bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro development of equine nuclear transfer embryos: effects of oocyte maturation media and amino acid composition during embryo culture
Authors: Choi Y.H. , Chung Y.G. , Walker S.C. , M.E. Westhusin , Hinrichs K.
Published: February 2003

This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei or average nucleus number at 96 h post-ICSI between the absence and presence of IGF-I in maturation medium, or between embryos cultured in G1.2 or a modified CZB medium (CZB-C). Embryos produced by interspecies NT (equine donor cells into bovine cytoplasts) also showed no difference in cleavage rate or average nucleus number whether cultured in G1.2 or in CZB-C. The rates of cleavage, cleavage with normal nuclei and average nucleus number of equine NT embryos were not significantly different among oocytes matured in M199 with FSH in the presence or absence of IGF-I, or in EMMI medium, which contains IGF-I, epidermal growth factor, steroid hormones, FSH and LH. There were no differences in development of equine NT embryos cultured in any of three amino acid treatments (with or without non-essential amino acids, or containing taurine, hypotaurine and cysteine only). The cleavage rate and average nucleus number of parthenogenetically activated oocytes (treated similarly to NT oocytes but not enucleated or subjected to donor cell injection) were significantly (p < 0.05) higher than those for NT embryos. These results indicate that the presence of IGF-I or of EMMI medium during in vitro maturation of equine oocytes does not have a beneficial effect on their developmental competence as assessed at 96 h. Presence or absence of non-essential amino acids in embryo culture medium does not affect development of NT embryos within the first 96 h of culture. Factors associated with enucleation or nuclear transfer decrease the developmental competence of equine NT embryos. CZB-C medium may be used for culture of equine embryos with results similar to those obtained with G1.2 medium, thus providing a base medium that may be modified for further study of culture requirements of equine embryos.
Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR
Authors: Lee Jong Ho , Park Joong Hoon , Choi Eun Joo , Yoon Jong Taek , Park Chang Sik , Lee Seong Ho , Im Kyung Soon , Jin Dong Il
Published: February 2003

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (>45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.
Tensions between the dispute settlement process and the diplomatic and treaty-making activities of the WTO
Authors: EHLERMANN CLAUS-DIETER
Published: February 2003

The tensions between the (quasi-judicial) dispute settlement process and the (political) diplomatic and treaty-making activities have intrigued me ever since I arrived in Geneva in December 2001. In the course of the six years during which I served as a member of the Appellate Body, my original doubts have grown into concerns. They are shared by prominent and well-informed observers. They have not only become the subject of a provocative and alarming book, but also found their way into one of the world's leading newspapers. It is therefore timely to discuss these issues widely, particularly in the context of the ‘Doha development agenda’ and more specifically the negotiations on improvements and clarifications of the Dispute Settlement Understanding (DSU).
Environment and trade: the implications of imperfect information and political economy
Authors: STURM DANIEL , ULPH ALISTAIR
Published: February 2003

The last ten years have seen an upsurge in interest in the nexus of trade and environmental policies. In part this reflects the need to deal with major global pollution problems, and in part a concern that globalization may have adverse impacts on the environment. Environmentalists worry that globalization may trigger a race-to-the-bottom in environmental standards. While they would like to see upward harmonization in environmental standards, they are sceptical about the ability of supra-national agencies to achieve this. Industrialists also raise concerns about the need for a ‘level playing field’ in environmental regulations because of fears about the impact of environmental regulations on competitiveness. However, developing countries question whether disputes over differences in environmental regulations simply reflect a covert form of ‘green protectionism’. In this paper we review what light recent developments in economic analysis (conceptual and empirical) can shed on these concerns. We quickly summarize conventional trade models in which government bodies have perfect information and are welfare maximizers, and show that this analysis does not provide much support for the concerns or proposed policy recommendations. We then turn to models of political economy and imperfect information to see whether they provide a better explanation for the concerns and policy recommendations.
WTO 2002: imaginary crisis, real problems
Authors: HENDERSON DAVID
Published: February 2003

This article contests the view advanced by Daniel Esty in World Trade Review, that the WTO is now facing ‘a crisis of legitimacy’, for which the remedy is to create for it ‘links with popular sovereignty’, to broaden its objectives and concerns, and to establish it as part of a stronger system of ‘global governance’. I argue that the legitimacy of an international agency derives first and foremost from its member governments; that democratic legitimacy and ‘popular sovereignty’ do not necessarily go together; that non-governmental organizations have no valid claim in their own right to participate in the activities of international agencies; that bringing these organizations into the WTO would weaken and divide it; that governments, in handling and deciding trade matters, already take account of wider issues and goals and are not uncritically committed to freer trade; that it is still appropriate to limit the concerns of the WTO to questions relating to trade and trade liberalization; that globalization has neither undermined the power and competence of national states nor given rise to a need for new forms of global governance; and that the WTO, despite its now more secure status and enlarged scope as compared with the GATT, is for good reasons neither a powerful instrument of global governance nor in course of becoming so. While the Organization has no ‘legitimacy crisis’, it faces substantial problems both old and new. Its potential for useful activity largely depends, as in the past, on the readiness of its member governments to defend and pursue the goal of freer trade.
Rejoinder
Authors: ESTY DANIEL C.
Published: February 2003

