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BBA - General Subjects (v.1770, #6)

Editorial Board (pp. ii).

Biomarkers for early detection of breast cancer: What, when, and where? by Victor V. Levenson (pp. 847-856).

Early detection of breast cancer reduces the suffering and cost to society associated with the disease. A sensitive assay to identify biomarkers that can accurately diagnose the onset of breast cancer using non-invasively collected clinical specimens is ideal for early detection. The earlier and more accurate the diagnostic biomarker can predict disease onset, the more valuable it becomes. Here, a brief review of existing and emerging approaches for breast cancer biomarker identification and analysis is presented. Those biomarkers found in biological fluids, blood in particular, apparently hold the best promise for fast development of screening assays. Autoantibodies and abnormal tumor-specific DNA methylation found in cell-free plasma DNA may provide the best opportunity for constructing multiplexed and highly redundant tests, which will be sufficiently specific and sensitive for early detection of breast cancer. It is expected that technologies developed for breast cancer detection will be useful for other types of cancer.

Keywords: Early detection; Breast cancer; Biomarker; DNA methylation; Autoantibody

Biomarkers for early detection of breast cancer: What, when, and where? by Victor V. Levenson (pp. 847-856).

Keywords: Early detection; Breast cancer; Biomarker; DNA methylation; Autoantibody

A database study that identifies genes whose expression correlates, negatively or positively, with 5-year survival of cancer patients by Wilfred D. Stein; Thomas Litman; Tito Fojo; Susan E. Bates (pp. 857-871).

A published microarray gene expression database containing data on 174 tumor samples from ten tissues was mined, enabling the identification of classes of genes whose expression correlates significantly with the intractability, or tractability, to therapy of tumors derived from such tissues. As a measure of tractability, the 5-year survival of patients presenting with distant (metastatic) tumors was used. Genes that encode proteins related to cell adhesion, and enzymes involved in metabolic oxidation or reduction, were upregulated in intractable cancers. Genes that encode proteins implicated in the control of DNA transcription were downregulated in the intractable cancers. We describe hypotheses with regard to cell functions that may help in designing new therapeutic modalities, aimed at improving survival of cancer patients.

Keywords: Microarray; SEER survivals; Cell adhesion; Drug resistance; Adhesion-mediated resistance

Sphingosine 1-phosphate stimulation of NADPH oxidase activity: Relationship with platelet-derived growth factor receptor and c-Src kinase by Serena Catarzi; Elisa Giannoni; Fabio Favilli; Elisabetta Meacci; Teresa Iantomasi; Maria T. Vincenzini (pp. 872-883).

This study demonstrates for the first time that sphingosine 1-phosphate (S1P) increases H₂O₂ production in NIH3T3 fibroblasts through NADPH oxidase activation, confirming the involvement of phosphoinositide-3-kinase and protein kinase C in the activation of this enzyme in non-phagocyte mammalian cells. The results demonstrate also that both platelet-derived growth factor (PDGF) and S1P-mediated NADPH oxidase activation and H₂O₂ production by Gi-protein coupled receptors (GPCRs) and c-Src kinase. Moreover, both PDGF and S1P activate c-Src kinase through GPCRs, indicating that this kinase can constitute a connection factor between PDGF and S1P signaling, confirming the cross-talk previously found between their receptors. Thus, Gi-protein-mediated NADPH oxidase activation with the consequent H₂O₂ increase constitutes an early event in the PDGF and S1P pathways. However, a different time course of H₂O₂ production in S1P-stimulated cells compared to that obtained in PDGF-stimulated cells has been observed, and this seems to be related to the different activation behavior of c-Src kinase induced after S1P or PDGF stimulation. Finally, these data demonstrate that S1P-induced H₂O₂ production is necessary to maximize c-Src kinase activation, confirming that this is a redox regulated kinase. After which, c-Src plays an important role both upstream and downstream from NADPH oxidase activation.

