bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary supplementation of (+)‐catechin protects against UVB‐induced skin damage by modulating antioxidant enzyme activities
Authors: Jeon S. E. , Choi‐Kwon S. , Park K. A. , Lee H. J. , Park M. S. , Lee J. H. , Kwon S. B. , Park K. C.
Published: August 2003

Purpose: The aim of this study was to investigate the effects of dietary supplementation with (+)‐catechin on cutaneous antioxidant enzymes and the skin damage caused by UVB irradiation.

Methods: BALB/c mice were divided into three groups. Each group was fed a regular diet (RD) or a 2% catechin‐supplemented diet for either 2 weeks (2CSD) or 4 weeks (4CSD) ad libitum prior to UVB irradiation. Skin was removed for the antioxidant enzyme assay, hematoxylin and eosin staining, and the TEM analysis before and at various time points after UVB (200 mJ/cm2) irradiation.

Results: Before UVB irradiation, the superoxide dismutase (SOD) and catalase (CAT) activities of the 2CSD and the 4CSD groups were found to be lower than those of the RD group, whereas the glutathione peroxidase (GPx) activity of the 4CSD group was higher than those of the RD and the 2CSD groups (P<0.05). The SOD and CAT activities of the RD group decreased after UVB irradiation, while those of the 2CSD and the 4CSD groups increased immediately after irradiation and then decreased (P<0.05). Immediately after UVB irradiation, the GPx activities of the 4CSD and the 2CSD groups increased, but that of the RD group decreased. The GPx activity of all three groups showed a tendency to return to pre‐UVB irradiation levels with time. Light microscopic findings of the RD group showed epidermal thinning and apoptotic cells at 24 h after UVB irradiation and mostly necrotic cells at 48 h, whereas only moderate thickening of the epidermis was observed in the 2CSD group at 48 h after irradiation. An electron microscopic examination produced similar findings. At 48 h after irradiation, nearly all epidermal cells seemed to be damaged in the RD group as compared to the 2CSD group.

Conclusion: These results demonstrate that dietary supplementation with (+)‐catechin could protect epidermal cells against UVB‐induced damage by modulating antioxidant enzyme activities.
Characterization of the bipartite nuclear localization signal of protein LANA2 from Kaposi's sarcoma-associated herpesvirus
Authors: MUÑOZ-FONTELA Cesar , RODRÍGUEZ Estefanía , NOMBELA Cesar , ARROYO Javier , RIVAS Carmen
Published: August 2003

LANA2 is a nuclear latent protein detected exclusively in Kaposi's sarcoma-associated herpesvirus-infected B cells. The protein inhibits p53-dependent transactivation and apoptosis, suggesting an important role in the transforming activity of the virus. To explore the molecular mechanisms of its nuclear localization, fusion proteins of green fluorescent protein (EGFP) and deletion constructs of LANA2 were expressed in HeLa cells. Only the fragment comprising amino acid residues 355–440 of LANA2 localized in the cell nucleus. This fragment contains two closely located basic domains and forms a putative bipartite nuclear localization signal (NLS). The putative LANA2 NLS was able to target EGFP to the nucleus consistently. Site-directed mutation analyses demonstrated that LANA2 contains a functional bipartite NLS between amino acid positions 367 and 384. In addition, analysis of cells transfected with a cytoplasmic LANA2 mutant revealed that an appropriate subcellular localization may be crucial to regulate p53 activity.
Oxidized low-density lipoprotein induces calpain-dependent cell death and ubiquitination of caspase 3 in HMEC-1 endothelial cells
Authors: PÖRN-ARES M. Isabella , SAIDO Takaomi C. , ANDERSSON Tommy , ARES Mikko P. S.
Published: August 2003

Oxidized low-density lipoprotein (oxLDL) is known to induce apoptosis in endothelial cells, and this is believed to contribute to the progression of atherosclerosis. In the present study we made the novel observation that oxLDL-induced death of HMEC-1 cells is accompanied by activation of calpain. The μ-calpain inhibitor PD 151746 decreased oxLDL-induced cytotoxicity, whereas the general caspase inhibitor BAF (t-butoxycarbonyl-Asp-methoxyfluoromethylketone) had no effect. Also, oxLDL provoked calpain-dependent proteolysis of cytoskeletal α-fodrin in the HMEC-1 cells. Our observation of an autoproteolytic cleavage of the 80 kDa subunit of μ-calpain provided further evidence for an oxLDL-induced stimulation of calpain activity. The Bcl-2 protein Bid was also cleaved during oxLDL-elicited cell death, and this was prevented by calpain inhibitors, but not by inhibitors of cathepsin B and caspases. Treating the HMEC-1 cells with oxLDL did not result in detectable activation of procaspase 3 or cleavage of PARP [poly(ADP-ribose) polymerase], but it did cause polyubiquitination of caspase 3, indicating inactivation and possible degradation of this protease. Despite the lack of caspase 3 activation, oxLDL treatment led to the formation of nucleosomal DNA fragments characteristic of apoptosis. These novel results show that oxLDL initiates a calpain-mediated death-signalling pathway in endothelial cells.
Regulation of alternative splicing of Gtf2ird1 and its impact on slow muscle promoter activity
Authors: TAY Enoch S. E. , GUVEN Kim L. , SUBRAMANIAM Nanthakumar , POLLY Patsie , ISSA Laura L. , GUNNING Peter W. , HARDEMAN Edna C.
Published: August 2003

