bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel function of tissue-type transglutaminase: protein disulphide isomerase
Authors: HASEGAWA Go , SUWA Motoi , ICHIKAWA Yasuo , OHTSUKA Tetsuro , KUMAGAI Satoru , KIKUCHI Masashi , SATO Yoshitaka , SAITO Yuji
Published: August 2003

We have found that tissue-type transglutaminase (tTG), also called TGc, TGase2 and Gαh, has the activity of protein disulphide isomerase (PDI). We have shown that tTG converts completely reduced/denatured inactive RNase A molecule to the native active enzyme. The PDI activity of tTG was strongly inhibited by bacitracin, which is a frequently used inhibitor of conventional PDI activity. It was substantially inhibited by the simultaneous presence of other potential substrate proteins such as completely reduced BSA, but not by native BSA. This activity was especially high in the presence of GSSG, but not GSH. The addition of GSH to the reaction mixture in the presence of GSSG at a fixed concentration up to at least 200-fold excess did not very substantially inhibit the PDI activity. It is possible that tTG can exert PDI activity in a fairly reducing environment like cytosol, where most of tTG is found. It is quite obvious from the following observations that PDI activity of tTG is catalysed by a domain different from that used for the transglutaminase reaction. Although the alkylation of Cys residues in tTG completely abolished the transglutaminase activity, as was expected, it did not affect the PDI activity at all. This PDI activity did not require the presence of Ca2+. It was not inhibited by nucleotides including GTP at all, unlike the other activity of tTG.
Regioselectivity of glucosylation of caffeic acid by a UDP-glucose:glucosyltransferase is maintained in planta
Authors: LIM Eng-Kiat , HIGGINS Gillian S. , LI Yi , BOWLES Dianna J.
Published: August 2003

Caffeic acid is a phenylpropanoid playing an important role in the pathways leading to lignin synthesis and the production of a wide variety of secondary metabolites. The compound is also an antioxidant and has potential utility as a general protectant against free radicals. Three glucosylated forms of caffeic acid are known to exist: the 3-O- and 4-O-glucosides and the glucose ester. This study describes for the first time a glucosyltransferase [UDP-glucose:glucosyltransferase (UGT)] that is specific for the 3-hydroxyl, and not the 4-hydroxyl, position of caffeic acid. The UGT sequence of Arabidopsis, UGT71C1, has been expressed as a recombinant fusion protein in Escherichia coli, purified and assayed against a range of substrates in vitro. The assay confirmed that caffeic acid as the preferred substrate when compared with other hydroxycinnamates, although UGT71C1 also exhibited substantial activity towards flavonoid substrates, known to have structural features that can be recognized by many different UGTs. The expression of UGT71C1 in transgenic Arabidopsis was driven by the constitutive cauliflower mosaic virus 35 S (CaMV35S) promoter. Nine independent transgenic lines were taken to homozygosity and characterized by Northern-blot analysis, assay of enzyme activity in leaf extracts and HPLC analysis of the glucosides. The level of expression of UGT71C1 was enhanced considerably in several lines, leading to a higher level of the corresponding enzyme activity and a higher level of caffeoyl-3-O-glucoside. The data are discussed in the context of the utility of UGTs for natural product biotransformations.
Small glutamine-rich tetratricopeptide repeat-containing protein (SGT) interacts with the ubiquitin-dependent endocytosis (UbE) motif of the growth hormone receptor
Authors: SCHANTL Julia A. , ROZA Marcel , De JONG Ad P. , STROUS Ger J.
Published: August 2003

Endocytosis of the growth hormone receptor (GHR) is regulated by the ubiquitin-conjugating system. A cytosolic 10 amino acid motif, referred to as the ubiquitin-dependent endocytosis (UbE) motif, is involved in the ubiquitination as well as in the endocytosis of the receptor. Proteins that are implicated in one of these processes have not been identified so far. Using a glutathione S-transferase (GST)-pulldown assay with a GST fusion protein encompassing the UbE motif of the GHR, a 35 kDa protein was purified. The protein was identified by MS as small glutamine-rich tetratricopeptide repeat (TPR)-containing protein (SGT). We found that GHR interacts with SGT. In vivo, both the precursor and the mature form of the receptor interacted with SGT. Inactivation of the ubiquitin-conjugating system did not affect the GHR–SGT interaction. Binding studies showed that the first TPR motif of SGT interacts with the UbE motif of the GHR. Taken together, these data show that SGT is a GHR-interacting protein, which binds independent of the ubiquitin-conjugating system.
Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells
Authors: CERBÓN Jorge , del Carmen LÓPEZ-SÁNCHEZ Rosa
Published: August 2003

We have investigated the effects of inhibiting sphingomyelin (SM) biosynthesis on cellular diacylglycerol (DAG) content and protein kinase C (PKC) activation during growth initiation in Madin–Darby canine kidney cells. We utilized β-chloroalanine (BCA) to inactivate serine C-palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway. This inactivation prevented growth, but did not affect viability. When the inhibitor was replaced with fresh culture medium, the cells continued their proliferation in a normal way. BCA (2 mM) inhibited [32P]Pi, [3H]palmitic acid and [methyl-3H]choline incorporation into SM, but did not influence the synthesis of other major phospholipids. SM synthesis and DAG generation were decreased by 51% and 47.6% respectively. Particulate PKC activity was not observed in cells incubated with BCA, in contrast with a 5-fold increase in control cells. BCA inhibited 75% of the [3H]thymidine incorporation, and the cells were arrested before the S phase of the cell cycle. Moreover, exogenous d-erythrosphingosine restored SM synthesis, DAG generation and cell proliferation. These data indicate that the contribution of DAG generated during SM synthesis plays an important role in PKC activation and cell proliferation.
Human aldose reductase and human small intestine aldose reductase are efficient retinal reductases: consequences for retinoid metabolism
Authors: CROSAS Bernat , HYNDMAN David J. , GALLEGO Oriol , MARTRAS Sílvia , PARÉS Xavier , FLYNN T. Geoffrey , FARRÉS Jaume
Published: August 2003