Keywords: Sphingosine 1-phosphate; NADPH oxidase activation; PDGF receptor; c-Src kinase

Altered expression of MUC2 and MUC5AC in response to Shigella infection, an in vivo study by Prakash Radhakrishnan; Devaraj Halagowder; S. Niranjali Devaraj (pp. 884-889).

Infection of mucosal epithelial cells by Shigella species leads to an intense and acute inflammatory bowel disease that is characterized by watery diarrhea and purulent discharge. Mucin production is a common defense mechanism to protect the underlying mucosa against pathogens. The molecular mechanism(s) underlying mucin induction is unknown in Shigellosis. In this study, we have evaluated the relationship between Shigella infection, the expression of MUC2 and MUC5AC and the participation of signaling molecules TNF-α, PKC and ERK1/2. Shigella infection up-regulated MUC2 and MUC5AC expression in 6–8 h, through activation of TNF-α, PKC and ERK1/2. These results confirm that, in response to Shigella infection, the normal expression pattern of MUC-2 and MUC-5AC is altered. This in vivo study brings new insights into the molecular pathogenesis of Shigellosis and new potential therapeutic targets for Shigellosis.

Keywords: Shigella; species; MUC2; MUC5AC; Ileal loop ligation assay; Shigellosis

Altered expression of MUC2 and MUC5AC in response to Shigella infection, an in vivo study by Prakash Radhakrishnan; Devaraj Halagowder; S. Niranjali Devaraj (pp. 884-889).

Keywords: Shigella; species; MUC2; MUC5AC; Ileal loop ligation assay; Shigellosis

Tissue distribution and functional analyses of the constitutively active orphan G protein coupled receptors, GPR26 and GPR78 by Philip G. Jones; Stanley P. Nawoschik; Kodangattil Sreekumar; Albert J. Uveges; Eugene Tseng; Lynn Zhang; Jeremy Johnson; Lan He; Janet E. Paulsen; Brian Bates; Mark H. Pausch (pp. 890-901).

GPR26 and GPR78 are orphan GPCRs (oGPCRs) that share 51% amino acid sequence identity and are widely expressed in selected tissues of the human brain as well as the developing and adult mouse brain. Investigation of the functional activity of GPR26 and GPR78 via expression in HEK293 cells showed that both proteins are constitutively active and coupled to elevated cAMP production. Accordingly, in yeast, GPR26 demonstrated apparent agonist-independent coupling to a chimeric Gpa1 protein in which the 5 C-terminal amino acids were from Gαs. A comparison of the proteins revealed an atypical glutamine residue in GPR78 in place of the conserved arginine residue (R3.50) in the so-called DRY box. Site-directed mutants R3.50 in GPR26 were constructed and retained their constitutive activity suggesting that these 2 receptors activate G proteins in a manner that is distinct from other group 1 GPCRs.

