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BBA - General Subjects (v.1790, #5)

Editorial Board (pp. i).

Marine pharmacology in 2005–6: Marine compounds with anthelmintic, antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action by Alejandro M.S. Mayer ⁎; Abimael D. Rodríguez; Roberto G.S. Berlinck; Mark T. Hamann (pp. 283-308).

The review presents the 2005–2006 peer-reviewed marine pharmacology literature, and follows a similar format to the authors' 1998–2004 reviews. The preclinical pharmacology of chemically characterized marine compounds isolated from marine animals, algae, fungi and bacteria is systematically presented.Anthelmintic, antibacterial, anticoagulant, antifungal, antimalarial, antiprotozoal, antituberculosis and antiviral activities were reported for 78 marine chemicals. Additionally 47 marine compounds were reported to affect the cardiovascular, immune and nervous system as well as possess anti-inflammatory effects. Finally, 58 marine compounds were shown to bind to a variety of molecular targets, and thus could potentially contribute to several pharmacological classes.Marine pharmacology research during 2005–2006 was truly global in nature, involving investigators from 32 countries, and the United States, and contributed 183 marine chemical leads to the research pipeline aimed at the discovery of novel therapeutic agents.Continued preclinical and clinical research with marine natural products demonstrating a broad spectrum of pharmacological activity will probably result in novel therapeutic agents for the treatment of multiple disease categories.

Keywords: Drug; Marine; Metabolite; Natural product; Pharmacology; Review; Toxicology

Cellular iron transport by Michael D. Garrick; Laura M. Garrick (pp. 309-325).

Iron has a split personality as an essential nutrient that also has the potential to generate reactive oxygen species. We discuss how different cell types within specific tissues manage this schizophrenia. The emphasis in enterocytes is on regulating the body's supply of iron by regulating transport into the blood stream. In developing red blood cells, adaptations in transport manage the body's highest flux of iron. Hepatocytes buffer the body's stock of iron. Macrophage recycle the iron from effete red cells among other iron management tasks. Pneumocytes provide a barrier to prevent illicit entry that, when at risk of breaching, leads to a need to handle the dangers in a fashion essentially shared with macrophage. We also discuss or introduce cell types including renal cells, neurons, other brain cells, and more where our ignorance, currently still vast, needs to be removed by future research.

Keywords: Divalent metal transporter (DMT1); Ferroportin; Transferrin; Ferric reductase; Iron responsive element (IRE); Iron regulatory protein (IRP)

Embryonic lethality of fortilin-null mutant mice by BMP-pathway overactivation by Yuichi Koide; Tomomi Kiyota; Moltira Tonganunt; Decha Pinkaew; Zhihe Liu; Yoichi Kato; Nongporn Hutadilok-Towatana; Amornrat Phongdara; Ken Fujise ⁎ (pp. 326-338).

Fortilin negatively regulates apoptosis and is overexpressed in cancer. However, the role of fortilin in mammalian development is not clear.In order to evaluate the physiological role of fortilin in vivo, we performed a targeted disruption of the fortilin gene in mice. Fortilin^+/− mice have the ability to survive and exhibit normal growth, while fortilin^−/− mice are embryonically lethal around the 3.5 days post-coital (dpc). Cultured blastocysts from fortilin^+/− embryos undergo normal outgrowth to produce inner cell mass (ICM) and trophoblasts (TB), while ICM of fortilin^−/− embryos either fails to outgrow or prematurely disintegrates. Mouse embryonic fibroblasts (MEF) derived from fortilin^+/− embryos are more susceptible to noxious stimuli than are wild type embryos. It has been consistently shown in Xenopus embryos that the depletion of fortilin's message severely compromises the formation of neural tissue, even in the brain, while overexpression of fortilin induces the partial double body axis in embryos and is capable of blocking BMP4-induced transcription of Vent1, Vent2, and Msx1 genes. This suggests that fortilin is an inhibitor of the BMP pathway. Strikingly, when fortilin levels are reduced by siRNA, BMP4 causes MEF to undergo extensive DNA-fragmentation, while DNA fragmentation is minimal in the presence of fortilin. In addition, BMP4 induces more Msx2 in the absence of fortilin than in its presence. Furthermore, Msx2 overexpression causes MEF to undergo apoptotic cell death.We conclude that in early phase of development, fortilin functions as an inhibitor of the BMP pathway. The presence of fortilin in the very early stages of development is required for the survival of embryos.Abnormalities in the fortilin gene may be associated with early pregnancy loss.