David Henderson misreads, misstates, and misunderstands my argument in The World Trade Organization's Legitimacy Crisis. He pejoratively refers to my suggestions for restoring the WTO's legitimacy as ‘radical’ no fewer than three times.
The WTO core agreement, non-trade issues and institutional integrity
Authors: JONES KENT
Published: February 2003

WTO member countries understand the core agreement of the organization to consist of binding reciprocal market access achieved through multilateral negotiation, and supported by a system of trade policy rules and dispute settlement. Attempts to introduce social chapters into the WTO would compromise the core agreement. Specifically, authorizing the use of trade sanctions to pursue non-trade goals would diminish the value of the WTO to its members, and undermine the global trading system. WTO agreements and rules can be reconciled with environmental goals, the improvement of labor standards and the promotion of human rights through the development and strengthening of international institutions dedicated to these issues. Efforts by governments to promote new global institutions and international agreements would thereby remove political barriers to trade liberalization.
On assessing the cost of TRIPS implementation
Authors: LYBBERT TRAVIS J.
Published: February 2003

Efforts to ensure that intellectual property rights are respected and protected world-wide have met increasing resistance by critics who see extreme imbalances in the costs and benefits of implementing stronger intellectual property protection. The WTO's Agreement on Trade-Related Aspects of Intellectual Property (TRIPS) attracts particular criticism as an enforceable multilateral embodiment of these efforts. While few disagree that developed countries stand to benefit more in the short term from TRIPS implementation than developing countries, precisely estimating associated costs and benefits is challenging. This paper comments on an approach to estimating the ‘indirect’ costs of implementing TRIPS proposed by McCalman (2001) and argues that the approach overestimates the costs born by developing countries. Specifically, this overestimation is due primarily to an inadequate representation of the TRIPS Agreement and a counterfactual assumption that countries would not have strengthened their intellectual property policies in the absence of the TRIPS Agreement.
p300/cAMP-response-element-binding-protein ('CREB')-binding protein (CBP) modulates co-operation between myocyte enhancer factor 2A (MEF2A) and thyroid hormone receptor-retinoid X receptor
Authors: LUCA Antonio De , SEVERINO Anna , PAOLIS Paola De , COTTONE Giuliano , LUCA Luca De , FALCO Maria De , PORCELLINI Antonio , VOLPE Massimo , CONDORELLI Gianluigi
Published: February 2003

Thyroid hormone receptors (TRs) and members of the myocyte enhancer factor 2 (MEF2) family are involved in the regulation of muscle-specific gene expression during myogenesis. Physical interaction between these two factors is required to synergistically activate gene transcription. p300/cAMP-response-element-binding-protein ('CREB')-binding protein (CBP) interacting with transcription factors is able to increase their activity on target gene promoters. We investigated the role of p300 in regulating the TR—MEF2A complex. To this end, we mapped the regions of these proteins involved in physical interactions and we evaluated the expression of a chloramphenicol acetyltransferase (CAT) reporter gene in U2OS cells under control of the α-myosin heavy chain promoter containing the thyroid hormone response element (TRE). Our results suggested a role of p300/CBP in mediating the transactivation effects of the TR—retenoid X receptor (RxR)—MEF2A complex. Our findings showed that the same C-terminal portion of p300 binds the N-terminal domains of both TR and MEF2A, and our in vivo studies demonstrated that TR, MEF2A and p300 form a ternary complex. Moreover, by the use of CAT assays, we demonstrated that adenovirus E1A inhibits activation of transcription by TR—RxR—MEF2A—p300 but not by TR—RxR—MEF2A. Our data suggested that p300 can bind and modulate the activity of TR—RxR—MEF2A at TRE. In addition, it is speculated that p300 might modulate the activity of the TR—RxR—MEF2A complex by recruiting a hypothetical endogenous inhibitor which may act like adenovirus E1A.
Proteomic response to physiological fermentation stresses in a wild-type wine strain of Saccharomyces cerevisiae
Authors: TRABALZINI Lorenza , PAFFETTI Alessandro , SCALONI Andrea , TALAMO Fabio , FERRO Elisa , CORATZA Grazietta , BOVALINI Lucia , LUSINI Paola , MARTELLI Paola , SANTUCCI Annalisa
Published: February 2003

We report a study on the adaptive response of a wild-type wine Saccharomyces cerevisiae strain, isolated from natural spontaneous grape must, to mild and progressive physiological stresses due to fermentation. We observed by two-dimensional electrophoresis how the yeast proteome changes during glucose exhaustion, before the cell enters its complete stationary phase. On the basis of their identification, the proteins representing the S. cerevisiae proteomic response to fermentation stresses were divided into three classes: repressed proteins, induced proteins and autoproteolysed proteins. In an overall view, the proteome adaptation of S. cerevisiae at the time of glucose exhaustion seems to be directed mainly against the effects of ethanol, causing both hyperosmolarity and oxidative responses. Stress-induced autoproteolysis is directed mainly towards specific isoforms of glycolytic enzymes. Through the use of a wild-type S. cerevisiae strain and PMSF, a specific inhibitor of vacuolar proteinase B, we could also distinguish the specific contributions of the vacuole and the proteasome to the autoproteolytic process.
Inhibition of phosphatidylcholine synthesis induces expression of the endoplasmic reticulum stress and apoptosis-related protein CCAAT/enhancer-binding protein-homologous protein (CHOP/GADD153)
Authors: SANDEN Michiel H.M. van der , HOUWELING Martin , GOLDE Lambert M.G. van , VAANDRAGER Arie B.
Published: February 2003