A human MusTRD [muscle TFII-I repeat domain (RD)-containing protein] isoform was originally identified in a yeast one-hybrid screen as a protein that binds the slow fibre-specific enhancer of the muscle gene troponin I slow [O'Mahoney, Guven, Lin, Joya, Robinson, Wade and Hardeman (1998) Mol. Cell. Biol. 18, 6641–6652]. MusTRD shares homology with the general transcription factor TFII-I by the presence of diagnostic I-RDs [Roy (2001) Gene 274, 1–13]. The human gene encoding MusTRD, GTF2IRD1 (WBSCR11/GTF3), was subsequently located on chromosome 7q11.23, a region deleted in the neurodegenerative disease, Williams–Beuren Syndrome [Osborne, Campbell, Daradich, Scherer, Tsui, Franke, Peoples, Francke, Voit, Kramer et al. (1999) Genomics 57, 279–284; Franke, Peoples and Francke (1999) Cytogenet. Cell. Genet. 86, 296–304; Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur. J. Hum. Genet. 7, 737–747]. The haploinsufficiency of MusTRD has been implicated in the myopathic aspect of this disease, which manifests itself in symptoms such as lowered resistance to fatigue, kyphoscoliosis, an abnormal gait and joint contractures [Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur. J. Hum. Genet. 7, 737–747]. Here, we report the identification of 11 isoforms of MusTRD in mouse skeletal muscles. These isoforms were isolated from a mouse skeletal muscle cDNA library and reverse transcription–PCR on RNA from various adult and embryonic muscles. The variability in these isoforms arises from alternative splicing of a combination of four cassettes and two mutually exclusive exons, all in the 3′ region of the primary transcript of Gtf2ird1, the homologous mouse gene. The expression of some of these isoforms is differentially regulated spatially, suggesting individual regulation of the expression of these isoforms. Co-transfection studies in C2C12 muscle cell cultures reveal that isoforms differentially regulate muscle fibre-type-specific promoters. This indicates that the presence of different domains of MusTRD influences the activity exerted by this molecule on multiple promoters active in skeletal muscle.
Subsite structure of the endo-type chitin deacetylase from a Deuteromycete, Colletotrichum lindemuthianum: an investigation using steady-state kinetic analysis and MS
Authors: HEKMAT Omid , TOKUYASU Ken , WITHERS Stephen G.
Published: August 2003

The endo-type chitin deacetylase (EC 3.5.1.41) from a Deuteromycete, Colletotrichum lindemuthianum (ATCC 56676), catalyses the hydrolysis of the acetamido group of GlcNAc (2-acetamido-2-deoxy-d-glucose) residues in chitin or chito-oligosaccharides with a degree of polymerization (n) equal to or greater than 2. The steady-state kinetic parameters for the initial deacetylation reactions of (GlcNAc)2–6 were determined using a direct, continuous spectrophotometric assay in combination with ESI-MS (electrospray ionization MS) analysis of the products. The dependence of the observed Km and kcat/Km on n suggests the presence of four enzyme subsites (−2, −1, 0 and +1) that interact with GlcNAc residues from the non-reducing end to the reducing end of the substrate. The turnover number (kcat, 7 s−1) is independent of n and represents the intrinsic rate constant (kint) for the hydrolysis of the acetamido group in subsite 0. The subsite affinities for the GlcNAc residues were calculated from the observed kcat/Km values (A−2, −11.0; A−1, −1.5; A0, −7.7; A+1, −12.5 kJ·mol−1). The increments in the subsite affinities due to the recognition of the acetamido groups were calculated [ΔΔG(N-acetyl)=3.3, 0, 4.0 and 0 kJ·mol−1 for subsites −2, −1, 0 and +1 respectively]. The steady-state kinetic parameters for the second deacetylation reaction of (GlcNAc)4 were also determined using (GlcNAcGlcNAcGlcNGlcNAc) as the substrate. The comparison of the experimental and theoretical values (calculated using the subsite affinities) suggests that the mono-deacetylated substrate binds strongly in a non-productive mode occupying all four subsites, thereby inhibiting the second deacetylation reaction.
ATP-binding sites in brain p97/VCP (valosin-containing protein), a multifunctional AAA ATPase
Authors: ZALK Ran , SHOSHAN-BARMATZ Varda
Published: August 2003

VCP (valosin-containing protein) or p97 is a member of the AAA family (ATPases associated with a variety of cellular activities family), a diverse group of proteins sharing a key conserved AAA module containing duplicate putative ATP-binding sites. Although the functions of the AAA family are related to their putative ATP-binding sites, the binding of ATP to these sites has not yet been demonstrated. In the present study, the ATP-binding site(s) of brain VCP was characterized using the photoreactive ATP analogue, BzATP [3′-O-(4-benzoylbenzoyl)ATP]. Photo-activation of Bz-[α-32P]ATP resulted in its covalent binding to a 97-kDa purified soluble or membrane-associated protein, identified by amino acid sequencing as VCP. Bz-[α-32P]ATP covalently bound to the purified homo-hexameric VCP with an apparent high affinity (74–111 nM). A molar stoichiometry of 2.23±0.14 BzATP bound per homo-hexameric VCP (n=6) was determined using different methods for analysis of radiolabelling and protein determination. Nucleotides inhibited the binding of Bz-[α-32P]ATP to VCP with the following efficiency: BzATP>ATP>ADP≫adenosine 5′-[β,γ-imido]triphosphate≥adenosine 5′-[β,γ-methylene]triphosphate, whereas AMP, GTP and CTP were ineffective. VCP was observed to possess very low ATPase activity, with nucleotide specificity similar to that for BzATP binding. Conformational changes induced by an alternating site mechanism for ATP binding are suggested as a molecular mechanism for coupling ATP binding to the diverse activities of the AAA family.
Expression of melanotransferrin isoforms in human serum: relevance to Alzheimer's disease
Authors: DESROSIERS Richard R. , BERTRAND Yanick , NGUYEN Quynh-Tran , DEMEULE Michel , GABATHULER Reinhard , KENNARD Malcolm L. , GAUTHIER Serge , BÉLIVEAU Richard
Published: August 2003