Keywords: Fortilin; Knockout; Targeted gene disruption; Apoptosis; Msx2; BMP

Cell cycle arrest triggered by conjugated eicosapentaenoic acid occurs through several mechanisms including G1 checkpoint activation by induced RPA and ATR expression by Yuko Kumamoto-Yonezawa; Ryohei Sasaki; Yosuke Ota; Yoko Suzuki; Shoji Fukushima; Takahiko Hada; Keisuke Uryu; Kazuro Sugimura; Hiromi Yoshida; Yoshiyuki Mizushina ⁎ (pp. 339-346).

Conjugated eicosapentaenoic acid (cEPA) containing conjugated double bonds, which is prepared by alkaline treatment of eicosapentaenoic acid (EPA), selectively inhibited the activities of both mammalian DNA polymerases (pols) and human DNA topoisomerases (topos).Human colon carcinoma cell line, HCT116, was cultured and performed drug and small interfering RNA (siRNA) treatment, flow cytometry analysis, BrdU incorporation analysis, and western blot analysis.The levels of bromodeoxyuridine (BrdU) incorporation labeling during DNA synthesis were decreased in time- and dose-dependent manners in HCT116 cells, treated with cEPA. The level of chromatin association of RPA70, a subunit of the single-stranded DNA (ssDNA)-binding protein, was increased following cEPA exposure, suggesting that the replication forks were stalled in response to inhibition of replicative pol activity by cEPA in the cells. cEPA also induced the activation of ataxia-telangiectasia and Rad3-related (ATR) protein in HCT116 cells, and activated the G1 checkpoint pathway in the cells, which was down-regulated by a small interfering RNA (siRNA) against ATR protein. Moreover, caffeine, a known ATR kinase inhibitor, abrogated the cEPA-induced G1 checkpoint in HCT116 cells.cEPA could inhibit the activity of replicative pols, such as pols α, δ and ɛ, affect the DNA replication fork including ssDNA, and then activate the G1 checkpoint pathway by the induction of RPA and ATR expression levels in cancer cells.

Keywords: Abbreviations; EPA; eicosapentaenoic acid; cEPA; conjugated EPA; PUFA; polyunsaturated fatty acid; pol; DNA polymerase (EC 2.7.7.7); topo; DNA topoisomerase; ATR; ataxia-telangiectasia mutated- and Rad3-related protein kinase; ATRIP; ATR-interacting protein; RPA; replication protein A; DSB; double strand breaks; Cdk; cyclin-dependent kinase; Chk1; checkpoint kinase 1; Chk2; checkpoint kinase 2; Cyc E; cyclin E; dTTP; 2′-deoxythymidine 5′-triphosphate; siRNA; small interfering RNA; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DI; DNA Index; BrdU; bromodeoxyuridine; dsDNA; double-stranded DNA; ssDNA; single-stranded DNA; CPT; camptothecinConjugated eicosapentaenoic acid (cEPA); DNA polymerase (pol); DNA topoisomerase (topo); Enzyme inhibitor; DNA replication; Cell cycle arrest; Ataxia-telangiectasia mutated- and Rad3-related protein kinase (ATR); Replication protein A (RPA); RNA interference; Cancer chemotherapy agent

Mobility of the conserved glycine 155 is required for formation of the active plasmodial Pdx1 dodecamer by Julia Knöckel; Rositsa Jordanova; Ingrid B. Müller; Carsten Wrenger; Matthew R. Groves ⁎ (pp. 347-350).