Aldo–keto reductases (AKRs) are NAD(P)H-dependent oxidoreductases that catalyse the reduction of a variety of carbonyl compounds, such as carbohydrates, aliphatic and aromatic aldehydes and steroids. We have studied the retinal reductase activity of human aldose reductase (AR), human small-intestine (HSI) AR and pig aldehyde reductase. Human AR and HSI AR were very efficient in the reduction of all-trans-, 9-cis- and 13-cis-retinal (kcat/Km=1100–10300 mM−1·min−1), constituting the first cytosolic NADP(H)-dependent retinal reductases described in humans. Aldehyde reductase showed no activity with these retinal isomers. Glucose was a poor inhibitor (Ki=80 mM) of retinal reductase activity of human AR, whereas tolrestat, a classical AKR inhibitor used pharmacologically to treat diabetes, inhibited retinal reduction by human AR and HSI AR. All-trans-retinoic acid failed to inhibit both enzymes. In this paper we present the AKRs as an emergent superfamily of retinal-active enzymes, putatively involved in the regulation of retinoid biological activity through the assimilation of retinoids from β-carotene and the control of retinal bioavailability.
Functional characterization of the human phosphodiesterase 7A1 promoter
Authors: TORRAS-LLORT Mònica , AZORÍN Fernando
Published: August 2003

In this paper, the human phosphodiesterase 7A1 (hPDE7A1) promoter region was identified and functionally characterized. Transient transfection experiments indicated that a 2.9 kb fragment of the hPDE7A1 5′-flanking region, to position −2907, has strong promoter activity in Jurkat T-cells. Deletion analysis showed that the proximal region, up to position −988, contains major cis-regulatory elements of the hPDE7A1 promoter. This minimal promoter region contains a regulatory CpG island which is essential for promoter activity. The CpG island contains three potential cAMP-response-element-binding protein (CREB)-binding sites that, as judged by in vivo dimethyl sulphate (DMS) footprinting, are occupied in Jurkat T-cells. Moreover, over-expression of CREB results in increased promoter activity, but, on the other hand, promoter activity decreases when a dominant-negative form of CREB (KCREB) is over-expressed. In vivo DMS footprinting strongly indicates that other transcription factors, such Ets-2, nuclear factor of activated T-cells 1 (NFAT-1) and nuclear factor κB (NF-κB), might also contribute to the regulation of hPDE7A1 promoter. Finally, hPDE7A1 promoter was found to be induced by treatment with PMA, but not by treatment with dibutyryl cAMP or forskolin. These results provide insights into the factors and mechanisms that regulate expression of the hPDE7A gene.
p300/CBP-associated factor (P/CAF) interacts with nuclear respiratory factor-1 to regulate the UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyltransferase-3 gene
Authors: IZUMI Hiroto , OHTA Ryo , NAGATANI Gunji , ISE Tomoko , NAKAYAMA Yoshifumi , NOMOTO Minoru , KOHNO Kimitoshi
Published: August 2003

We demonstrated recently that expression of the UDP-N-acetyl-α-d-galactosamine: polypeptide N-acetylgalactosaminyltrans-ferase-3 (GalNAc-T3) gene is restricted to epithelial glands [Nomoto, Izumi, Ise, Kato, Takano, Nagatani, Shibao, Ohta, Imamura, Kuwano, Matsuo, Yamada, Itoh and Kohno (1999) Cancer Res. 59, 6214–6222]. In the present study, we show that sodium butyrate treatment of human breast cancer MCF-7 cells transcriptionally activates the GalNAc-T3 gene. Transient transfection of plasmids containing a reporter gene under the control of GalNAc-T3 indicated that several transcriptional elements are involved in response to sodium butyrate, with the nuclear respiratory factor-1 (NRF-1)-binding motif located between −88 and −77nt being the most important. Incubation of a labelled probe encompassing the NRF-1-binding motif with a nuclear extract of sodium butyrate-treated MCF-7 cells yielded a higher level of specific DNA–protein complex versus controls. Flag-tagged NRF-1 expressed in MCF-7 cells can bind to the NRF-1-binding motif of the GalNAc-T3 promoter. Nuclear content of NRF-1 remained constant in MCF-7 cells treated with or without sodium butyrate. Moreover, NRF-1 interacts with and is acetylated by p300/CBP-associated factor (P/CAF). Acetylation of NRF-1 enhances DNA binding. Co-transfection of the GalNAc-T3 reporter plasmid with either NRF-1 or P/CAF expression plasmid resulted in the activation of the GalNAc-T3 promoter. These results indicate a correlation between acetylation of NRF-1 by P/CAF and the butyrate-induced expression of the GalNAc-T3 gene. Additionally, induced expression of P/CAF may be a component of the adenocarcinoma differentiation process.
Complexes between the nonsense-mediated mRNA decay pathway factor human upf1 (up-frameshift protein 1) and essential nonsense-mediated mRNA decay factors in HeLa cells
Authors: SCHELL Thomas , KÖCHER Thomas , WILM Matthias , SERAPHIN Bertrand , KULOZIK Andreas E. , HENTZE Matthias W.
Published: August 2003

