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BBA - General Subjects (v.1780, #4)
Reduction of hypervalent states of myoglobin and hemoglobin to their ferrous forms by thymoquinone: The role of GSH, NADH and NADPH by K.H. Khalife; G. Lupidi (pp. 627-637).
The reactivity of thymoquinone towards different redox states of hemoglobin and myoglobin in the presence of GSH, NADH, and NADPH was evaluated by optical spectral analysis. Thymoquinone reduces the ferryl forms (HbIV/MbIV) of both met-hemoglobin (HbIII) and met-myoglobin (MbIII) to oxy-hemoglobin (HbIIO2) and oxy-myoglobin (MbIIO2) under physiological conditions. The reaction is mediated by the intermediate quinone forms of TQ, that is, glutathionyl-dihydrothymoquinone (DHTQ-GS) and dihydrothymoquinone (DHTQ), formed from direct interaction of TQ with GSH or NADH (NADPH). In vitro incubation of oxidized human erythrocytes with TQ, DHTQ, and the GSH/TQ mixture reduces the intracellular met-Hb at different rates. In the present study, we report that TQ and its reduced derivatives can also prevent lipid peroxidation induced by the MbFeIII/H2O2 system. In this system, lipid peroxidation is induced by MbIV or a putative MbIV/·MbVI composite; it is plausible that the antioxidant function of TQ derivatives is related to their ability to reduce these oxidizing species. This is of particular biological significance, as natural quinones may participate in reducing processes that lead to recovery of hemoglobin and myoglobin during oxidative stress.
Keywords: Abbreviations; TQ; Thymoquinone; DHTQ; dihydrothymoquinone; DHTQ-GS; glutathionyl-dihydrothymoquinone; H; 2; O; 2; hydrogen peroxide; DTPA; diethylene triaminepentaacetic acid; GSH; Glutathione; NADH; Nicotinamide adenine dinucleotide reduced form; NADPH; nicotinamide adenine dinucleotide phosphate reduced form; 9,11,13,15-; cis,trans,trans,cis; -octadecatetraenoic acid; PnA; cis; -parinaric acid met-myoglobin; Mb; III; met-hemoglobin; Hb; III; oxy-myoglobin; Mb; II; O; 2; oxy-hemoglobin; Hb; II; O; 2; ferrylmyoglobin; Mb; IV; or MbFe(IV); O; ferrylhemoglobin Hb; IV; or HbFe(IV); OThymoquinone; Dihydrothymoquinone; Nigella sativa; L.; redox regulation; Glutathione; Met-hemoglobin; Ferryl hemoglobin; Oxidative damage
The nitric oxide–iron interplay in mammalian cells: Transport and storage of dinitrosyl iron complexes by Des R. Richardson; Hiu Chuen Lok (pp. 638-651).
Nitrogen monoxide (NO) is a vital effector and messenger molecule that plays roles in a variety of biological processes. Many of the functions of NO are mediated by its high affinity for iron (Fe) in the active centres of proteins. Indeed, NO possesses a rich coordination chemistry with this metal and the formation of dinitrosyl–dithiolato–Fe complexes (DNICs) is well known to occur intracellularly. In mammals, NO produced by activated macrophages acts as a cytotoxic effector against tumour cells by binding and releasing cancer cell Fe that is vital for proliferation. Glucose metabolism and the subsequent generation of glutathione (GSH) are critical for NO-mediated Fe efflux and this process occurs by active transport. Our previous studies showed that GSH is required for Fe mobilisation from tumour cells and we hypothesized it was effluxed with Fe as a dinitrosyl–diglutathionyl–Fe complex (DNDGIC). It is well known that Fe and GSH release from cells induces apoptosis, a crucial property for a cytotoxic effector like NO. Furthermore, NO-mediated Fe release is mediated from cells expressing the GSH transporter, multi-drug resistance protein 1 (MRP1). Interestingly, the glutathione- S-transferase (GST) enzymes act to bind DNDGICs with high affinity and some members of the GST family act as storage intermediates for these complexes. Since the GST enzymes and MRP1 form a coordinated system for removing toxic substances from cells, it is possible to hypothesize these molecules regulate NO levels by binding and transporting DNDGICs.
