bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of fatty acid α-hydroperoxide-forming enzyme (α-oxygenase) in rice plants
Authors: Koeduka T. , Matsui K. , Akakabe Y. , Kajiwara T.
Published: January 2005

Genes encoding an α-oxygenase, in Nicotiana tabacum and Arabidopsis thaliana have been recently isolated. However, the reaction mechanism of the enzyme has not so far been elucidated. In this study, a cDNA encoding the fatty acid α-oxygenase gene in rice plants was isolated. The deduced amino acid sequence showed high similarity (63.6%) to that of N. tabacum. The gene was cloned into an expression vector system, pQE-30, and expressed in Escherichia coli as a host cell. Palmitic acid as a substrate was incubated with the lysate of the cells, and the products were analysed by HPLC. A compound formed predominantly by the recombinant enzyme was shown to be n-pentadecanal. By incubating the mixture at 0 °C, 2-hydroperoxypalmitic acid was detected as a primary product and little formation of n-pentadecanal was detected. Furthermore, uptake of molecular oxygen was observed with an oxygen electrode. This indicated that the gene in rice plants encodes the α-oxygenase.
Microbial degradation of the plant sulpholipid
Authors: Roy A. B. , Ellis A. J. , White G. F. , Harwood J. L.
Published: January 2005

Five bacterial strains capable of using sulphoquinovose (6-deoxy-6-sulpho-D-glucopyranose) as a sole source of carbon have been isolated and partially characterized.
Non-mevalonate isoprenoid biosynthesis: enzymes, genes and inhibitors
Authors: Lichtenthaler H. K.
Published: January 2005

The essential steps of the novel non-mevalonate pathway of isopentenyl diphosphate and isoprenoid biosynthesis in plants are described. The first five enzymes and genes of this 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway are known. The herbicide fosmidomycin specifically blocks the second enzyme, the DOXP reductoisomerase. The DOXP/MEP pathway is also present in several pathogenic bacteria and the malaria parasite. Hence, all herbicides and inhibitors blocking this novel isoprenoid pathway in plants are also potential drugs against malaria and diseases caused by pathogenic bacteria.
The non-mevalonate isoprenoid biosynthesis of plants as a test system for drugs against malaria and pathogenic bacteria
Authors: Zeidler J. , Schwender J. , Mueller C. , Lichtenthaler H. K.
Published: January 2005

Two plant test systems are presented in the search for new inhibitors of the non-mevalonate isoprenoid pathway. A derivative of clomazone appears to be an inhibitor of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate (DOXP/MEP) pathway of isoprenoid formation.
Phytosterols in the aleurone layer of corn kernels
Authors: Moreau R. A. , Singh V. , Nuñez A. , Hicks K. B.
Published: January 2005

Corn hulls are composed of two major layers: the outer layer, the pericarp, is made up of non-living cell walls, and an inner layer, the aleurone, consists of a single layer of living cells, surrounded by thick cell walls. Dissected pure pericarp and aleurone fractions were ground and extracted with hexane and the yields and compositions of the resulting oils were examined. This study revealed that the high levels of ferulate-phytosterol esters and the high concentration of sitostanol previously reported in corn-fibre oil actually originate in the aleurone cells.
Characterization of a phospholipase Dα cDNA from tomato fruit
Authors: Whitaker B. D. , Smith D. L. , Green K. C.
Published: January 2005