Keywords: Abbreviations; AcbC; accumbens nucleus core; AcbSh; accumbens nucleus shell; ACo; anterior cortical amygdaloid nucleus; AhiPM; amygdalohippocampal area posteromedial part; AOD; anterior olfactory nucleus dorsal part; AOL; anterior olfactory nucleus lateral part; AOM; anterior olfactory nucleus medial part; AP; area postrema; APir; amygdalopiriform transition area; BLA; basolateral amygdaloid nucleus anterior part; BLV; basolateral amygdaloid nucleus ventral part; BMP; basomedial amygdaloid nucleus posterior part; CC; central canal; Cg; cingulum; CPu; caudate putamen (striatum); DC; dorsal cochlear nucleus; DEn; dorsal endopiriform nucleus; DRI; dorsal raphe nucleus interfascicular part; FC; fasciola cinereum; GnRHR; Gonadotropin releasing hormone receptor; HDB; nucleus of the horizontal limb of the diagonal band; IC; inferior colliculus; ICj; islands of Calleja; ICjM; islands of Calleja major island; Icp; inferior cerebellar peduncle; La; lateral amygdaloid nucleus; LEnt; lateral entorhinal cortex; LPB; lateral parabrachial nucleus; LS; lateral septal nucleus; M1; primary motor cortex; M2; secondary motor cortex; MeA; medial amygdaloid nucleus anterior part; MePD; medial amygdaloid nucleus posterodorsal part; MePV; medial amygdaloid nucleus posteroventral part; MHb; medial habenular nucleus; MPB; medial parabrachial nucleus; MS; medial septal nucleus; Pir; piriform cortex; PLCo; posterolateral cortical amygdaloid nucleus; PMCo; posteromedial cortical amygdaloid nucleus; PRh; perirhinal cortex; PrS; presubiculum; PVA; paraventricular thalamic nucleus anterior part; qRT-PCR; quantitative reverse transcription-polymerase chain reaction; S; subiculum; scp; superior cerebellar peduncle; sm; stria medullaris of the thalamus; SNC; substantia nigra pars compacta; Sp5; spinal trigeminal nucleus; Tu; olfactory tubercle; VCP; ventral cochlear nucleus, ventral part; VDB; nucleus of the vertical limb of the diagonal band; VMH; ventromedial hypothalamic nucleus; VTA; ventral tegmental area; VTg; ventral tegmental nucleusConstitutive activity; Orphan receptor; Site-directed mutagenesis; In situ hybridization

Evaluation of the antioxidant effects of carotenoids from Deinococcus radiodurans through targeted mutagenesis, chemiluminescence, and DNA damage analyses by Bing Tian; Zhenjian Xu; Zongtao Sun; Jun Lin; Yuejin Hua (pp. 902-911).

Deinococcus radiodurans is highly resistant to reactive oxygen species (ROS). The antioxidant effect of carotenoids in D. radiodurans was investigated by using a targeted mutation of the phytoene synthase gene to block the carotenoid synthesis pathway and by evaluating the survival of cells under environmental stresses. The colorless mutant R1Δ crtB of D. radiodurans failed to synthesize carotenoids, and was more sensitive to ionizing radiation, hydrogen peroxide, and desiccation than the wild type, suggesting that carotenoids in D. radiodurans help in combating environmental stresses. Chemiluminescence analyses showed that deinoxanthin, a major product in the carotenoid synthesis pathway, had significantly stronger scavenging ability on H₂O₂ and singlet oxygen than two carotenes (lycopene and β-carotene) and two xanthophylls (zeaxanthin and lutein). Deinoxanthin also exhibited protective effect on DNA. Our findings suggest that the stronger antioxidant effect of deinoxanthin contribute to the resistance of D. radiodurans. The higher antioxidant effect of deinoxanthin may be attributed to its distinct chemical structure which has an extended conjugated double bonds and the presence of a hydroxyl group at C-1′ position, compared with other tested carotenoids.

Keywords: Deinococcus radiodurans; Reactive oxygen species; Antioxidant effect; Carotenoids; Phytoene synthase (CrtB); Deinoxanthin

Feasibility study using surface-enhanced Raman spectroscopy for the quantitative detection of tyrosine and serine phosphorylation by J. Moger; P. Gribbon; A. Sewing; C.P. Winlove (pp. 912-918).

We investigate the feasibility of colloid-based surface enhanced Raman scattering (SERS) as a highly sensitive technique for detecting peptide phosphorylation at serine and tyrosine residues. Using the recently reported drop-coating deposition Raman method we validate our SERS spectra against normal Raman spectra that would otherwise be unobtainable at such low concentrations. Compared with existing techniques for quantifying peptide phosphorylation, such as high-performance liquid chromatography (HPLC), the short scanning and processing time associated with SERS makes it an attractive alternative for near-real-time measurement at sub micro-molar concentrations. Following pre-processing by Savistky–Golay second derivative (SGSD), the degree of phosphorylation of synthetic peptides is determined using multivariate spectral classification, interval partial least squares (iPLS). Furthermore, our results show that the technique is robust to interference from complex proteins and other phosphorylated compounds present at concentrations typically found in a screening assay.