Keywords: Abbreviations; BSO; buthionine sulphoximine; CO; carbon monoxide; d; -Glc; d; -glucose; DMT1; divalent metal ion transporter 1; DNDGIC; dinitrosyl–diglutathionyl–Fe complex; DNIC; dinitrosyl–dithiolato–Fe complex; EPR; electron paramagnetic resonance spectroscopy; GSH; glutathione; GSNO; S; -nitroso-glutathione; GST; glutathione-; S; -transferase; HFE; haemochromatosis gene product; IRE; iron-responsive element; IRP; iron-regulatory protein; MRP1; multi-drug resistance protein 1; NO•; nitric oxide; NO; nitrogen monoxide; NO; +; nitrosonium ion; NO; −; nitroxyl anion; PPP; pentose phosphate pathway; SNAP; S; -nitroso-; N; -acetylpenicillamine; SNP; sodium nitroprusside; TCA; tricarboxylic acid cycle; Tf; transferrin; TfR1; transferrin receptor 1; UTR; untranslated regionNitrogen monoxide; Nitric oxide; Transferrin; Iron; Glutathione-; S; -transferase; Multi-drug resistance protein 1
Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804) by Rolando Perdomo-Morales; Vivian Montero-Alejo; Erick Perera; Zenia Pardo-Ruiz; Esther Alonso-Jiménez (pp. 652-658).
Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.
Keywords: Hemocyanin; Phenoloxidase; Tyrosinase; Hemolymph; Spiny lobster
Caffeic acid phenethyl ester and its related compounds limit the functional alterations of the isolated mouse brain and liver mitochondria submitted to in vitro anoxia–reoxygenation: Relationship to their antioxidant activities by Yun Feng; Ying-Wei Lu; Pei-Han Xu; Yuan Long; Wei-Min Wu; Wei Li; Rui Wang (pp. 659-672).
It is an important therapeutic strategy to protect mitochondria from oxidative stress, especially during ischemia–reperfusion. In the present study, an attempt has been made to evaluate the protective effects of caffeic acid phenethyl ester (CAPE) and its related phenolic compounds on mouse brain and liver mitochondria injury induced by in vitro anoxia–reoxygenation. Added before anoxia or reoxygenation, CAPE markedly protected coupled respiration with the decrease in state 4 and the increases in state 3, respiratory control ratio (RCR) and ADP/O ratio in a concentration-dependent manner. CAPE effectively protected mitochondria by inhibiting the mitochondrial membranes fluidity decrease, the lipoperoxidation and the protein carbonylation increase, which indicated its protective action against the mitochondrial oxidative damage. Meanwhile, CAPE blocked the enhanced release of cardiolipin (CL) and cytochrome c (Cyt c). The related phenolic compounds like caffeic acid (CA), ferulic acid (FA) and ethyl ferulate (EF) also had different-degree protective effects. CAPE and CA were more potent than FA and EF. Their structural differences played the key role in their activity levels. These results suggest that CAPE and its related phenolic compounds protect mitochondria mainly correlated to their antioxidative activities and may be of interest for the prevention and therapy of ischemia–reperfusion injuries.
Keywords: Caffeic acid phenethyl ester (CAPE); Phenolic compounds; Mitochondria; Anoxia–reoxygenation
Quantitative cellular uptake, localization and cytotoxicity of curcumin in normal and tumor cells by A. Kunwar; A. Barik; B. Mishra; K. Rathinasamy; R. Pandey; K.I. Priyadarsini (pp. 673-679).
Using absorption and fluorescence spectroscopic methods, quantitative cellular uptake of curcumin, an antioxidant and anti-tumor agent from Curcuma longa, was calculated in two types of normal cells: spleen lymphocytes, and NIH3T3 and two tumor cell lines: EL4 and MCF7. Both the uptake and fluorescence intensity of curcumin were significantly higher in tumor cells compared to the normal cells. A linear dependency on the uptake was observed with treatment concentration of curcumin. Using laser confocal microscopy, intracellular localization of curcumin was monitored and the results indicated that curcumin is located both in the cell membrane and the nucleus. Sub-cellular fractionation of curcumin-loaded MCF7 cells supported the differential distribution of curcumin in membrane, cytoplasm and nuclear compartments of cell with maximum localization in the membrane. Cytotoxicity studies in different cell lines indicated that the toxicity of curcumin increased with increasing uptake.