Inhibition of de novo synthesis of phosphatidylcholine (PC) by some anti-cancer drugs such as hexadecylphosphocholine leads to apoptosis in various cell lines. Likewise, in MT58, a mutant Chinese hamster ovary (CHO) cell line containing a thermo-sensitive mutation in CTP:phosphocholine cytidylyltransferase (CT), an important regulatory enzyme in the CDP-choline pathway, inhibition of PC synthesis causes PC depletion. Cellular perturbations like metabolic insults and unfolded proteins can be registered by the endoplasmic reticulum (ER) and result in ER stress responses, which can lead eventually to apoptosis. In this study we investigated the effect of PC depletion on the ER stress response and ER-related proteins. Shifting MT58 cells to the non-permissive temperature of 40°C resulted in PC depletion via an inhibition of CT within 24h. Early apoptotic features appeared in several cells around 30h, and most cells were apoptotic within 48h. The temperature shift in MT58 led to an increase of pro-apoptotic CCAAT/enhancer-binding protein-homologous protein (CHOP; also known as GADD153) after 16h, to a maximum at 24h. Incubation of wild-type CHO-K1 or CT-expressing MT58 cells at 40°C did not induce differences in CHOP protein levels in time. In contrast, expression of the ER chaperone BiP/GRP78, induced by an increase in misfolded/unfolded proteins, and caspase 12, a protease specifically involved in apoptosis that results from stress in the ER, did not differ between MT58 and CHO-K1 cells in time when cultured at 40°C. Furthermore, heat-shock protein 70, a protein that is stimulated by accumulation of abnormal proteins and heat stress, displayed similar expression patterns in MT58 and K1 cells. These results suggest that PC depletion in MT58 induces the ER-stress-related protein CHOP, without raising a general ER stress response.
The C-terminal segment of the 1,3-beta-glucanase Ole e 9 from olive (Olea europaea) pollen is an independent domain with allergenic activity: expression in Pichia pastoris and characterization
Authors: PALOMARES Oscar , VILLALBA Mayte , RODRÍGUEZ Rosalía
Published: February 2003

Several allergenic proteins, such as the 1,3-β-glucanases, have been associated with plant defence responses. Ole e 9 (46kDa) is a 1,3-β-glucanase and major allergen from olive pollen, which is a principal cause of allergy in Mediterranean countries. Its C-terminal segment (101 amino acid residues) has been produced as a recombinant polypeptide in the yeast Pichia pastoris. The cDNA encoding the polypeptide was inserted into the plasmid vector pPICZα-A and overexpressed in KM71 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reversed-phase HPLC. Edman degradation, MS and CD were used to determine molecular properties of the recombinant polypeptide, which exhibited 16% α-helix and 30% β-sheet as regular elements of secondary structure. Disulphide bridges of the molecule were determined at positions Cys-14—Cys-76, Cys-33—Cys-94 and Cys-39—Cys-48. The high IgE-binding capability of the recombinant C-terminal segment of Ole e 9 against sera from Ole e 9-sensitive individuals, which was determined by immunoblotting and ELISA inhibition, supported the proper folding of the polypeptide and the maintenance of antigenic properties that it exhibits as a part of the whole allergen. These data indicated that this portion of Ole e 9 constitutes an independent domain, which could be used to study its three-dimensional structure and function, as well as for clinical purposes such as diagnosis and specific immunotherapy. Since it shows sequence similarity with portions of 1,3-β-glucanases from plant tissues and the Gas/Phr/Epd protein families involved in yeast morphogenesis, we suggest that this domain could play an equivalent functional role within these enzymes.
Urea signalling to immediate-early gene transcription in renal medullary cells requires transactivation of the epidermal growth factor receptor
Authors: ZHAO Hongyu , TIAN Wei , XU Hongshi , COHEN David M.
Published: February 2003

Signalling by physiological levels of urea (e.g. 200mM) in cells of the mammalian renal medulla is reminiscent of activation of a receptor tyrosine kinase. The epidermal growth factor (EGF) receptor may be transactivated by a variety of G-protein-coupled receptors, primarily through metalloproteinase-dependent cleavage of a membrane-anchored EGF precursor. In the murine inner medullary collecting duct (mIMCD3) cell line, urea (200mM) induced prompt (1—5min) tyrosine phosphorylation of the EGF receptor. Pharmacological inhibition of EGF receptor kinase activity with AG1478 or PD153035 blocked urea-inducible transcription and expression of the immediate-early gene, Egr-1. AG1478 blocked, either fully or partially, other hallmarks of urea signalling including Elk-1 activation and extracellular signal-regulated kinase phosphorylation. EGF receptor kinase inhibition also blocked the cytoprotective effect of urea observed in the context of hypertonicity-inducible apoptosis. EGF receptor transactivation was likely to be attributable to metalloproteinase-dependent ectodomain shedding of an EGF receptor agonist because both specific and non-specific inhibitors of metalloproteinases blocked the urea effect. Heparin-binding EGF (HB-EGF), in particular, was implicated because the diphtheria toxin analogue and highly specific antagonist of HB-EGF, CRM197, also blocked urea-inducible transcription. In aggregate, these data indicate that signalling in response to urea in renal medullary cells requires EGF receptor transactivation, probably through autocrine action of HB-EGF.
Role of Cys41 in the N-terminal domain of lumican in ex vivo collagen fibrillogenesis by cultured corneal stromal cells
Authors: CARLSON Eric C. , MAMIYA Kazuhisa , LIU Chia-Yang , GENDRON Robert L. , BIRK David E. , FUNDERBURGH James L. , KAO Winston W.-Y.
Published: February 2003

