BBA - General Subjects (v.1780, #10)

Intestinal epithelial CD98: An oligomeric and multifunctional protein by Yutao Yan; Sona Vasudevan; Hang Thi Thu Nguyen; Didier Merlin (1087-1092).
The intestinal epithelial cell-surface molecule, CD98 is a type II membrane glycoprotein. Molecular orientation studies have demonstrated that the C-terminal tail of human CD98 (hCD98), which contains a PDZ-binding domain, is extracellular. In intestinal epithelial cells, CD98 is covalently linked to an amino-acid transporter with which it forms a heterodimer. This heterodimer associates with β1-integrin and intercellular adhesion molecular 1 (ICAM-1) to form a macromolecular complex in the basolateral membranes of polarized intestinal epithelial cells. This review focuses on the multifunctional roles of CD98, including involvement in extracellular signaling, adhesion/polarity, and amino-acid transporter expression in intestinal epithelia. A role for CD98 in intestinal inflammation, such as Intestinal Bowel Disease (IBD), is also proposed.
Keywords: Intestinal epithelial CD98; Extracellular PDZ-binding domain; Adhesion; Cell polarity; Amino-acid transporter; Intestinal inflammation;

Age-related upregulation of Drosophila caudal gene via NF-κB in the adult posterior midgut by Yoon-Jeong Choi; Mi-Sun Hwang; Joung-Sun Park; Soo-Kyung Bae; Young-Shin Kim; Mi-Ae Yoo (1093-1100).
The Drosophila midgut has emerged as a powerful model system for the investigation of fundamental cellular pathways relevant to intestinal stem cell biology. Understanding the age-related changes in the adult Drosophila midgut may provide insights into the molecular mechanisms that link aging to the modulation of adult stem cell population. The caudal-related homeobox genes encode intestine-specific transcription factors required for normal intestinal development and maintenance. Here, we demonstrate that caudal gene expression is upregulated in the adult posterior midgut in response to age and oxidative stress, and that overexpression of Caudal can stimulate cell proliferation in the adult posterior midgut. We further demonstrate that the age- and oxidative-stress-related upregulation of the caudal gene is mediated by the NF-κB binding site located in the 5′-flanking region of the caudal gene. Our results may contribute to an understanding of the mechanisms of age-related changes in the number and activity of intestinal stem cells and progenitors in the Drosophila adult midgut.
Keywords: caudal; Aging; Oxidative stress; NF-κB; Proliferation; Drosophila gut;

Effects of peroxisome proliferator-activated receptor α (PPARα) agonists on leucine-induced phosphorylation of translational targets in C2C12 cells by Naoya Nakai; Fuminori Kawano; Masahiro Terada; Yoshihiko Oke; Takashi Ohira; Yoshinobu Ohira (1101-1105).
Effect of peroxisome proliferator-activated receptor α (PPARα) agonists, WY-14,643 (WY) and/or clofibrate, on the leucine-induced phosphorylation of translational targets in C2C12 myoblasts was studied. C2C12 cells were treated with WY or clofibrate for 24 h prior to stimulation with leucine. Western blot analyses revealed that the leucine-induced phosphorylation of p70 S6 kinase (p70S6K), a key regulator of translation initiation, was significantly higher in WY-treated cells than in control and clofibrate-treated cells. Phosphorylation of extracellular-regulated kinase (ERK1/2) was higher in WY-treated cells. WY treatment also increased the leucine-induced phosphorylation of ribosomal protein S6 and eukaryotic initiation factor 4B. In contrast, eukaryotic elongation factor 2, a marker for peptide chain elongation process, was significantly activated (dephosphorylated) only in leucine-stimulated control cells. Pre-treatment of the cells with PD98059 (ERK1/2 kinase inhibitor) prevented the phosphorylation of ERK1/2 and decreased the leucine-induced phosphorylation of p70S6K. It is concluded that WY increased the leucine-induced phosphorylation of target proteins involving in translation initiation via ERK/p70S6K pathway, but impaired the signaling for elongation process, suggesting that p70S6K phosphorylation may be essential, but not sufficient for the activation of entire targets for protein translation in WY-treated cells.
Keywords: Peroxisome proliferator-activated receptor α; WY-14,643; Myoblast; Leucine; Protein translation initiation and elongation;