Keywords: Curcumin; Cellular uptake; Tumor cell; Nuclear localization; Fluorescence; Cytotoxicity
Modification of protein with BGL06, a novel branched oligoglycerol derivative by Hiroyuki Yamaguchi; Toshiyuki Suzawa; Takaaki Araki; Masaki Kamiya; Hisao Nemoto; Motoo Yamasaki (pp. 680-686).
A branched oligoglycerol derivative, BGL06, with a cascade-like structure of glycerol units was used as a novel reagent for protein modification. Modification reaction with a recombinant human granulocyte-colony stimulating factor derivative, ND28, was carried out successfully in aqueous conditions to obtain a coupled form, BGL06-ND28. Characterization of the modified ND28 suggests that two types of products were obtained by controlling the reaction; one was H(BGL06)-ND28, a highly modified version coupled with 4.34 molecules of BGL06 units on average, and the other was L(BGL06)-ND28, a moderately modified version coupled with 2.58 molecules of BGL06 units on average, respectively. The properties of these products were compared to the known polyethylene glycol (PEG)-modified ND28. In the cell proliferation assays, unlike PEGylation, modification with BGL06 did not produce a significant loss of biological activity even when the modification extent was elevated. Under such conditions, 76.0% of the activity was in fact maintained for H(BGL06)-ND28, while PEGylated ND28 retained only 24.6% of biological activity in vitro even though the extent of modification was smaller. In addition, H(BGL06)-ND28 showed comparable thermostability to a 20 kDa PEG-modified counterpart. Therefore, the BGL06 derivative will be a useful alternative as a protein modification reagent where PEGylation is not effective.
Keywords: Abbreviations; G-CSF; recombinant human granulocyte-colony stimulating factor; ND28; a derivative of G-CSF; PEG; polyethylene glycolConjugate; Chemical modification; G-CSF; Glycerol; PEG; Oligomer
Expression of sulfotransferases involved in the biosynthesis of chondroitin sulfate E in the bone marrow derived mast cells by Shiori Ohtake; Sachiko Kondo; Toshiko Morisaki; Kaori Matsumura; Koji Kimata; Osami Habuchi (pp. 687-695).
Bone marrow-derived mast cells (BMMCs) contain chondroitin sulfate (CS)-E comprised of GlcA-GalNAc(4SO4) units and GlcA-GalNAc(4,6-SO4) units. GalNAc 4-sulfate 6- O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO4) residues of CS. On the basis of the specificity of GalNAc4S-6ST, it is thought that CS-E is synthesized in BMMC through the sequential sulfation by chondroitin 4-sulfotransferase (C4ST)-1 and GalNAc4S-6ST. In this paper, we investigated whether GalNAc4S-6ST and C4ST-1 are actually expressed in BMMCs in which CS-E is actively synthesized. As the bone marrow cells differentiate to BMMCs, level of C4ST-1 and GalNAc4S-6ST messages increased, whereas chondroitin 6-sulfotransferase (C6ST)-1 message decreased. In the extract of BMMCs, activity of GalNAc4S-6ST and C4ST but not C6ST were detected. The recombinant mouse GalNAc4S-6ST transferred sulfate to both nonreducing terminal and internal GalNAc(4SO4) residues; the activity toward nonreducing terminal GalNAc(4SO4) was increased with increasing pH. When CS-E synthesized by BMMCs was metabolically labeled with35SO4 in the presence of bafilomycin A, chloroquine or NH4Cl, the proportion of the nonreducing terminal GalNAc(4,6-SO4) was increased compared with the control, suggesting that GalNAc4S-6ST in BMMC may elaborate CS-E in the intracellular compartment with relatively low pH where sulfation of the internal GalNAc(4SO4) by GalNAc4S-6ST preferentially occurs.