The keratan sulphate proteoglycan lumican regulates collagen fibrillogenesis to maintain the integrity and function of connective tissues such as cornea. We examined the role of a highly conserved cysteine-containing domain proximal to the N-terminus of lumican in collagen fibrillogenesis using site-specific mutagenesis to prepare plasmid DNA encoding wild-type murine lumican (Cys37-Xaa3-Cys41-Xaa-Cys-Xaa9-Cys) and a Cys→Ser (C/S) mutant (Cys37-Xaa3-Ser41-Xaa-Cys-Xaa9-Cys). cDNAs were cloned into the pSecTag2A vector, and cultures of MK/T-1 cells (an immortalized cell line from mouse keratocytes) were transfected with the cDNAs. Stable transformants were selected and cloned in the presence of Zeocin. All stable transformants maintained a dendritic morphology and growth rate similar to those of parental MK/T-1 cells. Western blot analysis with anti-lumican antibody detected a 42kDa lumican protein secreted into the culture medium of both wild-type and C/S mutant lumican cell lines. Ultrastructural analyses by transmission electron microscopy showed both cell lines to form a multi-layered stroma ex vivo, but the matrix assembled by the two cell lines differed. Compared with the mutant cell line, the wild-type cells assembled a more organized matrix with regions containing orthogonal collagen fibrils. In addition, the fibrils in the extracellular matrix formed by the mutant cell line exhibited alterations in fibril packing and structure. Immunostaining analysed by confocal microscopy showed a further difference in this matrix, with the marked occurrence of lumican and collagen I co-localization in the lumican wild-type cells, but a lack thereof in the lumican C/S mutant cells. The results indicate that the cysteine-rich domain of lumican is important in collagen fibrillogenesis and stromal matrix assembly.
CD38-dependent ADP-ribosyl cyclase activity in developing and adult mouse brain
Authors: CENI Claire , POCHON Nathalie , BRUN Virginie , MULLER-STEFFNER Hélène , ANDRIEUX Annie , GRUNWALD Didier , SCHUBER Francis , WAARD Michel DE , LUND Frances , VILLAZ Michel , MOUTIN Marie-Jo
Published: February 2003

CD38 is a transmembrane glycoprotein that is expressed in many tissues throughout the body. In addition to its major NAD+-glycohydrolase activity, CD38 is also able to synthesize cyclic ADP-ribose, an endogenous calcium-regulating molecule, from NAD+. In the present study, we have compared ADP-ribosyl cyclase and NAD+-glycohydrolase activities in protein extracts of brains from developing and adult wild-type and Cd38-/- mice. In extracts from wild-type brain, cyclase activity was detected spectrofluorimetrically, using nicotinamide—guanine dinucleotide as a substrate (GDP-ribosyl cyclase activity), as early as embryonic day 15. The level of cyclase activity was similar in the neonate brain (postnatal day 1) and then increased greatly in the adult brain. Using [14C]NAD+ as a substrate and HPLC analysis, we found that ADP-ribose is the major product formed in the brain at all developmental stages. Under the same experimental conditions, neither NAD+-glycohydrolase nor GDP-ribosyl cyclase activity could be detected in extracts of brains from developing or adult Cd38-/- mice, demonstrating that CD38 is the predominant constitutive enzyme endowed with these activities in brain at all developmental stages. The activity measurements correlated with the level of CD38 transcripts present in the brains of developing and adult wild-type mice. Using confocal microscopy we showed, in primary cultures of hippocampal cells, that CD38 is expressed by both neurons and glial cells, and is enriched in neuronal perikarya. Intracellular NAD+-glycohydrolase activity was measured in hippocampal cell cultures, and CD38-dependent cyclase activity was higher in brain fractions enriched in intracellular membranes. Taken together, these results lead us to speculate that CD38 might have an intracellular location in neural cells in addition to its plasma membrane location, and may play an important role in intracellular cyclic ADP-ribose-mediated calcium signalling in brain tissue.
Two Dictyostelium ribosomal proteins act as RNases for specific classes of mRNAs
Authors: MANGIAROTTI Giorgio
Published: February 2003

Phosphorylation of ribosomal protein S6 leads to the stabilization of pre-spore specific mRNAs during development of Dictyostelium discoideum. The purification of S6 kinase has allowed the identification of protein S11 as the mRNase specific for pre-spore mRNAs. Methylation of ribosomal protein S31 leads to the destabilization of ribosomal protein mRNAs. The purification of S31 methyltransferase has allowed the identification of protein S29 as the mRNAse specific for ribosomal protein mRNAs.
Molecular cloning and characterization of CIDE-3, a novel member of the cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector family
Authors: LIANG Liang , ZHAO Mujun , XU Zhenhua , YOKOYAMA Kazunari K. , LI Tsaiping
Published: February 2003