Levels of soluble melanotransferrin in serum have been reported to be higher in patients with Alzheimer's disease than in control subjects. The present study investigated melanotransferrin in human body fluids in the light of these findings. To clarify the correlation between melanotransferrin and Alzheimer's disease, the melanotransferrin content was determined by non-reducing, denaturing SDS/PAGE and Western blotting. Under these conditions, serum melanotransferrin migrated at 79 and 82 kDa. Melanotransferrin antigenicity and the relative proportions of the two forms were very sensitive to factors that altered its conformation, including disulphide bridges, pH and bivalent cations. Serum melanotransferrin levels were not significantly different between control subjects and patients with Alzheimer's disease using whole serum, EDTA-supplemented serum or serum immunoglobulin-depleted by Protein G–Sepharose and enriched by affinity precipitation with the lectin from Asparagus pea. Glycosylated forms of serum melanotransferrin bound to Asparagus lectin manifested similar patterns on two-dimensional gel electrophoresis in samples from controls and Alzheimer's disease subjects. Melanotransferrin was also present in saliva and at a high level in urine, but contents were similar in controls and patients with Alzheimer's disease. Together, these results demonstrate that serum melanotransferrin exists in various conformations depending on the binding of bivalent cations or following post-translational modification. These data also indicate that human serum melanotransferrin levels are unchanged in subjects with Alzheimer's disease.
Phosphorylation of calcipressin 1 increases its ability to inhibit calcineurin and decreases calcipressin half-life
Authors: GENESCÀ Lali , AUBAREDA Anna , FUENTES Juan J. , ESTIVILL Xavier , DE LA LUNA Susana , PÉREZ-RIBA Mercè
Published: August 2003

Calcipressin 1 is an endogenous inhibitor of calcineurin, which is a serine/threonine phosphatase under the control of Ca2+ and calmodulin. Calcipressin 1 is encoded by DSCR1, a gene on human chromosome 21 with seven exons, exons 1–4 are alternative first exons (isoforms 1–4). We show that calcipressin 1 isoform 1 has an N-terminal coding region longer than that previously described, and this generates a new polypeptide of 252 amino acids. This polypeptide is able to interact with calcineurin A and to inhibit NF-AT-mediated transcriptional activation. We demonstrate for the first time that endogenous calcipressin 1 exists as a complex together with the calcineurin A and B heterodimer. Calcipressin 1 is a phosphoprotein that increases its capacity to inhibit calcineurin when phosphorylated at the FLISPP motif, and this phosphorylation also controls the half-life of calcipressin 1 by accelerating its degradation. Additionally, we have also detected further phosphorylation sites outside the FLISPP motif and these contribute to the complex phosphorylation pattern of calcipressin 1. Taking all these results into consideration we suggest that phosphorylation of calcipressin 1 is involved in the regulation of the phosphatase activity of calcineurin and can therefore act as a modulator of calcineurin-dependent cellular pathways.
Polyamines reverse non-steroidal anti-inflammatory drug-induced toxicity in human colorectal cancer cells
Authors: HUGHES Alun , SMITH Nicholas I. , WALLACE Heather M.
Published: August 2003

Naproxen, sulindac and salicylate, three NSAIDs (non-steroidal anti-inflammatory drugs), were cytotoxic to human colorectal cancer cells in culture. Toxicity was accompanied by significant depletion of intracellular polyamine content. Inhibition of ornithine decarboxylase (the first enzyme of the polyamine biosynthetic pathway), induction of polyamine oxidase and spermidine/spermine N1-acetyltransferase (the enzymes responsible for polyamine catabolism) and induction of polyamine export all contributed to the decreased intracellular polyamine content. Morphological examination of the cells showed typical signs of apoptosis, and this was confirmed by DNA fragmentation and measurement of caspase-3-like activity. Re-addition of spermidine to the cells partially prevented apoptosis and recovered the cell number. Thus polyamines appear to be an integral part of the signalling pathway mediating NSAID toxicity in human colorectal cancer cells, and may therefore also be important in cancer chemoprevention in humans.
An antitumour lectin from the edible mushroom Agrocybe aegerita
Authors: ZHAO Chenguang , SUN Hui , TONG Xin , QI Yipeng
Published: August 2003

An antitumour lectin (named AAL) consisting of two identical subunits of 15.8 kDa was isolated from the fruiting bodies of the edible mushroom Agrocybe aegerita using a procedure which involved precipitating the extract by addition of (NH4)2SO4, ion exchange chromatography on DEAE-Sepharose Fast Flow, gel filtration chromatography on Sephacryl S-200 HR and finally purification on a GF-250 HPLC column. Amino acid analysis of the N-terminus and an internal fragment indicated that the sequences of the two fragments were QGVNIYNI and Q(K)PDGPWLVEK(Q)R respectively. AAL showed strong inhibition of the growth of human tumour cell lines HeLa, SW480, SGC-7901, MGC80-3, BGC-823, HL-60 and mouse sarcoma S-180. AAL also inhibited the viability of S-180 tumour cells in vivo. Analysis by Hoechst 33258 staining, MitoSensor Kit and flow cytometry showed that AAL induced apoptosis in HeLa cells. TUNEL (terminal transferase deoxytidyl uridine end labelling) analysis of slides of tumour tissues excised from BALB/c mice also demonstrated the apoptosis-induction activity of the lectin. Furthermore, AAL was shown to possess DNase activity in assays using plasmid pCDNA3 and salmon sperm DNA. Based on the results obtained in these assays, we conclude that AAL exerts its antitumour effects via apoptosis-inducing and DNase activities.
Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+
Authors: WERRY Tim D. , WILKINSON Graeme F. , WILLARS Gary B.
Published: August 2003