Keywords: Surface enhanced Raman scattering; Peptide; Phosphorylation; Serine; Tyrosine

Feasibility study using surface-enhanced Raman spectroscopy for the quantitative detection of tyrosine and serine phosphorylation by J. Moger; P. Gribbon; A. Sewing; C.P. Winlove (pp. 912-918).

Keywords: Surface enhanced Raman scattering; Peptide; Phosphorylation; Serine; Tyrosine

Interactions of enolase isoforms with tubulin and microtubules during myogenesis by A. Keller; J. Peltzer; G. Carpentier; I. Horváth; J. Oláh; A. Duchesnay; F. Orosz; J. Ovádi (pp. 919-926).

Enolase is a glycolytic enzyme, expressed as cell-type specific isoforms in higher vertebrates. Herein we demonstrated for the first time that enolase isoforms interact with microtubules during muscle satellite cell differentiation. While in undifferentiated myoblasts the ubiquitous αα enolase isoform, expressed at high level, exhibited extensive co-localization with microtubules, the muscle-specific ββ isoform, expressed at low level, did not. During differentiation, the level of β subunit increased significantly; the α and β enolase immunoreactivities were detected both in cytosol and along the microtubules. We identified tubulin from muscle extract as an interacting protein for immobilized ββ enolase. ELISA and surface plasmon resonance measurements demonstrated the direct binding of enolase isoforms to tubulin with an apparent K_D below the micromolar range, and indicated that the presence of 0.8 mM 2-phosphoglycerate abolished the interaction. Our data showed that, at various stages of myogenic differentiation, microtubules were decorated by different enolase isoforms, which was controled by the abundance of both partners. We suggest that the binding of enolase to microtubules could contribute to the regulation of the dynamism of the cytoskeletal filaments known to occur during the transition from myoblast to myotubes.

Keywords: Confocal microscopy; Enolase isoform; Microtubule; Tubulin; Glycolysis; Myogenesis

Interactions of enolase isoforms with tubulin and microtubules during myogenesis by A. Keller; J. Peltzer; G. Carpentier; I. Horváth; J. Oláh; A. Duchesnay; F. Orosz; J. Ovádi (pp. 919-926).

Keywords: Confocal microscopy; Enolase isoform; Microtubule; Tubulin; Glycolysis; Myogenesis

Phosphatidylinositol 3-kinase regulation of gastrin-releasing peptide-induced cell cycle progression in neuroblastoma cells by Titilope A. Ishola; JungHee Kang; Jingbo Qiao; B. Mark Evers; Dai H. Chung (pp. 927-932).

Gastrin-releasing peptide (GRP), the mammalian equivalent of bombesin (BBS), is an autocrine growth factor for neuroblastoma; its receptor is up-regulated in undifferentiated neuroblastomas. Phosphatidylinositol 3-kinase (PI3K) is a critical cell survival pathway; it is negatively regulated by the PTEN tumor suppressor gene. We have recently found that poorly differentiated neuroblastomas express decreased PTEN protein levels. Moreover, overexpression of the GRP receptor, a member of the G-protein coupled receptor family, down-regulates PTEN expression, resulting in increased neuroblastoma cell growth. Therefore, we sought to determine whether GRP or BBS activates PI3K in neuroblastoma cells (BE(2)-C, LAN-1, SK-N-SH). GRP or BBS treatment rapidly increased phosphorylation of Akt and GSK-3β in neuroblastoma cells. Inhibition of GRP receptor, with antagonist GRP-H2756 or siRNA, attenuated BBS-induced phosphorylation of Akt. LY294002, a PI3K inhibitor, also abrogated BBS-stimulated phospho-Akt as well as its cell cycle targets. GRP increased G1/S phase progression in SK-N-SH cells. BBS-mediated BrdU incorporation was blocked by LY294002. Our findings identify PI3K as an important signaling pathway for GRP-mediated neuroblastoma cell growth. A novel therapy targeted at GRP/GRP receptor may prove to be an effective treatment option to inhibit PI3K in neuroblastomas.

