Check out our New Publishers' Select for Free Articles

BBA - General Subjects (v.1790, #3)

Editorial Board (pp. i).

Oxidative inactivation of lactonase activity of purified human paraoxonase 1 (PON1) by Su Duy Nguyen; Nguyen Dang Hung; Park Cheon-Ho; Kim Mee Ree; Sok Dai-Eun ⁎ (pp. 155-160).

Paraoxonase1 (PON1), one of HDL-associated antioxidant proteins, is known to lose its activity in vivo systems under oxidative stress. Here, we examined the effect of various oxidants on lactonase activity of PON1, and tried to protect the lactonase activity from oxidative inactivation. Among the oxidative systems tested, the ascorbate/Cu²⁺ system was the most potent in inactivating the lactonase activity of purified PON1; in contrast to a limited role of Fe²⁺, Cu²⁺ (0.05–1.0 µM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.02–0.1 mM). Moreover, Cu²⁺ alone inhibited the lactonase activity at concentrations as low as 1 µM. The ascorbate/Cu²⁺-mediated inactivation of PON1 lactonase activity was prevented by catalase, but not general hydroxyl radical scavengers, suggesting the implication of Cu²⁺-bound hydroxyl radicals in the oxidative inactivation. Compared to arylesterase activity, lactonase activity appears to be more sensitive to Cu²⁺-catalyzed oxidation. Separately, ascorbate/Cu²⁺-mediated inactivation of lactonase activity was prevented by oleic acid as well as phoshatidylcholine. Taken together, our data demonstrate that Cu²⁺-catalyzed oxidation may be a primary factor to cause the decrease of PON1 lactonase activity under oxidative stress and that lactonase activity of PON1 is most susceptible to ascorbate/Cu²⁺ among PON1 activities. In addition, we have showed that radical-induced inactivation of lactonase activity is prevented by some lipids.

Keywords: Abbreviations; PON1; paraoxonase 1; HDL; high density lipoprotein; LDL; low density lipoprotein; DOPC; dioleoyl phosphatidylcholineLactonase; Arylesterase; Paraoxonase; Oxidative inactivation; Reactive oxygen specie; Antioxidant

Conserved sequence in the aggrecan interglobular domain modulates cleavage by ADAMTS-4 and ADAMTS-5 by Hazuki E. Miwa; Thomas A. Gerken; Tru D. Huynh; Lori R. Duesler; Meghan Cotter; Thomas M. Hering ⁎ (pp. 161-172).

Cleavage of aggrecan by ADAMTS proteinases at specific sites within highly conserved regions may be important to normal physiological enzyme functions, as well as pathological degradation.To examine ADAMTS selectivity, we assayed ADAMTS-4 and -5 cleavage of recombinant bovine aggrecan mutated at amino acids N-terminal or C-terminal to the interglobular domain cleavage site.Mutations of conserved amino acids from P18 to P12 to increase hydrophilicity resulted in ADAMTS-4 cleavage inhibition. Mutation of Thr, but not Asn within the conserved N-glycosylation motif Asn-Ile-Thr from P6 to P4 enhanced cleavage. Mutation of conserved Thr residues from P22 to P17 to increase hydrophobicity enhanced ADAMTS-4 cleavage. A P4′ Ser377Gln mutant inhibited cleavage by ADAMTS-4 and -5, while a neutral Ser377Ala mutant and species mimicking mutants Ser377Thr, Ser377Asn, and Arg375Leu were cleaved normally by ADAMTS-4. The Ser377Thr mutant, however, was resistant to cleavage by ADAMTS-5.We have identified multiple conserved amino acids within regions N- and C-terminal to the site of scission that may influence enzyme–substrate recognition, and may interact with exosites on ADAMTS-4 and ADAMTS-5.Inhibition of the binding of ADAMTS-4 and ADAMTS-5 exosites to aggrecan should be explored as a therapeutic intervention for osteoarthritis.

Keywords: Abbreviations; CS; chondroitin sulfate; ECM; extracellular matrix; EDTA; ethylene diamine tetraacetic acid; G1 domain; N-terminal globular domain of aggrecan; G2 domain; second globular domain of aggrecan; G3 domain; C-terminal globular domain of aggrecan; GAG; glycosaminoglycan; HA; hyaluronan; IGD; interglobular domain; KS; keratan sulfate; MMP; matrix metalloproteinases; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF; polyvinylidine fluoride; ECL; enhanced chemiluminescenceAggrecan; Interglobular domain; ADAMTS-4; ADAMTS-5; Aggrecanase; Proteinase; Exosite

Purification, characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells by Jure Pohleven; Nataša Obermajer; Jerica Sabotič; Sabina Anžlovar; Kristina Sepčić; Janko Kos; Bogdan Kralj; Borut Štrukelj; Jože Brzin (pp. 173-181).

Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric ( Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc ( N, N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.