Alteration in [Ca2+]i (the intracellular concentration of Ca2+) is a key regulator of many cellular processes. To allow precise regulation of [Ca2+]i and a diversity of signalling by this ion, cells possess many mechanisms by which they are able to control [Ca2+]i both globally and at the subcellular level. Among these are many members of the superfamily of GPCRs (G-protein-coupled receptors), which are characterized by the presence of seven transmembrane domains. Typically, those receptors able to activate PLC (phospholipase C) enzymes cause release of Ca2+ from intracellular stores and influence Ca2+ entry across the plasma membrane. It has been well documented that Ca2+ signalling by one type of GPCR can be influenced by stimulation of a different type of GPCR. Indeed, many studies have demonstrated heterologous desensitization between two different PLC-coupled GPCRs. This is not surprising, given our current understanding of negative-feedback regulation and the likely shared components of the signalling pathway. However, there are also many documented examples of interactions between GPCRs, often coupling preferentially to different signalling pathways, which result in a potentiation of Ca2+ signalling. Such interactions have important implications for both the control of cell function and the interpretation of in vitro cell-based assays. However, there is currently no single mechanism that adequately accounts for all examples of this type of cross-talk. Indeed, many studies either have not addressed this issue or have been unable to determine the mechanism(s) involved. This review seeks to explore a range of possible mechanisms to convey their potential diversity and to provide a basis for further experimental investigation.
Membrane disruption and cytotoxicity of hydrophobic N-alkylated imino sugars is independent of the inhibition of protein and lipid glycosylation
Authors: MELLOR Howard R. , PLATT Frances M. , DWEK Raymond A. , BUTTERS Terry D.
Published: August 2003

The N-alkyl moiety of N-alkylated imino sugars is crucial for therapeutic activities of these compounds as inhibitors of glycosphingolipid (GSL) biosynthesis and as antivirals. The improved potency afforded by a long N-alkyl moiety is coincident with increased compound-induced cytotoxicity. Therefore, in the present study, we examined the mechanism of this cytotoxicity in detail. Despite N-butyl-deoxynojirimycin and N-butyl-deoxygalactonojirimycin inhibiting the glycosylation of ceramide to glucosylceramide, ceramide levels did not increase in HL60 cells treated with these compounds. Long-chain N-alkylated imino sugars were toxic to cells at concentrations considerably lower than the critical micellar concentrations for these compounds and consequently did not solubilize radioactively labelled cellular proteins and lipids. However, membrane disruption and cell fragmentation did increase in a concentration- and chain-length-dependent manner. These results are consistent with previously proposed interactions between surface-active amphiphiles and protein-containing lipid membranes when drug concentrations are below the critical micellar concentration. Taken together, these results demonstrate that the cellular toxicity of hydrophobic N-alkylated imino sugars is due to cell lysis and cell fragmentation and, most importantly, is not related to the beneficial therapeutic effects of these compounds on protein and in lipid glycosylation. This information will aid in the future development of more selective imino sugar therapeutics for the treatment of human disease.
Hsp90 is a direct target of the anti-allergic drugs disodium cromoglycate and amlexanox
Authors: OKADA Miki , ITOH Hideaki , HATAKEYAMA Takashi , TOKUMITSU Hiroshi , KOBAYASHI Ryoji
Published: August 2003

Hsp90 (heat-shock protein 90) alone can act to prevent protein aggregation and promote refolding in vitro, but in vivo it operates as a part of a multichaperone complex, which includes Hsp70 and cohort proteins. Since the physiological function of Hsp90 is not yet fully understood, the development of specific antagonists might open new lines of investigation on the role of Hsp90. In an effort to discover Hsp90 antagonists, we screened many drugs and found that the anti-allergic drugs DSCG (disodium cromoglycate) and amlexanox target Hsp90. Both drugs were found to bind directly wild-type Hsp90 via the N- and C-terminal domains. Both drugs strongly suppressed the in vitro chaperone activity of native Hsp90 towards citrate synthase at 1.5–3.0 μM. Amlexanox suppressed C-terminal chaperone activity in vitro, but not N-terminal chaperone activity, and inhibited the association of cohort proteins, such as cyclophilin 40 and Hsp-organizing protein, to the C-terminal domain of Hsp90. These data suggest that amlexanox might disrupt the multichaperone complex, including Hsp70 and cohort proteins, both in vitro and in vivo. Although DSCG inhibited the in vitro chaperone activity of the N-terminal domain, the drug had no effect either on the C-terminal chaperone activity or on the association of the cohort proteins with the C-terminus of Hsp90. The physiological significance of these interactions in vivo remains to be investigated further, but undoubtedly must be taken into account when considering the pharmacology of anti-allergic drugs. DSCG and amlexanox may serve as useful tools for evaluating the physiological significance of Hsp90.
Monitoring disappearance of monomers and generation of resistance to proteolysis during the formation of the activation domain of human procarboxypeptidase A2 (ADA2h) amyloid fibrils by matrix-assisted laser-desorption ionization–time-of-flight-MS
Authors: VILLANUEVA Josep , VILLEGAS Virtudes , QUEROL Enrique , AVILÉS Francesc X. , SERRANO Luis
Published: August 2003