DNA fragmentation is one of the critical steps in apoptosis, which is induced by DNA fragmentation factor (DFF). DFF is composed of two subunits, a 40kDa caspase-activated nuclease (DFF40) and a 45kDa inhibitor (DFF45). Recently a novel family of cell-death-inducing DFF45-like effectors (CIDEs) has been identified. Among CIDEs, two from human (CIDE-A and CIDE-B) and three from mouse (CIDE-A, CIDE-B and FSP27) have been reported. In this study human CIDE-3, a novel member of CIDEs, was identified upon sequence analysis of a previously unidentified cDNA that encoded a protein of 238 amino acids. It was shown to be a human homologue of mouse FSP27, and shared homology with the CIDE-N and CIDE-C domains of CIDEs. Apoptosis-inducing activity was clearly shown by DNA-fragmentation assay of the nuclear DNA of CIDE-3 transfected 293T cells. The expression pattern of CIDE-3 was different from that of CIDE-B. As shown by Northern-blot analysis, CIDE-3 was expressed mainly in human small intestine, heart, colon and stomach, while CIDE-B showed strong expression in liver and small intestine and at a lower level in colon, kidney and spleen. Green-fluorescent-protein-tagged CIDE-3 was revealed in some cytosolic corpuscles. Alternative splicing of the CIDE-3 gene was also identified by reverse transcription PCR, revealing that two transcripts, CIDE-3 and CIDE-3α, were present in HepG2 and A375 cells. CIDE-3 comprised a full-length open reading frame with 238 amino acids; in CIDE-3α exon 3 was deleted and it encoded a protein of 164 amino acids. Interestingly the CIDE-3α isoform still kept the apoptosis-inducing activity and showed the same pattern of subcellular localization as CIDE-3. Consistent with its chromosome localization at 3p25, a region associated with high frequency loss of heterozygosity in many tumours, CIDE-3 may play an important role in prevention of tumorigenesis.
Triosidines: novel Maillard reaction products and cross-links from the reaction of triose sugars with lysine and arginine residues
Authors: TESSIER Frederic J. , MONNIER Vincent M. , SAYRE Lawrence M. , KORNFIELD Julia A.
Published: February 2003

The role of the highly reactive triose sugars glyceraldehyde and glyceraldehyde-3-phosphate in protein cross-linking and other amino acid modifications during the Maillard reaction was investigated. From the incubation of glyceraldehyde with Nα-acetyl-l-lysine and Nα-acetyl-l-arginine, we isolated four new Maillard reaction pyridinium compounds named ‘triosidines'. Two of them, ‘lys-hydroxy-triosidine’ {1-(5-amino-5-carboxypentyl)-3-[(5-amino-5-carboxypentylamino)methyl]-5-hydroxypyridinium} and ‘arg-hydroxy-triosidine’ {2-(4-amino-4-carboxybutylamino)-8-(5-amino-5-carboxypentyl)-6-hydroxy-3,4-dihydro-pyrido[2,3-d]pyrimidin-8-ium} are fluorescent, UV-active Lys—Lys and Lys—Arg cross-links respectively. Their structures were identified by NMR and MS. In addition, two UV-active lysine adducts, ‘trihydroxy-triosidine’ [1-(5-amino-5-carboxypentyl)-3,4-dihydroxy-5-(hydroxymethyl)pyridinium] and ‘triosidine carbaldehyde’ [1-(5-amino-5-carboxypentyl)-3-formylpyridinium] were tentatively identified by MS. All structures involve six sugar-derived carbons as part of the heterocyclic ring. Of the two novel cross-links, only arg-hydroxy-triosidine was formed by glyceraldehyde-3-phosphate, an intermediate metabolite of the glycolytic pathway. Lys-hydroxy-triosidine and arg-hydroxy-triosidine were detected in human and porcine corneas treated with glyceraldehyde. The HPLC-fluorescence identification was confirmed by MS. Triosidines were also formed from dihydroxyacetone, a widely used artificial sun-tanning agent. Triosidines are expected to be useful tools in tissue engineering, where the utilization of highly reactive sugars is needed to stabilize the loose matrix. In addition, they are expected to be present in selected biological conditions, such as on consumption of a high fructose diet, and syndromes associated with high glyceraldehyde excretion, such as Fanconi Syndrome, fructose-1,6-diphosphatase deficiency and tyrosinaemia.
A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers
Authors: BERGMAN Marina R. , CHENG Sunfa , HONBO Norman , PIACENTINI Lucia , KARLINER Joel S. , LOVETT David H.
Published: February 2003