Vanadium and 1, 25 (OH)2 vitamin D3 combination in inhibitions of 1,2, dimethylhydrazine-induced rat colon carcinogenesis by Shaonly Samanta; Mary Chatterjee; Balaram Ghosh; M. Rajkumar; Ajay Rana; Malay Chatterjee (1106-1114).
The current investigation demonstrates the antitumor effects of combined supplementations of vanadium (V) (4.27 µmol/L drinking water ad libitum) and1α, 25-dihydroxy vitamin D3 (Vitamin D3) (0.3 μg/100 μL propylene glycol per os twice a week) on 1, 2 dimethylhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. There was a significant reduction in incidence (70%), multiplicity (P  < 0.0001) and volume (P  < 0.01) of colon tumors. HPLC-fluorescence assay detected the combinatorial actions of V and Vitamin D3 against DMH-induced colonic O 6-methylguanine DNA adducts formation (at four sequential time points; ANOVA, F = 13.56, P  < 0.01). Simultaneous inhibition of DNA single strand breaks (P  < 0.001) indicates the potency of the combination regimen in limiting the initiation event of colon carcinogenesis. Immunohistochemical analysis revealed that the effect of V and vitamin D3 occurred through suppression of cell proliferation (BrdU-LI: P  < 0.001) along with an induction of apoptosis (TUNEL-LI: P  < 0.01). The immunoexpression of tumor suppressor p53 and downregulation of antiapoptotic protein BCl-2 in subsequent immunofluorescence assay further provide strong evidence for the combinatorial inhibitory actions of vanadium and vitamin D3 against DMH-induced rat colon carcinogenesis.
Keywords: Vanadium; 1, 25 (OH)2 vitamin D3; DMH; DNA adduct; Cell proliferation;

Branched-chain amino acids and arginine suppress MaFbx/atrogin-1 mRNA expression via mTOR pathway in C2C12 cell line by Elizabeth Henny Herningtyas; Yasuhiko Okimura; Anastasia Evi Handayaningsih; Daisuke Yamamoto; Taiki Maki; Keiji Iida; Yutaka Takahashi; Hidesuke Kaji; Kazuo Chihara (1115-1120).
The effect of amino acid on muscle protein degradation remains unclear. Recent studies have elucidated that proteolysis in catabolic conditions occurs through ubiquitin–proteasome proteolysis pathway and that muscle-specific ubiquitin ligases (atrogin-1 and MuRF1) play an important role in protein degradation. In the present study, we examined the direct effect of 5 mM amino acids (leucine, isoleucine, valine, glutamine and arginine) on atrogin-1 and MuRF1 levels in C2C12 muscle cells and the involved intracellular signal transduction pathway. Leucine, isoleucine and valine suppressed atrogin-1 and MuRF1 mRNA levels (≈ 50%) at 6 and 24 h stimulations. Arginine showed a similar effect except at 24 h-treatment for atrogin-1 mRNA. However, glutamine failed to reduce atrogin-1 and MuRF1 mRNA levels. The inhibitory effect of leucine, isoleucine or arginine on atrogin-1 mRNA level was reversed by rapamycin, although wortmannin did not reverse the effect. PD98059 and HA89 reduced basal atrogin-1 level without influencing the inhibitory effects of those amino acids. The inhibitory effect of leucine, isoleucine or arginine on MuRF1 mRNA levels was not reversed by rapamycin. Taken together, these findings indicated that leucine, isoleucine and arginine decreased atrogin-1 mRNA levels via mTOR and that different pathways were involved in the effect of those amino acids on MuRF1 mRNA levels.
Keywords: Atrogin-1; MuRF1; BCAA; Arginine; mTOR;