The term ‘amyloidosis’ is used to represent a group of protein misfolding diseases characterized by the polymerization of normally innocuous and soluble proteins or peptides into insoluble proteinaceous deposits. One of the several questions that remain unclear regarding the process of amyloid fibril formation is related to the status of the protein when such a process begins. Protein engineering is one of the selected approaches to study amyloidosis. Characterization of many variants of a protein can give information about why a soluble protein aggregates to form fibrils. In the present study, we report information on the conformational changes that precede the formation of fibrils, monitored by the complementary use of exoproteolysis and matrix-assisted laser-desorption ionization–time-of-flight-MS. This is a novel application of an easy and fast approach. In addition, we used it to evaluate the ability of the model protein ADA2h (activation domain of human procarboxypeptidase A2) and their mutants to generate amyloid fibrils. It could be a useful test to screen protein variants and to study to what extent some physicochemical parameters affect fibrillogenesis.
Differential spatial and temporal phosphorylation of the visual receptor, rhodopsin, at two primary phosphorylation sites in mice exposed to light
Authors: ADAMS Ryan A. , LIU Xinran , WILLIAMS David S. , NEWTON Alexandra C.
Published: August 2003

Phosphorylation of rhodopsin critically controls the visual transduction cascade by uncoupling it from the G-protein transducin. The kinase primarily responsible for this phosphorylation is rhodopsin kinase, a substrate-regulated kinase that phosphorylates light-activated rhodopsin. Protein kinase C has been implicated in controlling the phosphorylation of both light-activated and dark-adapted rhodopsin. Two of the major rhodopsin phosphorylation sites in vivo, Ser334 and Ser338, are effective protein kinase C phosphorylation sites in vitro, while the latter is preferentially phosphorylated by rhodopsin kinase in vitro. Using phosphospecific antibodies against each of these two sites, we show that both sites are under differential spatial and temporal regulation. Exposure of mice to light results in rapid phosphorylation of Ser338 that is evenly distributed along the rod outer segment. Phosphorylation of Ser334 is considerably slower, begins at the base of the rod outer segment, and spreads to the top of the photoreceptor over time. In addition, we show that phosphorylation of both sites is abolished in rhodopsin kinase−/− mice, revealing an absolute requirement for rhodopsin kinase to phosphorylate rhodopsin. This requirement may reflect the need for priming phosphorylations at rhodopsin kinase sites allowing for subsequent phosphorylation by protein kinase C at Ser334. In this regard, treatment of mouse retinas with phorbol esters results in a 4-fold increase in phosphorylation on Ser334, with no significant effect on the phosphorylation of Ser338. Our results are consistent with light triggering rapid priming phosphorylations of rhodopsin by rhodopsin kinase, followed by a slower phosphorylation on Ser334, which is regulated by protein kinase C.
Hydride transfer during catalysis by dihydrofolate reductase from Thermotoga maritima
Authors: MAGLIA Giovanni , JAVED Masood H. , ALLEMANN Rudolf K.
Published: August 2003

DHFR (dihydrofolate reductase) catalyses the metabolically important reduction of 7,8-dihydrofolate by NADPH. DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR), which shares similarity with DHFR from Escherichia coli, has previously been characterized structurally. Its tertiary structure is similar to that of DHFR from E. coli but it is the only DHFR characterized so far that relies on dimerization for stability. The midpoint of the thermal unfolding of TmDHFR was at approx. 83 °C, which was 30 °C higher than the melting temperature of DHFR from E. coli. The turnover and the hydride-transfer rates in the kinetic scheme of TmDHFR were derived from measurements of the steady-state and pre-steady-state kinetics using absorbance and stopped-flow fluorescence spectroscopy. The rate constant for hydride transfer was found to depend strongly on the temperature and the pH of the solution. Hydride transfer was slow (0.14 s−1 at 25 °C) and at least partially rate limiting at low temperatures but increased dramatically with temperature. At 80 °C the hydride-transfer rate of TmDHFR was 20 times lower than that observed for the E. coli enzyme at its physiological temperature. Hydride transfer depended on ionization of a single group in the active site with a pKa of 6.0. While at 30 °C, turnover of substrate by TmDHFR was almost two orders of magnitude slower than by DHFR from E. coli; the steady-state rates of the two enzymes differed only 8-fold at their respective working temperatures.
Protein S-thiolation targets glycolysis and protein synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae
Authors: SHENTON Daniel , GRANT Chris M.
Published: August 2003