mRNAs harbouring premature translation-termination codons are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway. Human up-frameshift protein 1 (Hupf1) is an NMD factor that is conserved between yeast and mammals. To isolate cellular complexes that are formed with Hupf1 and to explore the role of cellular proteins in NMD, we generated a HeLa cell line that stably expresses Hupf1 bearing a double-affinity tag (termed Hupf1-2tag). Hupf1-2tag is localized in the cytoplasm similar to the endogenous Hupf1 protein, and the Hupf1-2tag cell line is fully NMD-competent. Using affinity chromatography, Hupf1-2tag-associated proteins were isolated. MS and immunoblotting identified the NMD factors Hupf2 and Hupf3a/b as interaction partners of Hupf1. Size-exclusion chromatography indicates that the NMD factors Hupf1, Hupf2 and the large isoform of Hupf3a might exist in a stable, high-molecular-mass complex of approx. 1.3 MDa. Interestingly, the poly(A)-binding protein was also identified by MS to be associated specifically with Hupf1-2tag. In contrast with the interaction with Hupf2 and Hupf3a/b, the association of poly(A)-binding protein with Hupf1 is highly sensitive to treatment of the isolated complexes with RNase. Components of the exon–exon junction complex or the translational eukaryotic release factor (eRF) 3 were not identified in complexes associated with Hupf1-2tag. We discuss these findings in the context of current models of NMD.
Dictyostelium discoideum protein phosphatase-1 catalytic subunit exhibits distinct biochemical properties
Authors: ANDRIOLI Luiz P. M. , ZAINI Paulo A. , VIVIANI Wladia , da SILVA Aline M.
Published: August 2003

Protein phosphatase-1 (PP1) is expressed ubiquitously and is involved in many eukaryotic cellular functions, although PP1 enzyme activity could not be detected in the social amoeba Dictyostelium discoideum cell extracts. In the present paper, we show that D. discoideum has a single copy gene that codes for the catalytic subunit of PP1 (DdPP1c). DdPP1c is expressed throughout the D. discoideum life cycle with constant levels of mRNA, and its protein and amino acid sequence show a mean identity of 80% with other PP1c enzymes. However, it has a distinctive difference: the substitution of a phenylalanine residue (Phe269 in the DdPP1c) for a highly conserved cysteine residue (Cys273 in rabbit PP1c) in a region that was shown to have a critical role in the interaction of rabbit PP1c with toxin inhibitors. Wild-type DdPP1c and an engineered mutant form in which Phe269 was replaced by a cysteine residue were expressed in Escherichia coli. Both recombinant activities were similarly inhibited by okadaic acid, tautomycin and microcystin. However, the Phe269→Cys mutation resulted in a large increase in enzyme activity towards phosphorylase a and a higher sensitivity to calyculin A. These results, together with the molecular modelling of DdPP1c structure, indicate that the Phe269 residue, which occurs naturally in D. discoideum, confers distinct biochemical properties on this enzyme.
A novel cytochrome c peroxidase from Neisseria gonorrhoeae: a lipoprotein from a Gram-negative bacterium
Authors: TURNER Susan , REID Eleanor , SMITH Harry , COLE Jeffrey
Published: August 2003

A cytochrome c peroxidase (CCP) produced by Neisseria gonorrhoeae has been shown to have novel characteristics by investigating its location, expression and role in Neisseria gonorrhoeae and by expression in Escherichia coli. Analysis of the N-terminus of CCP indicated that it is a lipoprotein with a signal peptide for cleavage by signal peptidase II. Expression of the gonococcal CCP in E. coli revealed that it is first synthesized as a pro-apo-cytochrome that is translocated across the cytoplasmic membrane. The signal peptide is cleaved and haem is attached in the periplasm. The gonococcal CCP was associated with the membrane of both E. coli and N. gonorrhoeae. The expression of a MalE–CCP fusion protein has allowed characterization of CCP in vitro. Evidence is presented that CCP protects gonococci from hydrogen peroxide, presumably in the periplasmic compartment of the cell. The expression of CCP is dependent on the transcription factor FNR, but is repressed by nitrite, indicating that it could be most important in the stationary-phase response. These data support the hypothesis that the gonococcal lipoprotein CCP is anchored to the membrane in the periplasm, where it might be responsible for the reduction of hydrogen peroxide. Other putative CCP lipoproteins have been identified, representing a new subclass of bacterial CCP proteins.
N-linked oligosaccharides play a role in disulphide-dependent dimerization of intestinal mucin Muc2
Authors: BELL Sherilyn L. , XU Gongqiao , KHATRI Ismat A. , WANG Rongquan , RAHMAN Sameera , FORSTNER Janet F.
Published: August 2003

Within the C-terminal domain of many secretory mucins is a ‘cystine knot’ (CK), which is needed for dimer formation in the endoplasmic reticulum. Previous studies indicate that in addition to an unpaired cysteine, the three intramolecular cystine bonds of the knot are important for stability of the dimers formed by rat intestinal mucin Muc2. The present study was undertaken to determine whether the two N-glycans N9 and N10, located near the first and second cysteines of the knot, also play a role in dimer formation. The C-terminal domain of rat Muc2 (RMC), a truncated RMC mutant containing the CK, and mutants lacking N9 and N10 sites, were expressed in COS-1 cells and the products monitored by radioactive [35S]Met/Cys metabolic pulse–chase and immunoprecipitation. Mutation of N9, but not N10, caused increased synthesis of dimers over a 2-h chase period. The N9 mutant remained associated with calreticulin for a prolonged period. About 34–38% of the total labelled products of RMC and its mutants was secreted into the media by 2 h, but the proportion in dimer form was dramatically reduced for the N9 mutant, suggesting lower dimer stability relative to RMC or its N10 mutant. We conclude that under normal conditions the presence of the N9 glycan functions to maintain a folding rate for mucin monomers that is sufficiently slow to allow structural maturation and stability of Muc2 dimers. To our knowledge this report is the first demonstration that a specific N-glycan plays a definitive role in mucin dimer formation.
The peroxisome-proliferator-activated receptor alpha agonist ciprofibrate severely aggravates hypercholesterolaemia and accelerates the development of atherosclerosis in mice lacking apolipoprotein E
Authors: FU Tao , KASHIREDDY Papreddy , BORENSZTAJN Jayme
Published: August 2003