Import of hybrid forms of CYP11A1 into yeast mitochondria by A.N. Minenko; L.A. Novikova; V.N. Luzikov; I.E. Kovaleva (1121-1130).
Heterologous expression in yeast of mCYP11A1 fusions with different topogenic signals of yeast mitochondrial proteins for artificial channeling to different translocases of the inner membrane was used to gain insight in the mechanism of its topogenesis in mitochondria. To ensure insertion of the CYP11A1 domain into the inner mitochondrial membrane during the process of translocation, topogenic sequences containing transmembrane segments of Bcs1p(1–83), DLD(1–72), and full-sized AAC protein were used when constructing modified forms of CYP11A1, and the Su9(1–112) addressing signal was included to stimulate membrane insertion of CYP11A1 after its translocation to the matrix. Alternatively, to promote slippage of the hybrid molecules into the matrix, the hybrid of mCYP11A1 with the precursor of steroidogenic mitochondria matrix protein adrenodoxin (preAd) was designed. The extra sequences used for intramitochondrial sorting of CYP11A1 apparently ensured predicted topology of hybrid molecules in yeast mitochondria. All of the addressing sequences, containing transmembrane domains, provided effective insertion of the hybrid proteins AAC-mCYP11A1, Bcs1p(1–83)-mCYP11A1, DLD(1–72)-mCYP11A1 and Su9(1–116)-mCYP11A1 into the inner membrane. preAd-mCYP11A1 hybrid molecules were shown to be translocated across the inner membrane and tightly associated with the membrane on its matrix side but not membrane inserted. Measuring specific activities of hybrid proteins in the mitochondrial fractions upon addition of Ad and AdR showed that the hybrids predetermined for cotranslocational insertion of CYP11A1 into the inner membrane were more active in the reaction of cholesterol side-chain cleavage than those destined for insertion on the matrix side of the IM, the Ad-mCYP11A1 hybrid demonstrating only residual enzyme activity. The data obtained reinforce the proposal that complete transfer of the polypeptide chain into the matrix is not a necessary stage in its topogenesis, but rather persistent interaction of the polypeptide chain with the membrane during the process of translocation is of importance for heme binding, folding and membrane insertion.
Keywords: CYP11A1; Topogenic signal; Yeast mitochondria; Membrane insertion;

Caenorhabditis elegans galectins LEC-1–LEC-11: Structural features and sugar-binding properties by Yoko Nemoto-Sasaki; Ko Hayama; Hiroyuki Ohya; Yoichiro Arata; Mika Kato Kaneko; Naruya Saitou; Jun Hirabayashi; Ken-ichi Kasai (1131-1142).
Galectins form a large family of β-galactoside-binding proteins in metazoa and fungi. This report presents a comparative study of the functions of potential galectin genes found in the genome database of Caenorhabditis elegans. We isolated full-length cDNAs of eight potential galectin genes (lec-25 and 811) from a λZAP cDNA library. Among them, lec-2–5 were found to encode 31–35-kDa polypeptides containing two carbohydrate-recognition domains similar to the previously characterized lec-1, whereas lec-8–11 were found to encode 16–27-kDa polypeptides containing a single carbohydrate-recognition domain and a C-terminal tail of unknown function. Recombinant proteins corresponding to lec-1–4, -6, and 810 were expressed in Escherichia coli, and their sugar-binding properties were assessed. Analysis using affinity adsorbents with various β-galactosides, i.e., N-acetyllactosamine (Galβ1-4GlcNAc), lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and asialofetuin, demonstrated that LEC-1–4, -6, and -10 have a significant affinity for β-galactosides, while the others have a relatively lower affinity. These results indicate that the integrity of key amino acid residues responsible for recognition of lactose (Galβ1-4Glc) or N-acetyllactosamine in vertebrate galectins is also required in C. elegans galectins. However, analysis of their fine oligosaccharide-binding properties by frontal affinity chromatography suggests their divergence towards more specialized functions.
Keywords: Caenorhabditis elegans; Galectin; Lectin; Frontal affinity chromatography; Carbohydrate-binding property;

Tryptophan quenching as linear sensor for oxygen binding of arthropod hemocyanins by Wolfgang Erker; Rüdiger Hübler; Heinz Decker (1143-1147).
Oxygen binding of hemocyanins results in an absorption band around 340nm and a strong quenching of the intrinsic tryptophan fluorescence. Our study analyses in detail the fluorescence quenching within two hemocyanins, a hexamer (Panulirus interruptus) and a 4 × 6-mer (Eurypelma californicum). Based on the comparison of calculated and measured transfer efficiencies we could show that: (1) For both hemocyanins FRET (fluorescence resonance energy transfer) is exclusively responsible for quenching of the tryptophan fluorescence upon oxygen binding. (2) Tryptophan quenching by FRET is independent of the oxy- or deoxy conformation of the protein. (3) The quenching takes place at the subunit level only and the oligomerization of both hemocyanins has no influence on the amount of quenching. Therefore, tryptophan fluorescence is a linear sensor for bound oxygen. It can be used as a model-free signal to investigate oxygen binding of hemocyanins at all aggregation levels. Furthermore it may provide a new way to analyse oxygen binding of phenoloxidases.
Keywords: Fluorescence; Tarantula; Spiny lobster; Energy transfer; FRET; phenoloxidase;