The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, a process by which protein SH groups form mixed disulphides with low-molecular-mass thiols such as glutathione. We report here the target proteins which are modified in yeast cells in response to H2O2. In particular, a range of glycolytic and related enzymes (Tdh3, Eno2, Adh1, Tpi1, Ald6 and Fba1), as well as translation factors (Tef2, Tef5, Nip1 and Rps5) are identified. The oxidative stress conditions used to induce S-thiolation are shown to inhibit GAPDH (glyceraldehyde-3-phosphate dehydrogenase), enolase and alcohol dehydrogenase activities, whereas they have no effect on aldolase, triose phosphate isomerase or aldehyde dehydrogenase activities. The inhibition of GAPDH, enolase and alcohol dehydrogenase is readily reversible once the oxidant is removed. In addition, we show that peroxide stress has little or no effect on glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, the enzymes that catalyse NADPH production via the pentose phosphate pathway. Thus the inhibition of glycolytic flux is proposed to result in glucose equivalents entering the pentose phosphate pathway for the generation of NADPH. Radiolabelling is used to confirm that peroxide stress results in a rapid and reversible inhibition of protein synthesis. Furthermore, we show that glycolytic enzyme activities and protein synthesis are irreversibly inhibited in a mutant that lacks glutathione, and hence cannot modify proteins by S-thiolation. In summary, protein S-thiolation appears to serve an adaptive function during exposure to an oxidative stress by reprogramming metabolism and protecting protein synthesis against irreversible oxidation.
Induced expression of manganese superoxide dismutase by non-toxic concentrations of oxidized low-density lipoprotein (oxLDL) protects against oxLDL-mediated cytotoxicity
Authors: SHATROV Vladimir A. , BRÜNE Bernhard
Published: August 2003

Oxidized low-density lipoprotein (oxLDL) affects macrophages and plays a critical role in the development of atherosclerosis. In the present paper, we demonstrate that high concentrations of oxLDL provoked apoptosis of human Mono-Mac-6 cells, which was blocked by diphenylene-iodonium (DPI), an inhibitor of flavin-containing enzymes, such as NADPH oxidase, suggesting the involvement of reactive oxygen species (ROS). Importantly, pre-treatment of cells with low concentrations of oxLDL prevented apoptosis in response to high concentrations of oxLDL by up-regulating manganese superoxide dismutase (MnSOD). DPI prevented expression of MnSOD by oxLDL, whereas inhibitors of cytochrome P450 (methoxalen) or xanthine oxidase (allopurinol) did not, thus pointing to a role of NADPH-oxidase-derived ROS in oxLDL-induced MnSOD expression. Transfection of cells with MnSOD antisense, but not scrambled antisense, oligonucleotides significantly attenuated oxLDL-mediated MnSOD expression and hindered cytoprotective effects of non-toxic oxLDL concentrations. Our findings suggest that up-regulation of MnSOD by low concentrations of oxLDL is critical for protection towards oxLDL-mediated cytotoxicity.
Replacement of the catalytic nucleophile cysteine-296 by serine in class II polyhydroxyalkanoate synthase from Pseudomonas aeruginosa-mediated synthesis of a new polyester: identification of catalytic residues
Authors: AMARA Amro A. , REHM Bernd H. A.
Published: August 2003

The class II PHA (polyhydroxyalkanoate) synthases [PHAMCL synthases (medium-chain-length PHA synthases)] are mainly found in pseudomonads and catalyse synthesis of PHAMCLs using CoA thioesters of medium-chain-length 3-hydroxyfatty acids (C6–C14) as a substrate. Only recently PHAMCL synthases from Pseudomonas oleovorans and Pseudomonas aeruginosa were purified and in vitro activity was achieved. A threading model of the P. aeruginosa PHAMCL synthase PhaC1 was developed based on the homology to the epoxide hydrolase (1ek1) from mouse which belongs to the α/β-hydrolase superfamily. The putative catalytic residues Cys-296, Asp-452, His-453 and His-480 were replaced by site-specific mutagenesis. In contrast to class I and III PHA synthases, the replacement of His-480, which aligns with the conserved base catalyst of the α/β-hydrolases, with Gln did not affect in vivo enzyme activity and only slightly in vitro enzyme activity. The second conserved histidine His-453 was then replaced by Gln, and the modified enzyme showed only 24% of wild-type in vivo activity, which indicated that His-453 might functionally replace His-480 in class II PHA synthases. Replacement of the postulated catalytic nucleophile Cys-296 by Ser only reduced in vivo enzyme activity to 30% of wild-type enzyme activity and drastically changed substrate specificity. Moreover, the C296S mutation turned the enzyme sensitive towards PMSF inhibition. The replacement of Asp-452 by Asn, which is supposed to be required as general base catalyst for elongation reaction, did abolish enzyme activity as was found for the respective amino acid residue of class I and III enzymes. In the threading model residues Cys-296, Asp-452, His-453 and His-480 reside in the core structure with the putative catalytic nucleophile Cys-296 localized at the highly conserved γ-turns of the α/β-hydrolases. Inhibitor studies indicated that catalytic histidines reside in the active site. The conserved residue Trp-398 was replaced by Phe and Ala, respectively, which caused inactivation of the enzyme indicating an essential role of this residue. In the threading model this residue was found to be surface-exposed. No evidence for post-translational modification by 4-phosphopantetheine was obtained. Overall, these data suggested that in class II PHA synthases the conserved histidine which was found as general base catalyst in the catalytic triad of enzymes related to the α/β-hydrolase superfamily, was functionally replaced by His-453 which is conserved among all PHA synthases.
Mammalian NADH diphosphatases of the Nudix family: cloning and characterization of the human peroxisomal NUDT12 protein
Authors: ABDELRAHEIM Salama R. , SPILLER David G. , McLENNAN Alexander G.
Published: August 2003