Mice lacking apolipoprotein E (apoE) are characterized by severe hypercholesterolaemia, caused by an abnormal accumulation of apolipoprotein B-48 (apoB-48)-carrying remnants of chylomicrons and very-low-density lipoproteins (VLDL) in the plasma, and by the spontaneous development of atherosclerotic lesions. Ciprofibrate is a hypolipidaemic compound that acts primarily by enhancing the oxidation of fatty acids in the liver and, consequently, decreasing the production of hepatic VLDL. In the present study, homozygous apoE-deficient mice were fed with a normal chow diet, supplemented with ciprofibrate. We report that, as anticipated, ciprofibrate treatment (a) stimulated hepatic fatty acid oxidation, as indicated by an increase in the mRNA levels of peroxisomal fatty acyl-CoA oxidase (AOX) and peroxisomal bifunctional enzyme, and (b) decreased the hepatic secretion of VLDL into the plasma, as determined by treating the animals with Triton WR-1339. Paradoxically, the apoE-deficient mice developed a 3–4-fold increase in their plasma cholesterol levels. A similar effect was observed in apoE-deficient mice treated with other peroxisome-proliferator-activated receptor α agonists (fenofibrate, bezafibrate and WY14,643). By FPLC of the plasma and Western-blot analysis, we determined that the enhanced hypercholesterolaemia was due to an increased accumulation of apoB-48-carrying lipoprotein remnants in the plasma. Consistent with this finding, atherosclerotic lesions in animals treated with ciprofibrate for 90 days were considerably more advanced than in untreated animals. These results indicate that the ciprofibrate-induced accumulation of apoB-48-carrying remnants in apoE-deficient mice is caused by the inhibition of an as yet uncharacterized apoE-independent mechanism of removal of remnant from the circulation by the liver.
A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport
Authors: MITOMO Hideyuki , KATO Ryo , ITO Akiko , KASAMATSU Shiho , IKEGAMI Yoji , KII Isao , KUDO Akira , KOBATAKE Eiry , SUMINO Yasuhiro , ISHIKAWA Toshihisa
Published: August 2003

Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg482, Gly482, and Thr482 variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg482]ABCG2 transports [3H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly482 and Thr482). Transport of methotrexate by [Arg482]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S-octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg482]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg482 is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.
On the substrate specificity of bacterial DD-peptidases: evidence from two series of peptidoglycan-mimetic peptides
Authors: ANDERSON John W. , ADEDIRAN Suara A. , CHARLIER Paulette , NGUYEN-DISTÈCHE Martine , FRÈRE Jean-Marie , NICHOLAS Robert A. , PRATT Rex F.
Published: August 2003

The reactions between bacterial DD-peptidases and β-lactam antibiotics have been studied for many years. Less well understood are the interactions between these enzymes and their natural substrates, presumably the peptide moieties of peptidoglycan. In general, remarkably little activity has previously been demonstrated in vitro against potential peptide substrates, although in many cases the peptides employed were non-specific and not homologous with the relevant peptidoglycan. In this paper, the specificity of a panel of DD-peptidases against elements of species-specific d-alanyl-d-alanine peptides has been assessed. In two cases, those of soluble, low-molecular-mass DD-peptidases, high activity against the relevant peptides has been demonstrated. In these cases, the high specificity is towards the free N-terminus of the peptidoglycan fragment. With a number of other enzymes, particularly high-molecular-mass DD-peptidases, little or no activity against these peptides was observed. In separate experiments, the reactivity of the enzymes against the central, largely invariant, peptide stem was examined. None of the enzymes surveyed showed high activity against this structural element although weak specificity in the expected direction towards the one structural variable (d-γGln versus d-γGlu) was observed. The current state of understanding of the activity of these enzymes in vitro is discussed.
Effect of spermine synthase on the sensitivity of cells to anti-tumour agents
Authors: IKEGUCHI Yoshihiko , MACKINTOSH Caroline A. , McCLOSKEY Diane E. , PEGG Anthony E.
Published: August 2003

The role of spermine in the sensitivity of cells to various established and experimental anti-tumour agents was examined, using paired cell lines that possess or lack spermine synthase. All spermine-synthase-deficient cells had no detectable spermine, and elevated spermidine, content. Spermine content did not alter the cell growth rate. There was little or no difference in sensitivity of immortalized mouse embryonic fibroblasts to doxorubicin, etoposide, cisplatin, methylglyoxal bis(guanylhydrazone) or H2O2 and only a slight increase in sensitivity to vinblastine and nocodazole. However, the absence of spermine clearly increased the sensitivity to 1,3-bis(2-chloroethyl)-N-nitrosourea, suggesting that depletion of spermine may be a useful way to increase the anti-neoplastic effects of anti-tumour agents that form chloroethyl-mediated interstrand DNA cross-links. The effects of spermine on the response to polyamine analogues (which have been proposed to be useful anti-neoplastic agents) were complex, and depended on the compound examined and on the cells tested. Sensitivity to CHENSpm {N1-ethyl-N11-[(cycloheptyl)methyl]-4,8-diazaundecane} was substantially greater in immortalized fibroblasts that lack spermine. In contrast, BE-3-4-3 [N1,N12-bis(ethyl)spermine] and BE-3-3-3 [N1,N11-bis(ethyl)norspermine] were more active against cells that contained spermine. The presence of spermine correlated with a greater induction of spermidine/spermine-N1-acetyltransferase by BE-3-3-3, which is consistent with suggestions that this induction is important for the response to this drug. These findings support the concepts that different polyamine analogues have different sites of action and that CHENSpm has a different site of action from BE-3-3-3.
Free-oligosaccharide control in the yeast Saccharomyces cerevisiae: roles for peptide:N-glycanase (Png1p) and vacuolar mannosidase (Ams1p)
Authors: CHANTRET Isabelle , FRÉNOY Jean-Pierre , MOORE Stuart E. H.
Published: August 2003