Hypotonic stress (HTS) induces various responses in vascular endothelium, but the molecules involved in sensing HTS are not known. To investigate a possible role of heparan sulfate proteoglycan (HSPG) in sensing HTS, we compared the responses of control bovine aortic endothelial cells (BAECs) with those of cells treated with heparinase III, which exclusively degrades HSPG. Tyrosine phosphorylation of 125 kDa FAK induced by HTS (− 30%) in control cells was abolished in heparinase III-treated BAECs. The amplitude of the volume-regulated anion channel (VRAC) current, whose activation is regulated by tyrosine kinase, was significantly reduced by the treatment with heparinase III. Also, HTS-induced ATP release through the VRAC pore and the concomitant Ca2+ transients were significantly reduced in the heparinase III-treated BAECs. In contrast, exogenously applied ATP evoked similar Ca2+ transients in both control and heparinase III-treated BAECs. The transient formation of actin stress fibers induced by HTS in control cells was absent in heparinase III-treated BAECs. Lysophosphatidic acid (LPA) also induced FAK phosphorylation, actin reorganization and ATP release in control BAECs, but heparinase III did not affect these LPA-induced responses. We conclude from these observations that HSPG is one of the sensory molecules of hypotonic cell swelling in BAECs.
Keywords: Hyposmotic shock; Mechanosensitivity; ATP release; Actin; Anion channel; Tyrosine kinase;

Disintegration of amyloid fibrils of α-synuclein by dequalinium by Jae-Woo Park; In-Hwan Lee; Ji-Sook Hahn; Jongsun Kim; Kwang Chul Chung; Seung R. Paik (1156-1161).
α-Synuclein is the major amyloidogenic component observed in the Lewy bodies of Parkinson's disease. Amyloid fibrils of α-synuclein prepared in vitro were instantaneously disintegrated by dequalinium (DQ). Double-headed cationic amphipathic structure of DQ with two aminoquinaldinium rings at both ends turned out to be crucial to exert the disintegration activity. The defibrillation activity was shown to be selective toward the fibrils of α-synuclein and Aβ40 while the other β2-microglobulin amyloid fibrils were not susceptible so much. Besides the common cross β-sheet conformation of amyloid fibrils, therefore, additional specific molecular interactions with the target amyloidogenic proteins have been expected to be involved for DQ to exhibit its defibrillation activity. The disintegrating activity of DQ was also evaluated in vivo with the yeast system overexpressing α-synuclein-GFP. With the DQ treatment, the intracellular green inclusions turned into green smears, which resulted in the enhanced cell death. Based on the data, the previous observation that DQ led to the predominant protofibril formation of α-synuclein could be explained by the dual function of DQ showing both the facilitated self-oligomerization of α-synuclein and the instantaneous defibrillation of its amyloid fibrils. In addition, amyloidosis-related cytotoxicity has been demonstrated to be amplified by the fragmentation of mature amyloid fibrils by DQ.
Keywords: α-Synuclein; Amyloid fibril formation; Defibrillation; Dequalinium; Cytotoxicity;

Effects of new amphotericin analogues on the scrapie isoform of the prion protein by Lara Soler; Patrick Caffrey; Hilary E.M. McMahon (1162-1167).
Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of neurodegenerative disorders associated with the conversion of a normal host prion protein (PrPC) into a pathogenic isoform (PrPSc). Despite years of research, there is still no known cure for TSEs. Amphotericin B (AmB), an anti-fungal antibiotic, has antiprion activity but its usage is limited by its toxicity. This study assessed the antiprion properties of new amphotericin analogues in which the exocyclic carboxyl groups were replaced by methyl groups. These analogues reduced levels of the abnormal PrPSc isoform of the mouse prion protein in cultured cells. 16-descarboxyl-16-methyl-amphotericin B (16B) had antiprion activity equivalent to that of amphotericin B and was significantly less toxic to cells as determined by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide dye reduction assay. A non-anti-fungal analogue, 16-descarboxyl-16-methyl-19-O-(6-deoxyhexosyl)-19-O-desmycosaminyl-amphotericin (16-19B) had higher antiprion activity and significantly lower toxicity than AmB. Some of the new amphotericin analogues may have potential as antiprion drugs.
Keywords: Amphotericin; Analogues; Prion; Scrapie;