The human NUDT12 Nudix hydrolase has been expressed in insect cells from a baculovirus vector as a His-tagged recombinant protein. In vitro, it efficiently hydrolyses NAD(P)H to NMNH and AMP (2′,5′-ADP), and diadenosine diphosphate to AMP. It also has activity towards NAD(P)+, ADP-ribose and diadenosine triphosphate. Km values for NADH, NADPH and NAD+ are 11, 16 and 190 μM and kcat values are 11, 16 and 10.5 s−1 respectively. Thus, like other NADH diphosphatases of the Nudix family, NUDT12 has a marked substrate preference for the reduced nicotinamide nucleotides. Optimal activity was supported by 50 μM Mn2+ ions in vitro, with 3-fold lower activity at 0.4 mM Mg2+. Expression of NUDT12 as a C-terminal fusion to green fluorescent protein revealed that it was targeted to peroxisomes by the C-terminal tripeptide PNL acting as a novel type 1 peroxisomal targeting signal. Deletion of PNL resulted in diffuse cellular fluorescence. In addition, C-terminal, but not N-terminal, fusions with or without the PNL signal accumulated in large, unidentified cytoplasmic structures. NUDT12 may act to regulate the concentration of peroxisomal nicotinamide nucleotide cofactors required for oxidative metabolism in this organelle.
Association of human kinesin superfamily protein member 4 with BRCA2-associated factor 35
Authors: Mi LEE Young , KIM Wankee
Published: August 2003

A large portion of human kinesin superfamily protein member 4 (KIF4) is associated with the nuclear matrix during the interphase, while a small portion is found in the cytoplasm. During mitosis, it is associated with chromosomes throughout the entire process. In the present study, we identified a protein that interacts with KIF4 using a yeast two-hybrid system, co-immunoprecipitation and co-fractionation. This protein is BRCA2-associated factor 35 (BRAF35) containing a non-specific DNA binding high-mobility-group domain and a kinesin-like coiled-coil domain. It appeared that the interaction between the two proteins occurs through their respective α-helical coiled-coil domains. The co-fractionation experiment revealed that KIF4 and BRAF35 were present in a complex of approx. 540 kDa. The composition and biological significance of this complex should be studied further.
Phosphorylation of BATF regulates DNA binding: a novel mechanism for AP-1 (activator protein-1) regulation
Authors: DEPPMANN Christopher D. , THORNTON Tina M. , UTAMA Fransiscus E. , TAPAROWSKY Elizabeth J.
Published: August 2003

BATF is a member of the AP-1 (activator protein-1) family of bZIP (basic leucine zipper) transcription factors that form transcriptionally inhibitory, DNA binding heterodimers with Jun proteins. In the present study, we demonstrate that BATF is phosphorylated in vivo on multiple serine and threonine residues and at least one tyrosine residue. Reverse-polarity PAGE revealed that serine-43 and threonine-48 within the DNA binding domain of BATF are phosphorylated. To model phosphorylation of the BATF DNA binding domain, serine-43 was replaced by an aspartate residue. BATF(S43D) retains the ability to dimerize with Jun proteins in vitro and in vivo, and the BATF(S43D):Jun heterodimer localizes properly to the nucleus of cells. Interestingly, BATF(S43D) functions like wild-type BATF to reduce AP-1-mediated gene transcription, despite the observed inability of the BATF(S43D):Jun heterodimer to bind DNA. These data demonstrate that phosphorylation of serine-43 converts BATF from a DNA binding into a non-DNA binding inhibitor of AP-1 activity. Given that 40% of mammalian bZIP transcription factors contain a residue analogous to serine-43 of BATF in their DNA binding domains, the phosphorylation event described here represents a mechanism that is potentially applicable to the regulation of many bZIP proteins.
Effect of a null mutation of the oviduct-specific glycoprotein gene on mouse fertilization
Authors: ARAKI Yoshihiko , NOHARA Makoto , YOSHIDA-KOMIYA Hiromi , KURAMOCHI Takashi , ITO Mamoru , HOSHI Hiroyoshi , SHINKAI Yoichi , SENDAI Yutaka
Published: August 2003

The mammalian fertilization process takes place in a complex microenvironment within the female genital tract. A member of the chitinase protein family, oviduct-specific glycoprotein (OGP), has been identified in oviductal fluid from various mammalian species, including humans. Although OGP is widely believed to be involved in the process of mammalian fertilization, including spermatozoon function and gamete interactions, based on experimental results obtained in vitro, its physiological significance remains controversial. The present study established OGP gene-null (ogp−/−) mice, and primarily characterized their reproductive properties to study the physiological function(s) of OGP. Results obtained from studies using an in vivo or in vitro system showed that the fertility of ogp−/− females was within normal limits. These results indicate that OGP is not essential for the process of in vivo fertilization, at least in mice.
Rab5 and Rab11 mediate transferrin and anti-variant surface glycoprotein antibody recycling in Trypanosoma brucei
Authors: PAL Arun , HALL Belinda S. , JEFFRIES Tim. R. , FIELD Mark C.
Published: August 2003