Enhanced synthesis of a specific matrix metalloproteinase, MMP-2, has been demonstrated in experimental models of ventricular failure and in cardiac extracts from patients with ischaemic cardiomyopathy. Cultured neonatal rat cardiac fibroblasts and myocytes were used to analyse the determinants of MMP-2 synthesis, including the effects of hypoxia. Culture of rat cardiac fibroblasts for 24h in 1% oxygen enhanced MMP-2 synthesis by more than 5-fold and augmented the MMP-2 synthetic responses of these cells to endothelin-1, angiotensin II and interleukin 1β. A series of MMP-2 promoter—luciferase constructs were used to map the specific enhancer element(s) that drive MMP-2 transcription in cardiac cells. Deletion studies mapped a region of potent transactivating function within the 91bp region from −1433 to −1342bp, the activity of which was increased by hypoxia. Oligonucleotides from this region were cloned in front of a heterologous simian-virus-40 (SV40) promoter and mapped the enhancer activity to a region between −1410 and −1362bp that included a potential activating protein 1 (AP-1)-binding sequence, C-1394CTGACCTCC. Site-specific mutagenesis of the core TGAC sequence (indicated in bold) eliminated the transactivating activity within the −1410 to −1362bp sequence. Electrophoretic mobility shift assays (EMSAs) using the −1410 to −1362bp oligonucleotide and rat cardiac fibroblast nuclear extracts demonstrated specific nuclear-protein binding that was eliminated by cold competitor oligonucleotide, but not by the AP-1-mutated oligonucleotide. Antibody-supershift EMSAs of nuclear extracts from normoxic rat cardiac fibroblasts demonstrated Fra1 and JunB binding to the −1410 to −1362bp oligonucleotide. Nuclear extracts isolated from hypoxic rat cardiac fibroblasts contained Fra1, JunB and also included FosB. Co-transfection of cardiac fibroblasts with Fra1—JunB and FosB—JunB expression plasmids led to significant increases in transcriptional activity. These studies demonstrate that a functional AP-1 site mediates MMP-2 transcription in cardiac cells through the binding of distinctive Fra1—JunB and FosB—JunB heterodimers. The synthesis of MMP-2 is widely considered, in contrast with many members of the MMP gene family, to be independent of the AP-1 transcriptional complex. This report is the first demonstration that defined members of the Fos and Jun transcription-factor families specifically regulate this gene under conditions relevant to critical pathophysiological processes.
Pressure- and temperature-induced unfolding and aggregation of recombinant human interferon-gamma: a Fourier transform infrared spectroscopy study
Authors: GOOSSENS Koen , HAELEWYN Joost , MEERSMAN Filip , LEY Marc De , HEREMANS Karel
Published: February 2003

The effect of hydrostatic pressure on the secondary structure of recombinant human interferon-γ (rhIFN-γ) and its biologically inactive truncated form rhIFN-ΔC15 has been studied using Fourier-transform IR (FTIR) spectroscopy. In situ observation of the pressure-induced changes using the diamond anvil cell shows that the α-helical structure is mainly transformed into disordered structure at high pressure. Increasing pressure also induces the formation of a gel. Addition of 0.5M MgCl2 significantly reduces the pressure stability. Releasing the pressure below 300MPa results in the formation of intermolecular antiparallel β-sheets, which is seldom observed. This suggests that the intermolecular β-sheet of rhIFN-γ is stabilized by electrostatic interactions that are disrupted at high pressure. For comparison we also studied the effect of temperature. Temperature-induced changes reflect extensive transformation of α-helical structure into intermolecular antiparallel β-sheet, as is usually observed for most proteins.
Structure and function of the globin and globin gene from the Antarctic mollusc Yoldia eightsi
Authors: DEWILDE Sylvia , ANGELINI Elisa , KIGER Laurent , MARDEN Michael C. , BELTRAMINI Mariano , SALVATO Benedetto , MOENS Luc
Published: February 2003

The mechanism of adaptation of haemoglobin from the Antarctic mollusc Yoldia eightsi to its low-temperature environment is a decrease in the oxygen affinity via an increased ligand-dissociation rate. At 2°C this haemoglobin has an oxygen affinity similar to other haemoglobins at 25°C. At 25°C, Yoldia haemoglobin shows a low oxygen affinity, resembling that of human deoxyhaemoglobin. The mechanism involves a lower binding energy to oxygen, suggesting a loss or weakening of the usual hydrogen bond, leading to a higher oxygen-dissociation rate. However, Yoldia haemoglobin has the usual distal and proximal histidines, so the primary structure alone does not provide an obvious explanation for the low affinity. The CO-binding kinetics are biphasic, with the fraction of slow phase increasing at higher protein concentrations, indicating the formation of dimers or a higher level of polymerization. The protein—protein interaction appears to be of hydrophobic nature, since it can be partially reversed by addition of ethylene glycol as co-solvent. While the CO-association rates differ by a factor of 10, the oxygen equilibrium data could be simulated with a single affinity. The Yoldia haemoglobin gene contains three introns, interrupting the coding region at position NA1.2, B12.2 and G7.0. The conservation of the B12.2 and G7.0 introns is in contrast with the unprecedented NA1.2 intron. Phylogenetic analyses reveal a gene tree where the Yoldia haemoglobin gene is separated from other mollusc globin genes, confirming the specific adaptation of the Yoldia haemoglobin.
Disruption and overexpression of the Schizosaccharomyces pombe aps1 gene, and effects on growth rate, morphology and intracellular diadenosine 5′,5'-P1,P5-pentaphosphate and diphosphoinositol polyphosphate concentrations
Authors: INGRAM Stephen W. , SAFRANY Stephen T. , BARNES Larry D.
Published: February 2003