Free oligosaccharides (fOS) are generated during glycoprotein biosynthesis in mammalian cells. Here we report on the origin and fate of these structures in the yeast Saccharomyces cerevisiae. After metabolic radiolabelling with [2−3H]mannose ([2−3H]Man) for 30 min, Man8GlcNAc2 was identified as the predominant fOS in this organism, and radioactivity associated with this structure was found to correspond to ≈1% of that associated with the same structure N-linked to glycoprotein. Despite provoking a fourfold increase in radioactivity associated with lipid-linked oligosaccharide, the protein-synthesis inhibitor cycloheximide blocked [2−3H]Man incorporation into both endo-β-d-N-acetylglucosamine H-sensitive N-glycans and fOS. Peptide:N-glycanase, encoded by the PNG1 gene, was found to be required for the generation of a large proportion of yeast fOS during, and soon after, protein glycosylation. Use of an ams1Δ strain deficient in the vacuolar α-mannosidase revealed this enzyme to be responsible for the slow growth-associated catabolism of fOS. The present paper constitutes the first description of fOS formation in intact S. cerevisiae, and, with the demonstration that fOS are degraded by the vacuolar mannosidase, a novel function for this poorly understood enzyme has been identified.
Mouse matriptase-2: identification, characterization and comparative mRNA expression analysis with mouse hepsin in adult and embryonic tissues
Authors: HOOPER John D. , CAMPAGNOLO Luisa , GOODARZI Goodarz , TRUONG Tony N. , STUHLMANN Heidi , QUIGLEY James P.
Published: August 2003

We report the identification and characterization of mouse matriptase-2 (m-matriptase-2), an 811-amino-acid protein composed of an N-terminal cytoplasmic domain, a membrane-spanning domain, two CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains, three LDLR (low-density-lipoprotein receptor class A) domains and a C-terminal serine-protease domain. All m-matriptase-2 protein domain boundaries corresponded with intron/exon junctions of the encoding gene, which spans approx. 29 kb and comprises 18 exons. Matriptase-2 is highly conserved in human, mouse and rat, with the rat matriptase-2 gene (r-maltriptase-2) predicted to encode transmembrane and soluble isoforms. Western-blot analysis indicated that m-matriptase-2 migrates close to its theoretical molecular mass of 91 kDa, and immunofluorescence analysis was consistent with the proposed surface membrane localization of this protein. Reverse-transcription PCR and in-situ-hybridization analysis indicated that m-matriptase-2 expression overlaps with the distribution of mouse hepsin (m-hepsin, a cell-surface serine protease identified in hepatoma cells) in adult tissues and during embryonic development. In adult tissues both are expressed at highest levels in liver, kidney and uterus. During embryogenesis m-matriptase-2 expression peaked between days 12.5 and 15.5. m-hepsin expression was biphasic, with peaks at day 7.5 to 8.5 and again between days 12.5 and 15.5. In situ hybridization of embryonic tissues indicated abundant expression of both m-matriptase-2 and m-hepsin in the developing liver and at lower levels in developing pharyngo–tympanic tubes. While m-hepsin was detected in the residual embryonic yolk sac and with lower intensity in lung, heart, gastrointestinal tract, developing kidney tubules and epithelium of the oral cavity, m-matriptase-2 was absent in these tissues, but strongly expressed within the nasal cavity by olfactory epithelial cells. Mechanistic insight into the potential role of this new transmembrane serine protease is provided by its novel expression profile in embryonic and adult mouse.
A sensitive core region in the structure of glutathione S-transferases
Authors: WONGSANTICHON Jantana , HARNNOI Thasaneeya , KETTERMAN Albert J.
Published: August 2003

A variant form of an Anopheles dirus glutathione S-transferase (GST), designated AdGSTD4-4, possesses a single amino acid change of leucine to arginine (Leu-103-Arg). Although residue 103 is outside of the active site, it has major effects on enzymic properties. To investigate these structural effects, site-directed mutagenesis was used to generate mutants by changing the non-polar leucine to alanine, glutamate, isoleucine, methionine, asparagine, or tyrosine. All of the recombinant GSTs showed approximately the same expression level at 25° C. Several of the mutants lacked glutathione (GSH)-binding affinity but were purified by S-hexyl-GSH-based affinity chromatography. However the protein yields (70-fold lower), as well as the GST activity (100-fold lower), of Leu-103-Tyr and Leu-103-Arg purifications were surprisingly low and precluded the performance of kinetic experiments. Size-exclusion chromatography showed that both GSTs Leu-103-Tyr and Leu-103-Arg formed dimers. Using 1-chloro-2,4-dinitrobenzene (CDNB) and GSH substrates to determine kinetic constants it was demonstrated that the other Leu-103 mutants possessed a greater Km towards GSH and a differing Km towards CDNB. The Vmax ranged from 44.7 to 87.0 μmol/min per mg (wild-type, 44.7 μmol/min per mg). Substrate-specificity studies showed different selectivity properties for each mutant. The structural residue Leu-103 affects the active site through H-bond and van-der-Waal contacts with six active-site residues in the GSH binding site. Changes in this interior core residue appear to disrupt internal packing, which affects active-site residues as well as residues at the subunit–subunit interface. Finally, the data suggest that Leu-103 is noteworthy as a sensitive residue in the GST structure that modulates enzyme activity as well as stability.
The mcyF gene of the microcystin biosynthetic gene cluster from Microcystis aeruginosa encodes an aspartate racemase
Authors: SIELAFF Heike , DITTMANN Elke , TANDEAU de MARSAC Nicole , BOUCHIER Christiane , von DÖHREN Hans , BÖRNER Thomas , SCHWECKE Torsten
Published: August 2003