Keywords: PI3K; Akt; Cell cycle; GRP; Bombesin; Neuroblastoma

Formation of the molten globule-like state during prolonged glycation of human serum albumin by Naghmeh Sattarahmady; Ali A. Moosavi-Movahedi; Faizan Ahmad; Gholam H. Hakimelahi; Mehran Habibi-Rezaei; Ali A. Saboury; Nader Sheibani (pp. 933-942).

The interaction of reducing carbohydrates with proteins leads to a cascade of reactions that are known as glycation or Maillard reaction. We studied the impact of incubation of human serum albumin (HSA) with glucose, at various concentrations and incubation times, on the extent of HSA glycation and structural changes using circular dichroism (CD), fluorescence, and microviscometer techniques. The number of moles of glucose bound per mole of HSA ( r), the number of reacted lysine and arginine residues, and the Amadori product formation during glycation were determined using 3-(dansylamino) phenyl boronic acid, fluorescamine, 9, 10 phenanthrenequinone, and p-nitroblue tetrazoliumchloride, respectively. The formation of advanced glycation end products (AGE) was detected using the autofluorescence characteristic of samples. We identified three stages of Maillard reaction for HSA upon incubation with the physiological level of glucose (0–630 mg/dl): the early, intermediate and late stages, which occurred after 7–14, 21, and >28 days of incubation, respectively. Structural information, Stokes radius, and 1-anilinonaphthalene-8-sulfonate (ANS) binding data indicated the formation of a molten globule-like state of HSA after 21 days of incubation with 35 mM (630 mg/dl) glucose. Thus, the extent of the Maillard reaction was influenced by the concentration of glucose and incubation time, such that longer exposure of HSA to glucose may have a more deleterious effect on its structure and especially on its half-life and turnover in the circulation. Our results suggest that in acute diabetes mellitus patients, HSA, after 21 days of glycation, passes through a molten globule-like state and may contribute to the pathogenesis of diabetes, and perhaps other diseases.

Keywords: Abbreviations; AGE; Advanced Glycation End Product; HSA; Human Serum Albumin; DnsPBA; 3-(Dansylamino) phenyl boronic acid; ANS; 1-anilinonaphthalene-8-sulfonate; NBT; p; -nitroblue tetrazoliumchlorideAGE; Diabetes; Glycation; Human serum albumin; Molten globule

Molecularly imprinted poly β-cyclodextrin polymer: Application in protein refolding by Mohammad Ali Esmaeili; Razieh Yazdanparast (pp. 943-950).

Regarding our previous report on refolding of alkaline phosphatase [Yazdanparast and Khodagholi, 2005 Arch. Biochem. Biophys] it was found that in spite of the anti-aggregatory effect of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), a zwitteronic detergent, the recovered activity was almost the same as the recovered activity obtained through the unassisted approach. The low recovery yield is probably due to the bulky groups of the detergent that interfere with its entrance into the small cavity of the stripping agent, cyclodextrin, implying that the stripping of detergent molecules from the detergent–protein complexes plays a major role in successful refolding processes. To improve the efficiency of CHAPS stripping, we evaluated, for the first time, the stripping potential of a molecular imprinting polymer designed to replace β-CD. In this approach, CHAPS was used as the template and the refolding of GuHCl denatured alkaline phosphatase was studied. Our results indicated that under the optimally developed refolding environment and similar to stripping by soluble β-CD, a refolding yield of 79% was obtained for denatured alkaline phosphatase using 20 mg/ml of the molecularly imprinted poly (β-CD) polymer. The major advantage of the new stripping agent, besides of its recycling option and ease of separation from the finished product, is its high potential of preventing aggregate formation. Based on these results, it seems that the new stripping strategy can constitute an ideal approach for refolding of proteins at much lower industrial costs compared to stripping with soluble β-cyclodextrin.