Schizosaccharomyces pombe Aps1 is an enzyme that degrades both diadenosine oligophosphates (ApnA, n = 5 or 6) and diphosphoinositol polyphosphates {diphosphoinositol pentakisphosphate (PP-InsP5) and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4)} in vitro. The in vivo substrates of Aps1 are unknown. We report here the identification of Ap5A, PP-InsP5, [PP]2-InsP4 and a novel diphosphoinositol polyphosphate ([PP]x-InsPx) in S. pombe using HPLC methods. Ap5A was present at 0.06pmol/mg of protein (approx. 4nM). PP-InsP5, [PP]x-InsPx and [PP]2-InsP4 were present at 15pmol/mg (approx. 1.1μM), 15pmol/mg (approx. 1.1μM) and 30pmol/mg (approx. 2.2μM) respectively, while the intracellular concentration of InsP6 was 0.5nmol/mg of protein (approx. 36μM). Disruption of aps1 resulted in a 52% decrease in Ap6A hydrolase activity in vitro, no detectable change in the intracellular Ap5A concentration, and 3-fold increased intracellular concentrations of PP-InsP5 and [PP]x-InsPx. Disruption of aps1 resulted in no detectable change in morphology or growth rate in minimal or rich media at 30°C. Overexpression of aps1 via two different plasmids that resulted in 60% and 6-fold increases above wild-type enzymic activity in vitro caused no detectable changes in the intracellular concentrations of [PP]2-InsP4, [PP]x-InsPx or PP-InsP5, but paradoxical increases of approx. 2.5- and 55-fold respectively in the intracellular Ap5A concentration. Overexpression of aps1 also resulted in a reduced growth rate and in morphological changes, including swollen, rounded and multiseptate cells. No phenotypic changes or changes in intracellular Ap5A occurred upon overexpression of aps1E93Q, which encodes a mutated Aps1 lacking significant enzymic activity. We conclude that Aps1 degrades PP-InsP5 and [PP]x-InsPxin vivo.
Molecular identification of the long isoform of the human neuropeptide Y Y5 receptor and pharmacological comparison with the short Y5 receptor isoform
Authors: RODRIGUEZ Marianne , AUDINOT Valérie , DROMAINT Sandra , MACIA Christelle , LAMAMY Véronique , BEAUVERGER Philippe , RIQUE Hervé , IMBERT Jérôme , NICOLAS Jean Paul , BOUTIN Jean A. , GALIZZI Jean Pierre
Published: February 2003

The neuropeptide Y Y5 receptor gene generates two splice variants, referred to here as Y5L (long isoform) and Y5S (short isoform). Y5L mRNA differs from Y5S mRNA in its 5′ end, generating a putative open reading frame with 30 additional nucleotides upstream of the initiator AUG compared with the Y5S mRNA. The purpose of the present work was to investigate the existence of the Y5L mRNA. The authenticity of this transcript was confirmed by isolating part of its 5′ untranslated region through 5′ rapid amplification of cDNA ends and analysing its tissue distribution. To study the initiation of translation on Y5L mRNA, we cloned the Y5L cDNA and two Y5L cDNA mutants lacking the first or the second putative initiation start codon. Transient expression of the three plasmids in COS-7 cells and saturation binding experiments using 125I-labelled polypeptide YY (PYY) as a ligand showed that initiation of translation on Y5L mRNA could start at the first AUG, giving rise to a Y5L receptor with an N-terminal 10-amino-acid extension when compared with the Y5S receptor. The human Y5L and Y5S receptor isoforms displayed similar affinity constants (1.3nM and 1.5nM respectively). [125I]PYY binding to COS-7 cells expressing either the Y5L or the Y5S isoform was inhibited with the same rank order of potency by a selection of six chemically diverse compounds: PYY>neuropeptide Y>pancreatic polypeptide>CGP71683A>Synaptic 34>Banyu 6. Comparison of the tissue distribution of Y5L and Y5S mRNAs, as determined by reverse transcription—PCR analysis, indicated that expression of Y5L mRNA occurs in a tissue-specific manner. Finally, we have shown that the two AUG triplets contained in the 5′ untranslated region of Y5L mRNA did not affect receptor expression.
Anion- and pH-dependent activation of the soluble form of dopamine beta-hydroxylase
Authors: TERLAND Ole , FLATMARK Torgeir
Published: February 2003

The present study presents evidence for the conclusion that the catalytic activity of dopamine β-hydroxylase (DBH; dopamine β-mono-oxygenase, EC 1.14.17.1) is regulated independently by pH and by anions. In the absence of activating anions (i.e. in 50mM Mes buffer) the activity was essentially zero at low pH (5.1—5.3) when assayed with the artificial electron donors ferrocyanide (0.25mM), N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD, 2mM) or N,N-dimethyl-p-phenylenediamine (1mM) and tyramine (8mM) as the substrate to be hydroxylated. However, in the presence of activating anions (e.g. 0.05—0.6M Cl- in 50mM Mes buffer, 0.1M phosphate buffer or 0.2M acetate buffer) a high catalytic activity was observed at pH5.1—5.3. The pronounced effect of anions at this pH may be related to the postulated anion-induced conformational change of DBH [Syvertsen, Mel⊘ and Ljones (1987) Biochim. Biophys. Acta 914, 6—18] resulting in a facilitated access of the substrates to the active site(s). The anion-activated DBH was inhibited when assayed with ferrocyanide and activated when assayed with TMPD as electron donors by increasing the pH (5.1 to 6.0). By contrast, in the absence of anions the activity increased from essentially zero at pH5.1—5.3 to high values at pH6.0, irrespective of the electron donor used. The data suggest that the conformational change induced by anion activation exposes a negatively charged group at or near the electron-donor-binding site(s) imposing an electrostatic repulsion towards ferrocyanide (four negative charges) and an electrostatic attraction towards the positively charged TMPD, thus explaining the different pH-activity curves obtained for the two electron donors. In contrast to the artificial electron donors, the physiological donor ascorbate [Terland and Flatmark (1975) FEBS Lett. 59, 52—56] supports hydroxylation of tyramine at low pH also in the absence of Cl-, acetate or phosphate, confirming that ascorbate also functions as an anion activator.
Sorting of carboxypeptidase E to the regulated secretory pathway requires interaction of its transmembrane domain with lipid rafts
Authors: ZHANG Chun-Fa , DHANVANTARI Savita , LOU Hong , LOH Y. Peng
Published: February 2003