Microcystins are hepatotoxic, non-ribosomal peptides produced by several genera of freshwater cyanobacteria. Among other enzymic activities, in particular those of peptide synthetases and polyketide synthases, microcystin biosynthesis requires racemases that provide d-aspartate and d-glutamate. Here, we report on the cloning, expression and characterization of an open reading frame, mcyF, that is part of the mcy gene cluster involved in microcystin biosynthesis in the Microcystis aeruginosa strain PCC 7806. Conserved amino acid sequence motifs suggest a function of the McyF protein as an aspartate racemase. Heterologous expression of mcyF in the unicellular cyanobacterium Synechocystis PCC 6803 yielded an active His6-tagged protein that was purified to homogeneity by Ni2+-nitriloacetate affinity chromatography. The purified recombinant protein racemized in a pyridoxal-5′-phosphate-independent manner l-aspartate, but not l-glutamate. Furthermore, we have identified a putative glutamate racemase gene that is located outside the mcy gene cluster in the M. aeruginosa PCC 7806 genome. Whereas homologues of this glutamate racemase gene are present in all the Microcystis strains examined, mcyF could only be detected in microcystin-producing strains.
The role of serine/threonine protein phosphatases in exocytosis
Authors: SIM Alistair T. R. , BALDWIN Monique L. , ROSTAS John A. P. , HOLST Jeff , LUDOWYKE Russell I.
Published: August 2003

Modulation of exocytosis is integral to the regulation of cellular signalling, and a variety of disorders (such as epilepsy, hypertension, diabetes and asthma) are closely associated with pathological modulation of exocytosis. Emerging evidence points to protein phosphatases as key regulators of exocytosis in many cells and, therefore, as potential targets for the design of novel therapies to treat these diseases. Diverse yet exquisite regulatory mechanisms have evolved to direct the specificity of these enzymes in controlling particular cell processes, and functionally driven studies have demonstrated differential regulation of exocytosis by individual protein phosphatases. This Review discusses the evidence for the regulation of exocytosis by protein phosphatases in three major secretory systems, (1) mast cells, in which the regulation of exocytosis of inflammatory mediators plays a major role in the respiratory response to antigens, (2) insulin-secreting cells in which regulation of exocytosis is essential for metabolic control, and (3) neurons, in which regulation of exocytosis is perhaps the most complex and is essential for effective neurotransmission.
Characterization of SMOC-2, a modular extracellular calcium-binding protein
Authors: VANNAHME Christian , GÖSLING Silke , PAULSSON Mats , MAURER Patrik , HARTMANN Ursula
Published: August 2003

We have isolated the novel gene SMOC-2, which encodes a secreted modular protein containing an EF-hand calcium-binding domain homologous to that in BM-40. It further consists of two thyroglobulin-like domains, a follistatin-like domain and a novel domain found only in the homologous SMOC-1. Phylogenetic analysis of the calcium-binding domain sequences showed that SMOC-1 and −2 form a separate group within the BM-40 family. The human and mouse SMOC-2 sequences are coded for by genes consisting of 13 exons located on chromosomes 6 and 17, respectively. Analysis of recombinantly expressed protein showed that SMOC-2 is a glycoprotein with a calcium-dependent conformation. Results from Northern blots and reverse transcription PCR revealed a widespread expression in many tissues.
Identification of ubiquitination sites on the X-linked inhibitor of apoptosis protein
Authors: SHIN Hwain , OKADA Kazuya , WILKINSON John C. , SOLOMON Katherine M. , DUCKETT Colin S. , REED John C. , SALVESEN Guy S.
Published: August 2003

The execution phase of apoptosis is under the control of members of the inhibitor of apoptosis (IAP) family of zinc finger proteins. Several of these proteins contain a C-terminal RING (really interesting new gene) domain that has been postulated to regulate ubiquitination of themselves or their target proteins, thereby modulating thresholds for apoptosis. We demonstrate that the auto-ubiquitination sites of the X-linked IAP (XIAP) are Lys322 and Lys328, located in the third baculovirus IAP repeat domain of the protein. Modification of these sites to arginine dramatically reduces ubiquitination of XIAP, but has no measurable effect on the ability of ectopically expressed IAP to rescue cells from two independent apoptotic inducers. Our data firmly locate the auto-ubiquitination sites, and raise doubts regarding the importance of this event as a mechanism for regulating the levels of XIAP.
Expression of Plasmodium falciparum G6PD‐6PGL in laboratory parasites and in patient isolates in G6PD‐deficient and normal Nigerian children
Authors: Sodeinde Olugbemiro , Clarke Julia L. , Vulliamy Tom J. , Luzzatto Lucio , Mason Philip J.
Published: August 2003