Keywords: Abbreviations; ALP; Alkaline phosphatase; ANS; 1-anilinonaphtalene-8-sulfonate; β-CD; β-cyclodextrin; CHAPS; 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; CTAB; cetyltrimethylammonium bromide; pNPP; p-nitrophenyl phosphate; GuHCl; Guanidine hydrochloride; MIP; molecularly imprinted poly (β-CD); THF; tetrahydrofuran; TDI; Toluene 2, 4-diisocyanateAlkaline phosphatase; β-cyclodextrin; CHAPS; Molecularly imprinted poly (β-CD) polymer; Refolding Kinetic

Molecularly imprinted poly β-cyclodextrin polymer: Application in protein refolding by Mohammad Ali Esmaeili; Razieh Yazdanparast (pp. 943-950).

Identification of Vanabin-interacting protein 1 (VIP1) from blood cells of the vanadium-rich ascidian Ascidia sydneiensis samea by Tatsuya Ueki; Koki Shintaku; Yuki Yonekawa; Nariaki Takatsu; Hiroshi Yamada; Toshiyuki Hamada; Hiroshi Hirota; Hitoshi Michibata (pp. 951-957).

Several species of ascidians, the so-called tunicates, accumulate extremely high levels of vanadium ions in their blood cells. We previously identified a family of vanadium-binding proteins, named Vanabins, from blood cells and blood plasma of a vanadium-rich ascidian, Ascidia sydneiensis samea. The 3-dimensional structure of Vanabin2, the predominant vanadium-binding protein in blood cells, has been revealed, and the vanadium-binding properties of Vanabin2 have been studied in detail. Here, we used Far Western blotting to identify a novel protein that interacts with Vanabin2 from a blood cell cDNA library. The protein, named Vanabin-interacting protein 1 (VIP1), was localized in the cytoplasm of signet ring cells and giant cells. Using a two-hybrid method, we revealed that VIP1 interacted with Vanabins 1, 2, 3, and 4 but not with Vanabin P. The N-terminal domain of VIP1 was shown to be important for the interaction. Further, Vanabin1 was found to interact with all of the other Vanabins. These results suggest that VIP1 and Vanabin1 act as metal chaperones or target proteins in vanadocytes.

Keywords: Vanadium; Ascidian; Metal-binding protein; Protein-protein interaction

Flavonoids and their oxidation products protect efficiently albumin-bound linoleic acid in a model of plasma oxidation by Claire Dufour; Michèle Loonis (pp. 958-965).

Although LDL esterified polyunsaturated fatty acids (PUFA) contribute largely to the pool of oxidizable lipids in plasma, they coexist with a non-negligible content of free PUFA. In some pathological conditions, the free PUFA/albumin ratio becomes abnormally elevated. Modeling was performed in a system constituted of linoleic acid bound to human serum albumin (HSA) in which oxidation was initiated by hydrophilic AAPH. Inhibition of lipid peroxidation was evaluated for various flavonoids. The accumulations of hydroperoxyoctadecadienoic acids (HPODE), hydroxyoctadecadienoic acids (HODE) and ketooctadecadienoic acids (KODE) were similarly inhibited : isoquercitrin>quercetin>catechin=isorhamnetin≫kaempferol>quercetin-4′-β-d-glucoside=quercetin-3,4′-di-β-d-glucoside. Surprisingly, quercetin and isorhamnetin afforded a protection to linoleic acid long after their consumption. Elucidation by mass spectrometry and NMR of the quercetin oxidation products and assessment of their antioxidant capacity pointed out that 3,4-dihydroxybenzoic acid and 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2 H)-one are major contributors to the apparent quercetin antioxidant capacity.