Keywords: Abbreviations; CNL; Clitocybe nebularis; lectin; RP-HPLC; reversed-phase high-performance liquid chromatography; TFA; trifluoroacetic acid; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FPLC; fast protein liquid chromatography; PCR; polymerase chain reaction; ESI; electrospray ionization; 3′ RACE; 3′ rapid amplification of cDNA ends; RicB1; ricin B chain lectin domain 1; Abra1; abrin-a B chain lectin domain 1; RicB2; ricin B chain lectin domain 2; Abra2; abrin-a B chain lectin domain 2; Flav; flavastacin; MOA; Marasmius oreades; agglutinin; LSL; Laetiporus sulphureus; lectinMushroom; Clitocybe nebularis; Ricin B-like lectin; Antiproliferative effect; Leukemic T cells

Involvement of exon 6-mediated calpastatin intracellular movements in the modulation of calpain activation by Roberta De Tullio; Claudia Cantoni; Chiara Broggio; Carola Prato; Roberto Stifanese; Monica Averna; Renzo Antolini; Sandro Pontremoli; Edon Melloni (pp. 182-187).

To establish the physiological role of calpain, it is necessary to define how the protease can escape from the effect of its natural inhibitor calpastatin, since both proteins co-localize into the cell cytosol.To answer this question, we have overexpressed four fluorescent calpastatin constructs, differing in the composition of their XL- and L-domains, and the intracellular trafficking of this protein inhibitor has been followed by single cell fluorescence imaging.By the use of these calpastatin forms differing in the type of exon-derived sequences contained in the XL- and L-domains, we have demonstrated that the sequence coded by exon 6, containing multiple phosphorylation sites, is directly involved in determining the cell localization of calpastatin. In fact, exposure to cAMP promotes the recruitment into aggregates of those calpastatin forms containing the exon 6 sequence. These protein movements are directly related to the level of cytosolic inhibitory capacity and thereby to the extent of intracellular calpain activation.The recruitment of calpastatin into aggregates allows the translocation and activation of the protease to the membranes; on the contrary, the presence of large amounts of calpastatin in the cytosol prevents both processes, protecting the cell from undesired proteolysis.

Keywords: Calpain; Calpastatin; cAMP; Ca; ²⁺; homeostasis; Regulation of Ca; ²⁺; -dependent proteolysis; Intracellular protein trafficking

Biochemical characterization of tau protein and its associated syndapin 1 and protein kinase Cɛ for their functional regulation in rat brain by Kanzo Suzuki; Fumitaka Kawakami; Hisashi Sasaki; Hiroko Maruyama; Kenzo Ohtsuki ⁎ (pp. 188-197).

We recently reported that both sulfatide and cholesterol-3-sulfate (SCS) function as potent stimulators for the GSK-3β-mediated phosphorylation of tau protein (TP) in vitro [J. Biochem. 143 (2008) 359–367].By means of successive gel filtration on a Superdex 200 pg column and three distinct ion-exchange column chromatographies, TP and its associated proteins were highly purified from the extract of rat brain.We found that (i) syndapin 1 and novel protein kinase Cɛ (nPKCɛ) were identified as the TP-associated proteins; (ii) SCS highly stimulated the phosphorylation of TP and syndapin 1 by nPKCɛ as well as CK1; (iii) the full phosphorylation of TP and syndapin 1 by nPKCɛ in the presence of sulfatide resulted in their dissociation; (iv) TP primed by CK1 functioned as an effective phosphate acceptor for GSK-3β; (v) syndapin 1 highly stimulated the GSK-3β-mediated phosphorylation of TP; and (vi) TP isoforms were highly expressed in aged brain, whereas syndapin 1 was consistently detected in adult brain, but not in newborn brain.These results provided here suggest that (i) TP-associated nPKCɛ suppresses the GSK-3β-mediated phosphorylation of TP through the phosphorylation of GSK-3β by the kinase in vitro; and (ii) SCS act as effective sole mediators to induce the GSK-3β-mediated high phosphorylation of both TP and its associated syndapin 1 involved in the biochemical processes of neuronal diseases, including Alzheimer's disease.

Keywords: Abbreviations; AD; Alzheimer's disease; CDK5; cyclin-dependent kinase 5; CH-3S; cholesterol-3-sulfate; CK1; casein kinase 1; DTT; dithiothreitol; GSK-3β; glycogen synthase kinase-3β; NFT; neurofibrillary tangle; nPKC; novel Ca; ²⁺; -independent and diacylglycerol-dependent protein kinase; nPKCɛ; ɛ-isoform of nPKC; PMSF; phenylmethylsulfonyl fluoride; SCS; sulfatide and CH-3S; SCS-BP; SCS-binding protein; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TP; tau protein; 2DE; two-dimensional electrophoresisTau protein; Syndapin 1; Protein kinase Cɛ; Casein kinase 1; Glycogen synthase kinase-3β; Sulfatide; Cholesterol-3-sulfate; Rat brain

Bifunctional chimeric fusion proteins engineered for DNA delivery: Optimization of the protein to DNA ratio by Shan Gao; Melissa J. Simon; Barclay Morrison III; Scott Banta (pp. 198-207).

