We identified the inhibitory effect of streptonigrin against β-catenin/Tcf signaling in β-catenin activated cells. Streptonigrin inhibited the transcriptional activity of β-catenin/Tcf in SW480 cells and HEK293 cells transiently transfected with a constitutively active mutant β-catenin gene. The growth inhibitory effect of streptonigrin was more evident in β-catenin-activated cancer cells than in non-activated cancer cells. The electrophoresis mobility shift assay showed that the binding of Tcf complexes with their specific DNA-binding sites was suppressed by streptonigrin. Conclusion: Streptonigrin is a negative regulator of β-catenin/Tcf signaling, and their inhibitory mechanism is related to the proliferation inhibitory effect on β-catenin-activated cancer cells.This report reveals a molecular mechanism underlying the anti-tumor effect of streptonigrin from the perspective β-catenin/Tcf signaling. Given its function in inhibiting β-catenin/Tcf signaling, streptonigrin may be of interest as a leading compound for chemotherapeutic agent against β-catenin-activated tumorigenesis.Display Omitted► Streptonigrin as an inhibitor of β-catenin/Tcf transcription factor. ► Streptonigrin showed more evident cytotoxicity in β-catenin-activated cells. ► Inhibition effects depend on suppression of β-catenin/Tcf complexes with DNA.

Keywords: Abbreviations; APC; Axin-adenomatous polyposis coli; BCA; bicinchoninic acid; CBP; CREB binding protein; CRC; colorectal cancer; DMSO; dimethyl sulfoxide; Dsh; disheveled; EMSA; electrophoretic mobility shift assay; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; GSK; glycogen sythase kinase; IC; ₅₀; half maximal inhibitory concentration; Lef; lymphoid enhancer factor; NF-κB; nuclear factor-κB; PCR; polymerase chain reaction; SDS; sodium dodecyl sulfate; TBE; Tris base boric acid EDTA; TBS; Tris buffered saline; TBST; Tris buffered saline and Tween-20; Tcf; T-cell factorβ-catenin/Tcf signaling; Inhibitor; Streptonigrin

Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation by Shinjinee Sengupta; Sagar Lahiri; Shakri Banerjee; Bipasha Bashistha; Anil K. Ghosh (pp. 1346-1354).

Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP).In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100mMl-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC).An electrophoretically homogenous TPS that was purified was a 60kDa protein with 22.1 fold purification having a specific activity of 2.03U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1μg, at a temperature of 37°C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl₂, MgCl₂ and ZnSO₄, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V_max and lowest K_m values were calculated as 2.96U/mg and 1.36mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors.Substrate specificity, V_max and K_m values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis.► A 60kDa protein having TPS activity was purified to electrophoretic homogeneity ► The specific activity of purified TPS was 2.03U/mg protein. ► A new purification strategy was followed usingl-arginine. ► N-terminal analysis revealed the similarity of purified TPS with TPS from S. cerevisiae. ► Purified TPS showed more affinity towards ADPG than its normal substrate UDPG.

Keywords: Abbreviations; TPS; Trehalose-6-phophate synthase; TPP; Trehalose phosphate phosphatases; UDPG; Uridine diphosphaphate glucose; ADPG; Adenosine di phosphate synthase; G-6-P; Glucose-6-phophate; F-6-P; Fructose-6-phosphate Candida utilis; Trehalose; Trehalose-6-phosphate synthase; Arginine

Crystallographic survey of active sites of an unclassified glutathione transferase from Bombyx mori by Yoshimitsu Kakuta; Kazuhiro Usuda; Takashi Nakashima; Makoto Kimura; Yoichi Aso; Kohji Yamamoto (pp. 1355-1360).

Glutathione transferase (GST) catalyzes a major step in the xenobiotic detoxification pathway. We previously identified a novel, unclassified GST that is upregulated in an insecticide-resistant silkworm ( Bombyx mori) upon insecticide exposure. Here, we sought to further characterize this GST, bmGSTu, by solving and refining its crystal structure and identifying its catalytic residues.The structure of wild-type bmGSTu was determined with a resolution of 2.1Å by synchrotron radiation and molecular modeling. Potential catalytic residues were mutated to alanine by means of site-directed mutagenesis, and kinetic data determined for wild-type and mutated bmGSTu.We found that bmGSTu occurred as a dimer, and that, like other GSTs, each subunit displayed a G-site and an H-site in the active center. Bound glutathione could be localized at the G-site. Kinetic data of the mutated forms of bmGSTu show that Val55, Glu67, and Ser68 in the G-site are important for catalysis. Furthermore, the H-site showed some unique features.This is the first study to our knowledge to elucidate the molecular conformation of this B. mori GST. Our results indicate that residues Val55, Glu67, and Ser68, as well as Tyr7 and Ser12, in the glutathione-binding region of bmGSTu are critical for catalytic function.Our results, together with our previous finding that bmGSTu was preferentially induced in an insecticide-resistant strain, support the idea that bmGSTu functions in the transformation of exogenous chemical agents. Furthermore, the unique features observed in bmGSTu may shed light on mechanisms of insecticide resistance.► The crystal structure of bmGSTu has been determined with a resolution of 2.1Å. ► Bound glutathione could be localized at the G-site. ► Recombinant bmGSTu catalyzed transfer of glutathione to a synthetic GST substrate. ► Val55, Glu67, and Ser68 in the G-site are catalytically important. ► The H-site shows unique features that may have relevance to pest control.