Keywords: Abbreviations; HSA; human serum albumin; LA; linoleic acid; HPODE; hydroperoxyoctadecadienoic acid; HODE; hydroxyoctadecadienoic acid; KODE; ketooctadecadienoic acidSerum albumin; Flavonoid; Lipid peroxidation; Octadecadienyl hydroperoxide; Antioxidant; 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2; H; )-one

Flavonoids and their oxidation products protect efficiently albumin-bound linoleic acid in a model of plasma oxidation by Claire Dufour; Michèle Loonis (pp. 958-965).

Inhibitory effect of NPY on isoprenaline-induced osteoclastogenesis in mouse bone marrow cells by Shinobu Amano; Michitsugu Arai; Shigemi Goto; Akifumi Togari (pp. 966-973).

The effect of neuropeptide Y (NPY), a co-transmitter with noradrenaline in peripheral sympathetic nerve fibers, on the osteoclastogenesis in mouse bone marrow cell cultures treated with isoprenaline, a β-adrenergic receptor (β-AR) agonist, was examined. The mouse bone marrow cells constitutively expressed mRNAs for the NPY-Y1 receptor and β2-AR. NPY inhibited the formation of osteoclast-like cells induced by isoprenaline but not that by 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) or soluble receptor activator of nuclear factor-κB ligand (RANKL); and it suppressed the production of RANKL and cyclic AMP (cAMP) increased by isoprenaline but not those increased by 1α,25(OH)₂D₃. NPY also inhibited osteoclastogenesis induced by forskolin, an activator of adenylate cyclase; however, it did not inhibit that induced by exogenously supplied dibutyryl cAMP, a cell-permeable cAMP analog that activates cAMP-dependent protein kinase. These results demonstrate that NPY inhibited the isoprenaline-induced osteoclastogenesis by blocking the agonist-elicited increases in the production of cAMP and RANKL in mouse bone marrow cells, suggesting an interaction between NPY and β-AR agonist in bone resorption.

Keywords: Neuropeptide Y (NPY); Isoprenaline; Osteoclastogenesis; Mouse bone marrow cell

Inhibitory effect of NPY on isoprenaline-induced osteoclastogenesis in mouse bone marrow cells by Shinobu Amano; Michitsugu Arai; Shigemi Goto; Akifumi Togari (pp. 966-973).

Keywords: Neuropeptide Y (NPY); Isoprenaline; Osteoclastogenesis; Mouse bone marrow cell

A putative helical cytokine functioning in innate immune signalling in Drosophila melanogaster by D. Malagoli; D. Conklin; S. Sacchi; M. Mandrioli; E. Ottaviani (pp. 974-978).

In invertebrates and vertebrates, innate immunity is considered the first line of defense mechanism against non-self material. In vertebrates, cytokines play a critical role in innate immune signalling. To date, however, the existence of genes encoding for invertebrate helical cytokines has been anticipated, but never demonstrated. Here, we report the first structural and functional evidence of a gene encoding for a putative helical cytokine in Drosophila melanogaster. Functional experiments demonstrate that its expression, as well as that of the antimicrobial factors defensin and cecropin A1, is significantly increased after immune stimulation. These observations suggest the involvement of helical cytokines in the innate immune response of invertebrates.

Keywords: Helical cytokine; Insect; Immunity; Antimicrobial peptides

A putative helical cytokine functioning in innate immune signalling in Drosophila melanogaster by D. Malagoli; D. Conklin; S. Sacchi; M. Mandrioli; E. Ottaviani (pp. 974-978).

Keywords: Helical cytokine; Insect; Immunity; Antimicrobial peptides

Role of plasma and liver cholesterol- and lipoprotein-metabolism determinants in LpX formation in the mouse by Ignacio Bravo; Ludwig Amigo; David E. Cohen; Flavio Nervi; Attilio Rigotti; Omar Francone; Silvana Zanlungo (pp. 979-988).