Keywords: Abbreviations; BMMC; bone marrow derived mast cell; CS-E; chondroitin sulfate E; CS-A; chondroitin sulfate A; GalNAc4S-6ST; N; -acetylgalactosamine 4-sulfate 6-; O; -sulfotransferase; C4ST; chondroitin 4-sulfotransferase; C6ST; chondroitin 6-sulfotransferase; GalNAc(4SO; 4; ); 4-; O; -sulfo-; N; -acetylgalactosamine; GalNAc(6SO; 4; ); 6-; O; -sulfo-; N; -acetylgalactosamine; GalNAc(4,6-SO; 4; ); 4,6-bis-; O; -sulfo-; N; -acetylgalactosamine; GlcA; d; -glucuronic acid; Oligo I; GalNAc(4SO; 4; )-GlcA(2SO; 4; )-GalNAc(6SO; 4; ); Oligo II; GalNAc(4,6-SO; 4; )-GlcA(2SO; 4; )-GalNAc(6SO; 4; ); PAPS; 3′-phosphoadenosine 5′-phosphosulfate; 2AB; 2-aminobenzamide; ΔDi-0S; 2-acetamide-2-deoxy-3-; O; -(β-; d; -gluco-4-enepyranosyluronic acid)-; d; -galactose; ΔDi-6S; 2-acetamide-2-deoxy-3-; O; -(β-; d; -gluco-4-enepyranosyluronic acid)-6-; O; -sulfo-; d; -galactose; ΔDi-4S; 2-acetamide-2-deoxy-3-; O; -(β-; d; -gluco-4-enepyranosyluronic acid)-4-; O; -sulfo-; d; -galactose; ΔDi-diS; D; 2-acetamide-2-deoxy-3-; O; -(2-; O; -sulfo-β-; d; -gluco-4-enepyranosyluronic acid)-6-; O; -sulfo-; d; -galactose; ΔDi-diS; E; 2-acetamide-2-deoxy-3-; O; -(β-; d; -gluco-4-enepyranosyluronic acid)-4,6-bis-; O; -sulfo-; d; -galactose; ΔDi-TriS; 2-acetamide-2-deoxy-3-; O; -(2-; O; -sulfo-β-; d; -gluco-4-enepyranosyluronic acid) -4,6-bis-; O; -sulfo-; d; -galactose; RT-PCR; reverse trascription polymerase chain reaction; SAX-HPLC; strong anion ion exchange high performance liquid chromatographyChondroitin sulfate E; Bone marrow derived mast cells; Sulfotransferase; Bafilomycin A, GalNAc4S-6ST
Both the N-terminal fragment and the protein–protein interaction domain (PDZ domain) are required for the pro-apoptotic activity of presenilin-associated protein PSAP by Guozhang Mao; Jianxin Tan; Wei Gao; Yongchang Shi; Mei-Zhen Cui; Xuemin Xu (pp. 696-708).
Presenilin-associated protein (PSAP) was originally identified as a PS1-associated, PDZ domain protein. In a subsequent study, PSAP was found to be a mitochondrial apoptotic molecule. In this study, we cloned the PSAP gene and found that it is composed of 12 exons and localizes on chromosome 6. To better understand the structure and function of PSAP, we have generated a series of antibodies that recognize different regions of PSAP. Using these antibodies, we found that PSAP is expressed in four isoforms as a result of differential splicing of exon 8 in addition to the use of either the first or the second ATG codon as the start codon. We also found that all these isoforms are localized in the mitochondria and are pro-apoptotic. Furthermore, our data revealed that the PDZ domain and N-terminal fragment are required for the pro-apoptotic activity of PSAP.
Keywords: Abbreviations; AD; Alzheimer's disease; PS; presenilin; PSAP; presenilin-associated protein; siRNA; small interfering RNA; FISH; fluorescence in situ hybridization; SMART; Simple Modular Architecture Research ToolApoptosis; Mitochondria; Caspase; Cytochrome; c; Cell death; Alzheimer's disease
Involvement of heparanase in migration of microglial cells by Hisaaki Takahashi; Hiroaki Matsumoto; Anna Smirkin; Tomohide Itai; Yoshio Nishimura; Junya Tanaka (pp. 709-715).
Heparanase, a matrix-degrading enzyme that cleaves heparan sulfate side chains from heparan sulfate proteoglycans (HSPGs), has been shown to facilitate cell invasion, migration, and extravasation of metastatic tumor cells or immune cells. In this study, the expression and functions of heparanase were investigated using rat primary cultured microglia, the resident macrophages in the brain. The microglia were found to express heparanase mRNA and protein. Microglia treated with lipopolysaccharide (LPS) were activated, expressed induced nitric oxide synthase and elevated the expression of heparanase. Heparanase has two molecular weights: a 65 kDa latent form and an active 50 kDa. Both forms were expressed by LPS-treated activated microglia; however, untreated microglia primarily expressed the latent form. Cell lysates from microglia actually degraded Matrigel containing HSPG. Heparanase was colocalized with the actin cytoskeleton in microglial leading edges or ruffled membranes. Microglia transmigrated through a Matrigel-coated pored membrane. This process was inhibited by SF-4, a specific heparanase inhibitor, in a concentration-dependent manner. Degraded HSPG was generated when microglia transmigrated through the coated membrane, and this was also inhibited by SF-4. The results suggest the involvement of heparanase in the migration or invasion of microglia or brain macrophages across basement membrane around brain vasculature.