Keywords: Abbreviations; CDNB; 1-chloro-2,4-dinitrobenzene; GSH; glutathione; GST; glutathione transferase; GTX; S; -hexyl glutathione; SDS-PAGE; polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfateCrystal structure; Glutathione; Glutathione transferase; Lepidoptera; Site-directed mutagenesis

A new human dyskerin isoform with cytoplasmic localization by Alberto Angrisani; Mimmo Turano; Lorella Paparo; Concetta Di Mauro; Maria Furia (pp. 1361-1368).

The human DKC1 gene is causative of X-linked dyskeratosis congenita (X-DC), a syndrome characterized by mucocutaneous features, bone marrow failure, tumor susceptibility, perturbation of stem cell function, and premature aging. DKC1 is thought to produce a single protein, named dyskerin, which shows strict nucleolar localization and participates in at least two distinct nuclear functional complexes: the H/ACA small nucleolar ribonucleoproteic complex involved in RNA pseudouridylation and the active telomerase complex.By bioinformatics and molecular analyses we identified a DKC1 splice variant able to encode a truncated form of dyskerin, confirmed its active expression in diverse human tissues by RT-PCR, and showed by immunoblotting and immunocytochemistry experiments that it actually encodes a novel protein. Stably transfected clones over-expressing the new isoform were analyzed for growth, morphology and adhesion properties.Our results show that DKC1 encodes a new alternatively spliced mRNA able to direct the synthesis of a variant dyskerin with unexpected cytoplasmic localization. Intriguingly, when over-expressed in HeLa cells, the new isoform promotes cell to cell and cell to substratum adhesion, increases the cell proliferation rate and leads to cytokeratin hyper-expression.Our results highlight a novel degree of complexity and regulation of the human DKC1 gene and reveal that it can play a further, unpredicted role in cell adhesion. The identification of a dyskerin cytoplasmic variant reinforces the view that other mechanisms, in addition to telomere instability, can significantly contribute to the pathogenesis of the X-DC, and suggests that DKC1 nucleolar and cytoplasmic functions might cumulatively account for the plethora of manifestations displayed by this syndrome.► The human DKC1 gene encodes a novel protein isoform with cytoplasmic localization. ► Cells over-expressing this isoform show increased growth rate and adhesion. ► Over-expressing cells hyper-express cytokeratins.

Keywords: Abbreviations; DC; Dyskeratosis Congenita; X-DC; X-linked Dyskeratosis Congenita; snoRNA; small nucleolar RNA; snoRNPs; small nucleolar RNA ribonucleoproteins; hTERT; human telomerase reverse transcriptase; hTR; human telomerase RNA component; IRES; Internal Ribosome Entry Site; EST; expressed sequence tag; UTR; untranslated region; CDS; protein coding region; NLS; Nuclear Localization signal; DKLD; Dyskerin Like Domain; TruB_N; N-terminal catalytic domain of; E; .; coli; tRNA pseudouridine 55 synthase B; PUA; RNA binding domain from archaeal and eukaryotic pseudouridine synthases and archaeosine transglycosylase; LR; Lysine Rich domain; HeLa; human cervical carcinoma cell line; POLR2A; largest subunit of RNA polymerase II; HIER; Heat-Induced Epitope Retrieval DKC1; Dyskeratosis; Pseudouridine synthase; H/ACA snoRNPs; Alternative splicing; Cell adhesion

Corrigendum to “Hypertonicity-enhanced TNF-α release from activated human monocytic THP-1 cells requires ERK activation” [Biochim. Biophys. Acta 1810 (2011) 475–484] by Yung-Chen Chou; Joen-Rong Sheu; Chi-Li Chung; Che-Jen Hsiao; Po-Jen Hsueh; George Hsiao (pp. 1369-1369).

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BBA - General Subjects (v.1810, #12)