Cholestasis is characterized by hypercholesterolemia and the appearance of an abnormal lipoprotein, lipoprotein X (LpX), in plasma. The mechanisms responsible for this cholestatic plasma lipid phenotype are not fully understood. We used ATP-binding cassette A1 (ABCA1)(−/−) and scavenger receptor class B type I (SR-BI)(−/−) mice to test the hypothesis that hepatic sinusoidal cholesterol transporters contribute to LpX formation and hypercholesterolemia during cholestasis. Bile-duct ligation (BDL) of both ABCA1(−/−) and SR-BI(−/−) mice, as well as their respective controls, induced a dramatic increase in plasma cholesterol and phospholipid concentrations. Plasma fractionation revealed the presence of LpX in plasma of cholestatic mice, irrespective of their genetic background. We observed that the presence of HDL before cholestasis, a decrease in the activity of LCAT, and an increase in VLDL synthesis were not required for hypercholesterolemia and lipoprotein modifications induced by obstructive cholestasis in mice. In addition, murine cholestasis resulted in increased hepatic cholesterol synthesis that may contribute to the higher plasma free cholesterol levels found during the early hours after BDL. Together these findings indicate that hypercholesterolemia and LpX formation associated with obstructive cholestasis are correlated with an increase in hepatic cholesterol synthesis and are independent of plasma HDL levels, LCAT activity, VLDL synthesis, and ABCA1 and SR-BI expression.

Keywords: Abbreviations; LpX; lipoprotein X; BDL; bile-duct ligation; ABCA1; ATP-binding cassette A1; cyp7A1; cholesterol 7α-hydroxylase; SR-BI; scavenger receptor class B type I; IDL; intermediate-density lipoprotein; MTTP; microsomal triglyceride transfer protein; pI–pC; polyinosinic/polycytidylic ribonucleic acidCholestasis; Hypercholesterolemia; Lipoprotein X

Curcumin protects against acute liver damage in the rat by inhibiting NF-κB, proinflammatory cytokines production and oxidative stress by Karina Reyes-Gordillo; José Segovia; Mineko Shibayama; Paula Vergara; Mario G. Moreno; Pablo Muriel (pp. 989-996).

Curcumin, an anti-inflammatory and antioxidant compound, was evaluated for its ability to suppress acute carbon tetrachloride-induced liver damage. Acute hepatotoxicity was induced by oral administration of CCl₄ (4 g/kg, p.o.). Curcumin treatment (200 mg/kg, p.o.) was given before and 2 h after CCl₄ administration. Indicators of necrosis (alanine aminotransferase) and cholestasis (γ-glutamyl transpeptidase and bilirubins) resulted in significant increases after CCl₄ intoxication, but these effects were prevented by curcumin treatment. As an indicator of oxidative stress, GSH was oxidized and the GSH/GSSG ratio decreased significantly by CCl₄, but was preserved within normal values by curcumin. In addition to its antioxidants properties, curcumin is capable of preventing NF-κB activation and therefore to prevent the secretion of proinflammatory cytokines. Therefore, in this study we determined the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) mRNA, and NF-κB activation. CCl₄-administered rats depicted significant increases in TNF-α, IL-1β, and IL-6 production, while curcumin remarkably suppressed these mediators of inflammation in liver damage. These results were confirmed by measuring TNF-α, and IL-1β protein production using Western Blot analysis. Accordingly, these proteins were increased by CCl₄ and this effect was abolished by curcumin. Administration of CCl₄ induced the translocation of NF-κB to the nucleus; CCl₄ induced NF-κB DNA binding activity was blocked by curcumin treatment. These findings suggest that curcumin prevents acute liver damage by at least two mechanisms: acting as an antioxidant and by inhibiting NF-κB activation and thus production of proinflammatory cytokines.

Keywords: Curcumin; Cytokines; Liver injury; Carbon tetrachloride; Necrosis; NF-κB.

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