Cell penetrating peptides (CPPs) have been used to deliver nucleotide-based therapeutics to cells, but this approach has produced mixed results. Ionic interactions and covalent bonds between the CPPs and the cargos may inhibit the effectiveness of the CPPs or interfere with the bioactivity of the cargos.We have created a bifunctional chimeric protein that binds DNA using the p50 domain of the NF-κB transcription factor and is functionalized for delivery with the TAT CPP. The green fluorescent protein (GFP) has been incorporated for tracking delivery. The new chimeric protein, p50–GFP–TAT, was compared to p50–GFP, GFP–TAT and GFP as controls for the ability to transduce PC12 cells with and without oligonucleotide cargos.The p50–GFP–TAT construct can deliver 30 bp and 293 bp oligonucleotides to PC12 cells with an optimal ratio of 1.89 protein molecules per base pair of DNA length. This correlation was validated through the delivery of a fluorescent protein transgene encoded in a plasmid to PC12 cells. Thus, self-assembling CPP-based bifunctional fusion proteins can be engineered for the non-viral delivery of nucleotide-based cargos to mammalian cells.This work represents an important step forward in the rational design of protein-based systems for the delivery of macromolecular cargos.

Keywords: Cell penetrating peptide; TAT; p50 DNA-binding domain; Oligonucleotide delivery; Protein/DNA ratio

The effects of a ketogenic diet on ATP concentrations and the number of hippocampal mitochondria in Aldh5a1^−/− mice by Kirk Nylen ⁎; Jose Luis Perez Velazquez; Venus Sayed; K. Michael Gibson; W.M. Burnham; O. Carter Snead III (pp. 208-212).

Succinic semialdehyde dehydrogenase (SSADH) deficiency is an inborn error of GABA metabolism characterized clinically by ataxia, psychomotor retardation and seizures. A mouse model of SSADH deficiency, the Aldh5a1^−/− mouse, has been used to study the pathophysiology and treatment of this disorder. Recent work from our group has shown that the ketogenic diet (KD) is effective in normalizing the Aldh5a1^−/− phenotype, although the mechanism of the effect remains unclear.Here, we examine the effects of a KD on the number of hippocampal mitochondria as well as on ATP levels in hippocampus. Electron microscopy was performed to determine the number of mitochondria in the hippocampus of Aldh5a1^−/− mice. Adenosine triphosphate (ATP) levels were measured in hippocampal extracts.Our results show that the KD increases the number of mitochondria in Aldh5a1^−/− mice. We also show that Aldh5a1^−/− mice have significant reductions in hippocampal ATP levels as compared to controls, and that the KD restores ATP in mutant mice to normal levels.Taken together, our data suggest that the KD's actions on brain mitochondria may play a role in the diet's ability to treat murine SSADH deficiency.

Keywords: Ketogenic diet; Succinic semialdehyde dehydrogenasedeficiency; Mitochondria; ATP; Hippocampus

Endothelin-1 stimulates interleukin-6 secretion from 3T3-L1 adipocytes by Shin-Pei Chai; Yin-Nan Chang; Jim C. Fong ⁎ (pp. 213-218).

Since both endothelin-1 (ET-1) and interleukin-6 (IL-6) may induce insulin resistance and adipose tissue is a major contributor of circulating IL-6, we examined the effects of ET-1 on IL-6 secretion from 3T3-L1 adipocytes.IL-6 release was measured by ELISA. RT-PCR and real-time PCR analyses were used to determine cellular IL-6 mRNA levels. A luciferase reporter driven by promoter (−1310/+198) of mouse IL-6 gene was transfected into 3T3-L1 adipocytes to monitor IL-6 transcription.Treatment of adipocytes with ET-1 dose- and time-dependently increased IL-6 secretion. The stimulatory effect of ET-1 on IL-6 secretion was abolished by actinomycin D and ET-1 induced an increase in IL-6 mRNA levels. ET-1 was able to enhance the IL-6 promoter activity and its stimulatory effect was inhibited by GF109203X, U0126, salicylate, dominant negative CREB and mithramycin A. Thus it appears that ET-1 may stimulate IL-6 secretion mainly through an enhanced IL-6 transcription, by a mechanism involving both protein kinase C and p42/p44 mitogen-activated protein kinase, and probably downstream NF-κB, CREB and Sp1 transcription factors.This study demonstrates that ET-1 is able to increase IL-6 secretion from adipocytes and raises the possibility that ET-1-induced insulin resistance may be mediated by IL-6.

Keywords: Endothelin-1; Interleukin-6; Adipocyte; IL-6 promoter

Sections

Personal tools

BBA - General Subjects (v.1790, #3)

Sections

Personal tools

Local resources

BBA - General Subjects (v.1790, #3)