Vitamin B6 synthesis requires a functional Pdx1 assembly that is dodecameric in vivo. We have previously shown that mutation of a catalytic lysine in the plasmodial Pdx1 protein results in a protein that is both inactive and hexameric in vitro.Static and dynamic light scattering, circular dichroism, co-purification and enzyme assays are used to investigate the role of a glycine conserved in all Pdx1 family members.Static light scattering indicates that a glycine to alanine mutant is present as a hexamer in vitro. Subsequent circular dichroism experiments demonstrate that a significant change in secondary structure content is induced by this mutation. However, this mutant is still competent to bind and support Pdx2 activity.As the mutated glycine occupies an unrestricted region of the Ramachandran plot the additional stereo-chemical restrictions imposed on alanine residues strongly support our hypothesis that significant structural rearrangement of Pdx1 is required during the transition from hexamer to dodecamer.The presented results demonstrate that reduction in the mobility of this region in Pdx1 proteins is required for formation of the in vivo dodecamer, negatively affecting the activity of Pdx1, opening the possibility of allosteric Pdx1 inhibitors.

Keywords: Abbreviations; CD; circular dichroism; DLS; dynamic light scattering; Pf; Pdx1 or; Pf; Pdx2; P. falciparum; Pdx1 or Pdx2, PLP, pyridoxal 5-phosphate; SLS; static light scatteringMalaria; Pdx1 assembly; Static light scattering; Circular dichroism; Vitamin B6 biosynthesis

Bmi1 deficient neural stem cells have increased Integrin dependent adhesion to self-secreted matrix by Sophia W.M. Bruggeman; Danielle Hulsman; Maarten van Lohuizen ⁎ (pp. 351-360).

Neural cells deficient for Polycomb group (PcG) protein Bmi1 are impaired in the formation and differentiation of high grade glioma, an incurable cancer of the brain. It was shown that mechanisms involved in cell adhesion and migration were specifically affected in these tumors.Using biochemical and cell biological approaches, we investigated the adhesive capacities of Bmi1;Ink4a/Arf deficient primary neural stem cells (NSCs). Bmi1;Ink4a/Arf deficient NSCs have altered expression of Collagen-related genes, secrete increased amounts of extracellular matrix, and exhibit enhanced cell–matrix binding through the Beta-1 Integrin receptor. These traits are independent from the well described role of Bmi1 as repressor of the Ink4a/Arf tumor suppressor locus.In addition to proliferative processes, Bmi1 controls the adhesive capacities of primary NSCs by modulating extracellular matrix secretion.Since PcG protein Bmi1 is important for both normal development and tumorigenesis, it is vital to understand the complete network in which this protein acts. Whereas it is clear that control of Ink4a/Arf is a major Bmi1 function, there is evidence that other downstream mechanisms exist. Hence, our novel finding that Bmi1 also governs cell adhesion significantly contributes to our understanding of the PcG proteins.

Keywords: Polycomb group; Bmi1; Neural stem cells; Integrins; Adhesion; Brain cancer

Circulating hormone adrenomedullin and its binding protein protect neural cells from hypoxia-induced apoptosis by Stephanie M. Wang; Weng-Lang Yang ⁎ (pp. 361-367).