Summary. As the production of NADPH in the pentose phosphate pathway is the main antioxidant defence mechanism available to the Plasmodium falciparum, we have studied the expression of P. falciparum glucose 6‐phosphate dehydrogenase‐6‐phosphogluconolactonase (PfG6PD‐6PGL) in G6PD‐deficient and normal erythrocyte host cells. Both erythrocytes infected in vitro with a laboratory isolate and erythrocytes from natural human infections were used. Total RNA was prepared from parasites collected from five G6PD‐deficient and nine G6PD‐normal children in Ibadan, Nigeria, selected after screening 189 rural schoolchildren and 68 clinical malaria patients, and was subjected to Northern blot analysis. The probe was a cDNA fragment of the G6PD domain of the PfG6PD‐6PGL gene, with an internal control probe of P. falciparum 18S ribosomal RNA. Quantification was performed using a phosphoimager. Relative to internal control, the abundance of PfG6PD‐6PGL mRNA (mean ± standard deviation) was lower in parasites from G6PD‐deficient children (0·29 ± 0·27) than in G6PD‐normal control subjects (0·74 ± 0·26) (P = 0·014, Mann–Whitney U‐test). Although confirmation in a larger study is required, our results suggest a lower relative abundance of PfG6PD‐6PGL, and presumably antioxidant activity, in malaria parasites from G6PD‐deficient hosts, thus extending the current knowledge of the mechanism of G6PD‐deficiency related host protection.
A 2·6 Mb interval on chromosome 6q25.2–q25.3 is commonly deleted in human nasal natural killer/T‐cell lymphoma
Authors: Sun H. Sunny , Su Ih‐Jen , Lin Ya‐Chi , Chen Jia‐Shing , Fang Sheen‐Yie
Published: August 2003

Summary. Natural killer (NK)/T‐cell lymphoma is a special subtype of rare malignant lymphoma that is more prevalent in Asia than in America and Europe. This newly characterized haemato‐lymphoid malignancy is highly aggressive and frequently present in nasal and upper aerodigestive sites. Several studies have reported the commonly deleted region of chromosome 6q21–25 in this particular type of lymphoma. To refine the smallest region of overlapping (SRO) deletion for localization of potential tumour suppressor (TS) genes, we performed loss of heterozygosity (LOH) and homozygosity mapping of deletion (HOMOD) analyses on 37 nasal and nasal‐type NK/T‐cell lymphoma patients using a panel of 25 microsatellite markers, covering the 6q21–q25 region. In all patients studied, LOH was detected in eight (89%) paired‐sample patients, while hemizygous deletion was detected in three (11%) single‐sample patients. Combination of the LOH and HOMOD results defined a distinct 3 Mb SRO on chromosome 6q25. Quantitative multiplex polymerase chain reaction analysis of 10 sequence‐tagged sites further refined the putative TS‐gene‐containing region to a 2·6 Mb interval between TIAM2 and SNX9. Eighteen known genes/Unigene clusters and 25 hypothetical genes are located within this 2·6 Mb region, but none are previously identified TS genes. These results provide a framework for future positional cloning of novel TS gene(s) at 6q25.2–q25.3.
Activation of platelets in whole blood by recombinant factor VIIa by a thrombin‐dependent mechanism
Authors: Wilbourn Barry , Harrison Paul , Mackie Ian J. , Liesner Ri , Machin Samuel J.
Published: August 2003

Summary. Using a diluted whole blood method of flow cytometric analysis, we have shown that platelets could be activated in vitro in the presence of high concentrations (100 nmol/l) of recombinant factor (F) VIIa (rFVIIa; NovoSeven®) and 2·5 mmol/l calcium chloride. This was demonstrated by a significant increase in the mean percentage of platelets expressing CD62P and their mean fluorescent intensity (MFI) after 30 min versus platelets incubated with calcium or rFVIIa alone or diluted blood alone. The presence of rFVIIa and calcium increased the exposure of the PAC‐1 activation epitope of glycoprotein (Gp) IIb/IIIa. This effect was equally influenced by the presence of calcium alone but not by rFVIIa. The effect of rFVIIa was time and concentration dependent. Thrombin generation was also necessary, as the effect of rFVIIa was completely abrogated by the additional presence of hirudin. Furthermore, soy bean trypsin inhibitor (SBTI) but not corn trypsin inhibitor (CTI) abrogated CD62P exposure, suggesting that thrombin was derived via FX but not FXII activation. Exposure of CD62P demonstrated a significant lag phase, sometimes of the order of > 30 min, as well as large intersubject variation. Significant platelet activation was observed at a concentration as low as 25 nmol/l rFVIIa. Platelet–leucocyte aggregation was also increased in the presence of 25 nmol/l rFVIIa and calcium. No significant difference was observed between levels of CD62P in diluted whole blood and platelet‐rich plasma adjusted to an identical platelet count after their exposure to rFVIIa and calcium for 30 min.
Aberrant gene promoter methylation in acute promyelocytic leukaemia: profile and prognostic significance
Authors: Chim Chor S. , Wong Seung Y. , Kwong Yok L.
Published: August 2003

Summary.Acute promyelocytic leukaemia (APL) has distinct clinicopathological and molecular features. However, the profile of aberrant gene promoter methylation is undefined. In this study, methylation‐specific polymerase chain reaction (MSP) was used to define the methylation status of a panel of nine genes, comprisingp15,p16,RARβ,oestrogen receptor(ER),E‐cadherin (E‐CAD),p73,caspase 8 (CASP8),VHLandMGMT, in 29 patients with APL. Aberrant methylation ofp15,ER,RARβ,p16andE‐CADoccurred, respectively, in 23 (79%), 14 (48%), six (21%), six (21%) and two (7%) patients at diagnosis, butp73,VHL,CASP8andMGMTwere not methylated in any patients. There was methylation of one gene in 13 patients (45%), two genes in four patients (14%), three genes in six patients (21%) and four genes in three patients (10%). Concurrent methylation of two or more genes occurred in 13 patients (45%). No association was identified between gene methylation and presenting clinicopathological features. However,p15methylation was significantly associated with an inferior disease‐free survival (DFS,P = 0·008), and remained the only poor prognostic factor in multivariate analysis (P = 0·019). In APL,p15,p16,ERandRARβwere most frequently methylated. This profile is distinct from other types of myeloid leukaemias.p15methylation has a poor prognostic impact on DFS.
Increasing oxygen tension improves filtration of sickle trait donor blood
Authors: Byrne Karen M. , Leitman Susan F. , Schechter Alan N. , Stroncek David F.
Published: August 2003