Carboxypeptidase E (CPE) functions as a regulated secretory pathway sorting receptor for several prohormones, including pro-opiomelanocortin (POMC), proenkephalin and proinsulin. The association of CPE with lipid rafts in the trans-Golgi network and secretory granule membranes is necessary for its sorting receptor function. We now provide evidence that a domain within the C-terminal 25 residues of CPE functions as a signal for both raft association and the sorting of CPE to the regulated secretory pathway. A fusion protein containing the extracellular domain of the human interleukin-2 receptor Tac (N-Tac) and the C-terminal 25 amino acids of CPE was transfected into Neuro2A cells. This fusion protein floated in sucrose density gradients, indicating raft association, and co-localized with chromogranin A (CGA), a secretory granule marker. To define further a minimum sequence required for raft association and sorting, deletion mutants of CPE that lacked the C-terminal four or 15 residues (CPE-Δ4 and CPE-Δ15 respectively) were transfected into a clone of CPE-deficient Neuro2A cells. In contrast with full-length CPE, neither CPE-Δ4 nor CPE-Δ15 floated in sucrose density gradients. The sorting of both CPE-Δ4 and CPE-Δ15 to the regulated secretory pathway was impaired, as indicated by significantly increased basal secretion and a lack of response to stimulation. Additionally, there was a significant decrease in the co-localization of mutant CPE immunofluorescence with CGA when compared with full-length CPE. Finally, the sorting of the prohormone POMC to the regulated pathway was impaired in cells transfected with either CPE-Δ4 or CPE-Δ15. We conclude that the sorting of CPE to the regulated secretory pathway in endocrine cells is mediated by lipid rafts, and that the C-terminal four residues of CPE, i.e. Thr431-Leu-Asn-Phe434, are required for raft association and sorting.
Control of human carnitine palmitoyltransferase II gene transcription by peroxisome proliferator-activated receptor through a partially conserved peroxisome proliferator-responsive element
Authors: BARRERO María J. , CAMARERO Nuria , MARRERO Pedro F. , HARO Diego
Published: February 2003

The expression of several genes involved in fatty acid metabolism is regulated by peroxisome proliferator-activated receptors (PPARs). To gain more insight into the control of carnitine palmitoyltransferase (CPT) gene expression, we examined the transcriptional regulation of the human CPT II gene. We show that the 5′-flanking region of this gene is transcriptionally active and binds PPARα in vivo in a chromatin immunoprecipitation assay. In addition, we characterized the peroxisome proliferator-responsive element (PPRE) in the proximal promoter of the CPT II gene, which appears to be a novel PPRE. The sequence of this PPRE contains one half-site which is a perfect consensus sequence (TGACCT) but no clearly recognizable second half-site (CAGCAC); this part of the sequence contains only one match to the consensus, which seems to be irrelevant for the binding of PPARα. As expected, other members of the nuclear receptor superfamily also bind to this element and repress the activation mediated by PPARα, thus showing that the interplay between several nuclear receptors may regulate the entry of fatty acids into the mitochondria, a crucial step in their metabolism.
An ERG (ets-related gene)-associated histone methyltransferase interacts with histone deacetylases 1/2 and transcription co-repressors mSin3A/B
Authors: YANG Liu , MEI Qi , ZIELINSKA-KWIATKOWSKA Anna , MATSUI Yoshito , BLACKBURN Michael L. , BENEDETTI Daniel , KRUMM Anton A. , TABORSKY Gerald J. , CHANSKY Howard A.
Published: February 2003

Covalent modifications of histone tails play important roles in gene transcription and silencing. We recently identified an ERG (ets-related gene)-associated protein with a SET (suppressor of variegation, enhancer of zest and trithorax) domain (ESET) that was found to have the activity of a histone H3-specific methyltransferase. In the present study, we investigated the interaction of ESET with other chromatin remodelling factors. We show that ESET histone methyltransferase associates with histone deacetylase 1 (HDAC1) and HDAC2, and that ESET also interacts with the transcription co-repressors mSin3A and mSin3B. Deletion analysis of ESET reveals that an N-terminal region containing a tudor domain is responsible for interaction with mSin3A/B and association with HDAC1/2, and that truncation of ESET enhances its binding to mSin3. When bound to a promoter, ESET represses the transcription of a downstream luciferase reporter gene. This repression by ESET is independent of its histone methyltransferase activity, but correlates with its binding to the mSin3 co-repressors. In addition, the repression can be partially reversed by treatment with the HDAC inhibitor trichostatin A. Taken together, these data suggest that ESET histone methyltransferase can form a large, multi-protein complex(es) with mSin3A/B co-repressors and HDAC1/2 that participates in multiple pathways of transcriptional repression.