Brain ischemia is the underlying cause of neuron death during stroke and brain trauma. Neural cells exposed to ischemia can undergo apoptosis. Adrenomedullin (AM) in combination with its enhancing binding protein, AMBP-1, has been shown to reduce tissue damage in inflammation.To evaluate a beneficial effect of AM/AMBP-1 administration in brain ischemia, we employed an in vitro model of neuronal hypoxia using differentiated human neuroblastoma SH-SY5Y cells.After exposure to 1% O₂ for 20 h, neural cells were injured with decreased ATP levels and increased LDH release. Pre-administration of AM/AMBP-1 significantly reduced hypoxia-induced cell injury. Moreover, AM/AMBP-1 treatment reduced the number of TUNEL-positive cells and activation of caspase-3, compared to cells exposed to hypoxia alone. AM/AMBP-1 prevented a reduction of cAMP levels and protein kinase A (PKA) activity in neural cells after hypoxia exposure. Correspondingly, an elevation of cAMP levels by forskolin protected neural cells from hypoxia-induced injury. Inhibition of PKA by KT5720 abolished the protective effect of AM/AMBP-1 on hypoxia-induced apoptosis.AM/AMBP-1 elevates cAMP levels, followed by activating PKA, to protect neural cells from the injury caused by hypoxia.AM/AMBP-1 may be used as therapeutic agents to prevent neuron damage from brain ischemia.

Keywords: Adrenomedullin; Adrenomedullin binding protein-1; Neural cell; Hypoxia; Apoptosis; cAMP; Protein kinase A

Studies on substrate specificity and activity regulating factors of trehalose-6-phosphate synthase of Saccharomyces cerevisiae by Paramita Chaudhuri; Arghya Basu; Shinjinee Sengupta; Sagar Lahiri; Trina Dutta; Anil K. Ghosh ⁎ (pp. 368-374).

Purified trehalose-6-phosphate synthase (TPS) of Saccharomyces cerevisiae was effective over a wide range of substrates, although differing with regard to their relative activity. Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity, particularly when a pyrimidine glucose nucleotide like UDPG was used, rather than a purine glucose nucleotide like GDPG. A high V_max and a low K_m value of UDPG show its greater affinity with TPS than GDPG or TDPG. Among the glucosyl acceptors TPS showed maximum activity with G-6-P which was followed by M-6-P and F-6-P. Effect of heparin was also extended to the purification of TPS activity, as it helped to retain both stability and activity of the final purified enzyme. Metal co-factors, specifically MnCl₂ and ZnCl₂ acted as stimulators, while enzyme inhibitors had very little effect on TPS activity. Metal chelators like CDTA, EGTA stimulated enzyme activity by chelation of metal inhibitors. Temperature and pH optima of the purified enzyme were determined to be 40 °C and pH 8.5 respectively. Enzyme activity was stable at 0–40 °C and at alkaline pH.

Keywords: Abbreviations; ADPG; adenosine diphosphate glucose; F-6-P; fructose-6-phosphate; G-6-P; glucose-6-phosphate; M-6-P; mannose-6-phosphate; GDPG; guanosine diphosphate glucose; IAA; iodoacetic acid; IAM; iodoacetamide; NEM; N-ethylmaleimide; TDPG; thymidine diphosphate glucose; TPS; trehalose-6-phosphate synthase; UDPG; uridine diphosphate glucosePolyanion; Saccharomyces cerevisiae; Substrate specificity; Trehalose-6-phosphate synthase

Disulfide-bonded multimers of proteoglycan 4 (PRG4) are present in normal synovial fluids by Tannin A. Schmidt ⁎; Anna H.K. Plaas; John D. Sandy (pp. 375-384).

The proteoglycan 4 ( PRG4) gene encodes for a mucin-like O-linked glycosylated protein with several names, including lubricin and superficial zone protein. The objective of this study was to analyze PRG4 in normal bovine calf and steer synovial fluids for evidence of native multimers formed by intermolecular disulfide bonds.A combination of mucin biochemical techniques, with antibodies to both terminal domains and the mucin-like domain of PRG4, were used for analyses.Multimers were present in both calf and steer fluids, and reduction and alkylation converts the multimeric complex (likely dimeric) into monomeric subunits. Tandem mass spectrometry analyses supported the Western blot data and identified PRG4 in the reduced ∼345 kDa monomeric form. Interestingly, ∼70 kDa fragments released upon reduction contained peptides from both the N and C terminal regions, which most likely represent fragments of a sparsely glycosylated PRG4 population that are disulfide-linked to extensively glycosylated, intact monomers.The analyses described here have demonstrated the presence of native disulfide-bonded multimers of PRG4 in normal bovine synovial fluids.These structures are similar to those described for multimerization of mucins in general. Such multimerization and proteolytic cleavage of PRG4 may have functional significance in joint health and disease.