Summary. A major cause of filter failure of red cell (RBC) components from donors with sickle cell trait (HbAS) is the polymerization of haemoglobin. The oxygen saturation (sO2) of blood stored in various plastics and different volumes of air was assessed. Blood from 10 HbAS donors was collected and divided into two bags, one with air added, one without. Bags with added air had increased sO2 levels (from 49 ± 10% to 76 ± 6%). Filtration was successful for nine of 10 components with air, and one of 10 without air. Successful filtration of RBC components occurs when sO2 is increased.
Severe and selective deficiency of interferon‐γ‐producing invariant natural killer T cells in patients with myelodysplastic syndromes
Authors: Fujii Shin‐ichiro , Shimizu Kanako , Klimek Virginia , Geller Matthew D. , Nimer Stephen D. , V. Dhodapkar Madhav
Published: August 2003

Summary. Here we show that patients with myelodysplastic syndromes (MDS) have a severe deficiency of glycolipid reactive Vα24+/Vβ11+ natural killer T (NKT) cells, but not NK cells or CD4+ or CD8+ T cells. Neither the blood nor marrow of MDS patients had detectable interferon‐γ‐producing NKT cells in response to the NKT ligand, α‐galactosyl ceramide, although influenza‐virus‐specific effector T‐cell function was preserved. This severe and selective deficiency of an important immune regulatory cell may contribute to the pathogenesis of MDS.
CXCR4 chemokine receptors (CD184) and α4β1 integrins mediate spontaneous migration of human CD34+ progenitors and acute myeloid leukaemia cells beneath marrow stromal cells (pseudoemperipolesis)
Authors: Burger Jan A. , Spoo Anke , Dwenger Anne , Burger Meike , Behringer Dirk
Published: August 2003

Summary. Marrow stromal cells play an important role in regulating the development and proliferation of haematopoietic stem cells (HSC) within the marrow microenvironment. However, the molecular mechanisms of stem cell–stromal cell interactions are not fully understood. We observed that mobilized peripheral blood and cord‐blood‐derived CD34+ progenitor cells, or CD34+ acute myeloid leukaemia (AML) cells spontaneously migrated beneath marrow stromal cells, an in vitro migration phenomenon termed pseudoemperipolesis. In contrast, the CD34+ myeloid leukaemia cell line, Kasumi‐1, did not display pseudoemperipolesis. Cord blood CD34+ cells had a higher capacity than granulocyte‐colony‐stimulating‐factor‐mobilized CD34+ cells for pseudoemperipolesis (28·7 ± 12%vs 18·1 ± 6·1% of input cells within 24 h, mean ± SD, n = 8), whereas 9·4 ± 12·6% (mean ± SD, n = 10) of input AML cells displayed this phenomenon. Pseudoemperipolesis of CD34+ progenitor and AML cells was significantly inhibited by pertussis toxin and antibodies to the CXCR4 chemokine receptor (CXCR4, CD184), but not control antibodies. Moreover, CD34+ and AML cell migration was significantly inhibited by a CS1 peptide that blocks α4β1 integrin binding, but not by a control peptide, in which the fibronectin binding motif was scrambled. Pseudoemperipolesis was associated with an increased proliferation of migrated CD34+ progenitor cells but not AML cells within the stromal layer, demonstrated by cell cycle analysis and cell division tracking. We conclude that α4β1 integrin binding and CXCR4 chemokine receptor activation are prerequisites for the migration of CD34+ haematopoietic progenitors and AML cells beneath marrow stromal cells. These observations suggest a central role of marrow stromal cells for HSC trafficking and homing within the marrow microenvironment.
Graft‐versus‐host disease (GVHD)‐specific survival and duration of systemic immunosuppressive treatment in patients who developed chronic GVHD following allogeneic haematopoietic cell transplantation
Authors: Lee Jung‐Hee , Lee Je‐Hwan , Choi Seong‐Jun , Kim Shin , Seol Miee , Lee Young‐Shin , Lee Jung‐Shin , Kim Woo‐Kun , Lee Kyoo‐Hyung
Published: August 2003

Summary. We investigated graft‐versus‐host disease (GVHD)‐specific survival (GSS) and the duration of systemic immunosuppressive treatment (IST) in 82 patients who developed chronic GVHD following allogeneic haematopoietic cell transplantation (HCT). These two major study endpoints were calculated using the Kaplan–Meier method. Deaths solely due to the relapse of underlying disease or accidental deaths were censored at the time of occurrence for the analysis of GSS. The probability of GSS at 5 years was 74·2%. The median duration of systemic IST for chronic GVHD was 272 d (range: 7–1450), and the probability of withdrawal of systemic IST at 1, 2 and 3 years was 67·3%, 82·4% and 89·0% respectively. Analysis based on a multivariate model showed that a diagnosis other than leukaemia or myelodysplastic syndrome (P = 0·049), prior occurrence of grade III–IV acute GVHD (P = 0·021), onset of chronic GVHD before d 120 (P = 0·013), serum alkaline phosphatase over 120 IU/l (P = 0·034), and serum bilirubin over 34·2 μmol/l (P = 0·015) were independent adverse prognostic factors for GSS. Prior occurrence of grade III–IV acute GVHD significantly influenced the duration of systemic IST (P = 0·048). In conclusion, analyses of GSS and the duration of systemic IST will allow patients with different outcomes to be stratified for appropriate treatment application and will provide important parameters in prospective trials for the treatment of chronic GVHD.