The mammalian-infective bloodstream form of Trypanosoma brucei possesses a highly active endocytotic system. Evasion of the host immune response by T. brucei is dependent on antigenic variation of VSG (variant surface glycoprotein), but additional mechanisms for removal of surface-bound antibody also operate. Four Rab proteins, Tb (trypanosomal) RAB4, 5A, 5B and 11 are located to the endosomal system; TbRAB5A and TbRAB11 co-localize with internalized anti-VSG antibody and transferrin. A live cell assay was used to record a single cycle of endocytosis of anti-VSG IgG and transferrin, their subsequent degradation within the endosomal system and exocytosis of the products. TbRAB5A and TbRAB11 were involved in the overall process of endocytosis, degradation and exocytosis, whereas TbRAB5B and TbRAB4 were not implicated. The kinetics of anti-VSG IgG and transferrin recycling depend on the nucleotide state of TbRAB5A and TbRAB11. These data, together with previous work, suggest that IgG and transferrin initially enter a TbRAB5A sorting endosome and are most probably recycled subsequently via a TbRAB11-dependent step. Analysis of the recycled IgG and transferrin demonstrated extensive degradation of these recycled proteins. Degradation of transferrin was enhanced in cells expressing increased amounts of TbRAB5A or TbRAB11 with a Ser→Asn mutation, but was decreased when active TbRAB11 was overexpressed. The extent of degradation of anti-VSG IgG was found to be unaffected by mutant Rab protein expression. The presence of an efficient mechanism for the removal of IgG bound to the external surface of T. brucei and its subsequent proteolysis within the recycling system suggests a role for this pathway in immune evasion.
The role of two novel regulatory sites in the activation of the cGMP-dependent protein kinase from Plasmodium falciparum
Authors: DENG Wensheng , PARBHU-PATEL Asha , MEYER David J. , BAKER David A.
Published: August 2003

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) uniquely contains three cGMP binding sites, but also has a ‘degenerate’ fourth site. The role of each cGMP-binding site in PfPKG activation remains unknown. We have analysed the effect of mutation of each cGMP-binding site (individually and in combination) on PfPKG activation in vitro. The most striking result was that mutation of cGMP site 3 resulted in a 10–49-fold increase in the Ka(cGMP) value and a 45–55% decrease in maximal activity compared with wild-type. Mutations involving only cGMP-binding sites 1 and 2 had less effect on both the Ka(cGMP) values and the maximal activities. These results suggest that, although all three cGMP-binding sites are involved in PfPKG activation, cGMP-binding site 3 has the greatest influence on activation. A mutation in the fourth, degenerate cGMP-binding site decreased PfPKG maximal activity by 40%, but did not change the Ka(cGMP) value for the PfPKG mutant, suggesting that this site does not bind cGMP, but is required for full activation of PfPKG. The distinct activation properties of PfPKG from mammalian isoforms may be exploitable in the design of a parasite-specific inhibitor and development of a novel anti-malarial drug.
The requirement of cytosolic phospholipase A2 for the PMA activation of proton efflux through the N-terminal 230-amino-acid fragment of gp91phox
Authors: MANKELOW Tosti J. , PESSACH Etai , LEVY Rachel , HENDERSON Lydia M.
Published: August 2003

The absolute requirement for the 85 kDa cytosolic phospholipase A2 (cPLA2) in the PMA stimulation of proton efflux through the NADPH-oxidase-associated proton channel, has previously been demonstrated using a PLB-985 cell line deficient in cPLA2 (PLB-D). The flux of protons in Chinese-Hamster ovary (CHO) cells that express the N-terminal 230-amino-acid (NT) fragment of gp91phox is activated by arachidonic acid (AA) added externally. To investigate the physiological role of cPLA2, and the intracellular AA that it releases, in the activation of proton flux through the NT fragment of gp91phox, this fragment was stably expressed in PLB-985 cells (PLB-985 NT) and in PLB-D cells (PLB-D NT). The expression of the NT fragment of gp91phox by itself in PLB-985 did not initiate differentiation and did not alter their ability to undergo differentiation after the addition of DMSO. Addition of PMA induced a proton efflux from undifferentiated PLB-985 NT cells expressing the NT fragment of gp91phox, which was inhibited by zinc. In contrast, PMA failed to activate proton efflux in undifferentiated PLB-D NT cells, lacking the expression of cPLA2; however, addition of AA restored the efflux of protons in these cells. These results establish an essential and specific physiological requirement of cPLA2-generated AA in the activation of proton flux through the NT fragment of gp91phox.
dDYRK2: a novel dual-specificity tyrosine-phosphorylation-regulated kinase in Drosophila
Authors: LOCHHEAD Pamela A. , SIBBET Gary , KINSTRIE Ross , CLEGHON Tava , RYLATT Margie , MORRISON Deborah K. , CLEGHON Vaughn
Published: August 2003

Dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are an emerging family of protein kinases that have been identified in all eukaryotic organisms examined to date. DYRK family members are involved in regulating key developmental and cellular processes such as neurogenesis, cell proliferation, cytokinesis and cellular differentiation. Two distinct subgroups exist, nuclear and cytosolic. In Drosophila, the founding family member minibrain, whose human orthologue maps to the Down syndrome critical region, belongs to the nuclear subclass and affects post-embryonic neurogenesis. In the present paper, we report the isolation of dDYRK2, a cytosolic DYRK and the putative product of the smell-impaired smi35A gene. This is the second such kinase described in Drosophila, but the first to be characterized at the molecular and biochemical level. dDYRK2 is an 81 kDa dual-specificity kinase that autophosphorylates on tyrosine and serine/threonine residues, but appears to phosphorylate exogenous substrates only on serine/threonine residues. It contains a YXY motif in the activation loop of the kinase domain in the same location as the TXY motif in mitogenactivated protein kinases. dDYRK2 is tyrosine-phosphorylated in vivo, and mutational analysis reveals that the activation loop tyrosines are phosphorylated and are essential for kinase activity. Finally, dDYRK2 is active at all stages of fly development, with elevated levels observed during embryogenesis and pupation.
Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B
Authors: LIZCANO Jose M. , ALRUBAIE Saif , KIELOCH Agnieszka , DEAK Maria , LEEVERS Sally J. , ALESSI Dario R.
Published: August 2003

An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5)P3, which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5)P3 phosphatase dPTEN (Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5)P3 levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well dTOR, are required for the activation of dS6K by insulin.