Keywords: Heparanase; Microglial cell; Migration
Phosphorylated human galectin-3: Facile large-scale preparation of active lectin and detection of structural changes by CD spectroscopy by Dieter Kübler; Chien-Wen Hung; Tarun K. Dam; Jürgen Kopitz; Sabine André; Herbert Kaltner; Michaela Lohr; Joachim C. Manning; Lizhong He; Hui Wang; Anton Middelberg; C. Fred Brewer; Jennifer Reed; Wolf-Dieter Lehmann; Hans-Joachim Gabius (pp. 716-722).
Galectin-3 has a unique modular design. Its short N-terminal stretch can be phosphorylated, relevant for nuclear export and anti-anoikis/apoptosis activity. Enzymatic modification by casein kinase 1 at constant ATP concentration yielded mg quantities of mono- and diphosphorylated derivatives at Ser5/Ser11 in a 2:1 ratio. Their carbohydrate-inhibitable binding to asialofetuin, cell surfaces of three tumor lines, rabbit erythrocytes leading to haemagglutination and cytoplasmic sites in fixed tissue sections was not markedly altered relative to phosphate-free galectin-3. Spectroscopically, phosphorylation induced alterations in the far UV CD, indicative of an increase in ordered structure. This is accompanied by changes in the environment of aromatic amino acids signified by shifts in the near UV CD.
Keywords: Abbreviations; CD; circular dichroism; CK1; casein kinase 1; CRD; carbohydrate recognition domain; nanoESI-MS; nanoelectrospray ionization mass spectrometry; SPR; surface plasmon resonance; UV; ultravioletApoptosis; Histochemistry; Lectin; Serine phosphorylation; Titania chromatography
Distinct heparin binding sites on VEGF165 and its receptors revealed by their interaction with a non sulfated glycoaminoglycan (NaPaC) by Mélanie Di Benedetto; Anna Starzec; Roger Vassy; Gérard-Yves Perret; Michel Crépin (pp. 723-732).
We previously demonstrated that a non sulfated analogue of heparin, phenylacetate carboxymethyl benzylamide dextran (NaPaC) inhibited angiogenesis. Here, we observed that NaPaC inhibited the VEGF165 binding to both VEGFR2 and NRP-1 and abolished VEGFR2 activity. Further, we explored the effects of NaPaC on VEGF165 interactions with its receptors, VEGFR2 and NRP-1, co-receptor of VEGFR2. Surface plasmon resonance and affinity gel electrophoresis showed that NaPaC interacted directly with VEGF165, VEGFR2 and NRP-1 but not with heparin-independent factor such as VEGF121. NaPaC completely inhibited the heparin binding to VEGF165, NRP-1 and VEGFR2. We found that NaPaC bound to all three molecules, VEGF165, VEGFR2 and NRP-1, but was more effective in inhibiting heparin binding to VEGF165. These results suggested that heparin binding sites of VEGFR2 and NRP-1 were different from those of VEGF165.
Keywords: Heparin binding site; Interaction; VEGF; 165; VEGFR2; NRP-1
Inhibition of bfl-1/A1 by siRNA inhibits mycobacterial growth in THP-1 cells by enhancing phagosomal acidification by Rohan Dhiman; Mahesh Kathania; Manoj Raje; Sekhar Majumdar (pp. 733-742).