Keywords: Proteyglycan 4; Lubricin; Synovial fluid; Mucin; Cartilage

Lipoplex nanostructures reveal a general self-organization of nucleic acids by Alain R. Thierry; Vic Norris; Franck Molina; Marc Schmutz (pp. 385-394).

Lipid and plasmid DNA complexes (Lx) were designed for gene transfer and were studied comprehensibly to elucidate their formation and ultrastructure.We compared supramolecular self-assembly into stable Lx containing nucleic acids of various types and lengths using Cryo-Electron Microscopy, Small Angle X-ray Scattering and dynamic light scattering.Analysis of these complexes showed that they reproducibly formed monodisperse and spherical multilamellar particles. The same concentric and lamellar structure with different packing regimes was produced by circular double-stranded DNA, linear double-stranded DNA, single-stranded DNA, oligodeoxynucleotides or RNA. Strikingly, thousands of oligonucleotide molecules seem to align with one another and to behave as longer nucleic acid molecules in forming structurally similar particles. Neither excess cationic lipids nor excess DNA of different forms changed significantly the mean diameter and the size distribution of Lx particles. This suggests a role for Lx formation of steric size in addition to the conventional thermodynamic mechanism. The Lx ultrastructure is highly ordered and crystalline and is in a lamellar and/or hexagonal phase. Increasing NA size led to an increased proportion of Lx in a hexagonal structure phase as in the case of T4 phage virus DNA. These observations were made using two Lx made from different lipids exhibiting negative and positive charged surface. We also demonstrated structural similarities between the supramolecular auto-organization of Lx and that found in some viruses. In particular, both synthetic and viral particles have an ultrastructure that exhibits a phase transition between lamellar and hexagonal phases.Taken together, our data point towards the possible existence of a ubiquitous organization of genetic materials, at least with cationic lipids, that has implications for both therapy and the origins of life.

Keywords: DNA; Condensation; Supramolecular self-organization; Cationic lipid; Origins of life; Gene therapy

Identification and tissue-specific expression of a novel isoform of Semaphorin 3D by Kaoru Takahashi; Kayoko Tomizawa; Mami Ishida; Katsuiku Hirokawa; Hiroshi Takahashi ⁎ (pp. 395-400).

Semaphorins are a family of secreted and membrane-associated proteins involved in axon guidance in the developing brain as well as morphogenesis in various organs. There has been no report on the expression of different transcripts of the genes encoding Class 3 Semaphorins with different protein structures.Molecular cloning of rat Semaphorin 3D gene and the expression analysis at gene and protein levels were performed.We have isolated two cDNAs encoding rat Sema 3D, a Class 3 Semaphorin. One clone is predicted to encode a protein with a structure common to Class 3 Semaphorins. The other clone encodes a novel isoform of Sema 3D lacking half of the C2-type Ig domain and the entire basic region; this isoform is predicted to have a different structure from Class 3 Semaphorins. Analysis of protein expression using a cell culture system revealed that this splice variant isoform is not secreted into the media, whereas the classical Class 3 isoform is a secreted protein. The expression of each isoform shows tissue-specificity.Our present findings suggest that gene regulation, via an alternative splicing mechanism, affects not only the tissue-specificity of Sema 3D expression, but also the distance over which it can act.

Keywords: Sema 3D; Class 3 semaphorin; Alternative splicing

Corrigendum to “Glycobiology in the cytosol: The bitter side of a sweet world” [Biochim. Biophys. Acta 1790 (2009) 81–94] by Yoko Funakoshi; Tadashi Suzuki ⁎ (pp. 401-401).

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BBA - General Subjects (v.1790, #5)