Virulent tubercle bacilli inhibit apoptosis to establish a safe environment within the host cells. Here, we report that NF-κB dependent antiapoptotic protein bfl-1/A1 plays an important role in this process. Both virulent and avirulent mycobacteria bearing THP-1 cells expressed considerable amount of bfl-1/A1 after 4 h of infection. However, after 48 h of infection, bfl-1/A1 expression was evident only in Mycobacterium tuberculosis H37Rv but not in M. tuberculosis H37Ra infected cells. When parallel experiments were performed with Human monocyte-derived macrophages (MDMs), differential expression of bfl-1/A1 mRNA was observed in case of M. tuberculosis H37Rv and M. tuberculosis H37Ra infection. siRNA mediated inhibition of bfl-1/A1 induced apoptosis in M. tuberculosis H37Rv infected THP-1 and MDMs. Reduction in intracellular mycobacterial growth was observed in bfl-1/A1 siRNA transfected, M. tuberculosis H37Rv infected THP-1 cells. Enhancement of phagosome–lysosome fusion was observed in bfl-1/A1 siRNA treated and M. tuberculosis H37Rv infected THP-1 cells. These results clearly indicated that differential expression of bfl-1/A1 in M. tuberculosis H37Rv and M. tuberculosis H37Ra infected THP-1 cells probably account for the difference in infection outcome.
Keywords: Macrophages; Mycobacteria; bfl-1/A1; Apoptosis; Phagosome–lysosome fusion
Growth inhibition of human hepatic carcinoma HepG2 cells by fucoxanthin is associated with down-regulation of cyclin D by Swadesh K. Das; Takashi Hashimoto; Kazuki Kanazawa (pp. 743-749).
Fucoxanthin, a major carotenoid in brown sea algae, has recently been demonstrated by us to inhibit the proliferation of colon cancer cells, and this effect was associated with growth arrest. These results, taken together with previous studies with fucoxanthin, suggest that it may be useful in chemoprevention of other human malignancies. The present study was designed to evaluate the molecular mechanisms of fucoxanthin against hepatic cancer using the human hepatocarcinoma HepG2 cell line (HepG2). Fucoxanthin reduced the viability of HepG2 cells accompanied with the induction of cell cycle arrest during the G0/G1 phase at 25 μM. This concentration of fucoxanthin inhibited the phosphorylation of the retinoblastoma protein (Rb) at Serine 780 (Ser780) position 18 h after treatment. The kinase activity of cyclin D and cdk4 complex, responsible for the phosphorylation of Rb Ser780 site, was down-regulated 18 h after the treatment. Western blotting analysis revealed that the expression of cyclin D-type protein was suppressed by treatment of fucoxanthin. This reduction was partially blocked by concurrent treatment with the proteasome inhibitor MG132, indicating the involvement of the proteasome-mediated degradation. In addition, RT-PCR analysis revealed that fucoxanthin also appeared to repress cyclin D mRNA. Thus, both the protein degradation and transcriptional repression seem to be responsible for suppressed cyclin D level in fucoxanthin-treated HepG2 cells which may be related to the antitumorgenic activity.
Keywords: Fucoxanthin; Cyclin D; Cell cycle arrest at G; 0; /G; 1; phase; Proteasome; Hepatocarcinoma cell
A positive charge at the N-terminal segment of Discrepin increases the blocking effect of K+ channels responsible for the IA currents in cerebellum granular cells by Stefania Romeo; Gerardo Corzo; Attilio Vasile; Honoo Satake; Gianfranco Prestipino; Lourival D. Possani (pp. 750-755).
Discrepin is a scorpion peptide that blocks preferentially the IA currents of the voltage-dependent K+ channel of rat cerebellum granular cells. It was isolated from the venom of the buthid scorpion Tityus discrepans and contains 38 amino acid residues with a pyroglutamic acid at the N-terminal site. Discrepin has the lowest sequence identity (approx. 50%) among the six members of the α-KTx15 sub-family of scorpion toxins. In order to find out which residues are important for the blocking effects of Discrepin, six mutants were chemically synthesized (V6K, I19R, D20K, T35V, I19R-D20K, I19R-D20K-R21V), correctly folded and their physiological properties were examined. Substitution of residues V6 and D20 for basically charged amino acids increases the blocking activity of Discrepin, specially the mutation V6K at the N-terminal segment of the toxin. Analysis of 3D-structure models of the mutants V6K and D20K supports the idea that basic residues improve their blocking activities, similarly to what happens with BmTx3, a toxic peptide obtained from Buthus martensi scorpion, which has the highest known blocking effects of IA currents in K+ channels of rat cerebellum granular cells.
Keywords: A-currents; Discrepin; K; +; channel; Structure-function; Scorpion toxin