Phospholipase D (PLD) initiates phospholipid (PL) catabolism in plant cells and is also involved in signal transduction and retailoring of membrane PL. Total PL declines and phosphatidic acid increases in pericarp tissue during tomato fruit ripening, suggesting that increased PLD activity alters membrane structure. To assess the role of PLD in tomato ripening, we have begun a molecular genetic approach. Using a castor bean PLDα cDNA as a probe, a PLDα cDNA (LEPLD2) was isolated from our tomato fruit library. It has an open reading frame of 2421 nucleotides, encoding a polypeptide of 807 amino acids with a molecular mass of 92 kDa. The deduced LEPLD2 PLDα shares > 75% sequence identity with PLDαs from castor bean, tobacco and tomato. LEPLD2 transcript, detected by RNA gel-blot analysis, was very low in roots, low in stems, moderate in leaves, high in flowers, and increased in fruit during development and ripening. Expression of LEPLD2 in Escherichia coli yielded active enzyme, and a FLAG-PLDα fusion protein produced by transformed E. coli migrated close to the calculated 94 kDa on SDS/PAGE.
Lipoxygenase-catalysed degradation of carotenoids from tomato in the presence of antioxidant vitamins
Authors: Biacs P. A. , Daood H. G.
Published: January 2005

Carotenoid extract from ripe tomato fruit was subjected to a lipoxygenase-catalysed co-oxidation in the presence of vitamin C and vitamin E at different concentrations. Relative retention (%) of major carotenoids by the experimental mixture was used as an index of their degradation and interaction with the antioxidants. Oxidation-prevention activity of each antioxidant against pigment co-oxidation as impacted by their molar concentration was studied. β-Carotene was found to be the most sensitive pigment, followed by lycoxanthin and lycopene. Ascorbic acid when added in the range of 0–1.8 mM interacted with the different carotenoids by different modes. Evidence was given on regeneration, by ascorbic acid, of lycopene during the course of co-oxidation. The concentration required for α-tocopherol acetate to exhibit antioxidative effect was 10 times higher than that of ascorbic acid. β-Carotene was prevented, by α-tocopherol acetate, faster than lycoxanthin and lycopene. The latter carotenoids differed substantially in their interaction with the lipophilic antioxidant at only the lowest concentration (0.3 mM). It was established that under the given conditions there is no synergism between vitamin C and vitamin E that improves their oxidation prevention against co-oxidation of carotenoids. Moreover, the combined use of antioxidants caused more oxidative degradation of β-carotene.
Characterization of a Euphorbia lagascae epoxide hydrolase gene that is induced early during germination
Authors: Edqvist J. , Farbos I.
Published: January 2005

In Euphorbia lagascae the major fatty acid in triacylglycerol is the epoxidated fatty acid vernolic acid (cis- 12-epoxyoctadeca-cis-9-enoic acid). The enzymic reactions occurring during the catabolism of epoxidated fatty acids during germination are not known, but it seems likely that the degradation requires the activity of an epoxide hydrolase. Epoxide hydrolases are a group of functionally related enzymes that catalyse the cofactor-independent hydrolysis of epoxides to their corresponding vicinal diols by the addition of a water molecule. Here we report the cloning and characterization of an epoxide hydrolase gene from E. lagascae. The structure of the gene is unusual since it lacks introns. A detailed investigation of the transcription pattern of the epoxide hydrolase gene shows that the gene is induced during germination. We have used in situ hybridization to identify in which tissues the gene is expressed during germination. We speculate that this epoxide hydrolase enzyme is involved in the catabolism of epoxidated fatty acids during germination of E. lagascae seeds.
State of the lipid and fatty acid composition in chloroplasts of Nicotiana tabacum under the influence of an increased CO2 partial pressure of 700 p.p.m.
Authors: Radunz A. , Alfermann K. , Schmid G. H.
Published: January 2005

We analysed chloroplast lipids of Nicotiana tabacum var. John William's Broadleaf, cultivated under an increased Pco2 of 700 p.p.m. Glycolipids and phospholipids remain constant under these conditions, whereas the carotenoid content undergoes a quantitative change. The saturation degree of fatty acids increases due to an increase in palmitic acid and decreases in hexadecatrienoic acid and linolenic acid.
The tolerance of cyanobacterium Cylindrospermopsis raciborskii to low-temperature photo-inhibition affected by the induction of polyunsaturated fatty acid synthesis
Authors: Várkonyi Z. , Zsiros O. , Farkas T. , Garab G. , Gombos Z.
Published: January 2005

Acyl-lipid desaturation introduces double bonds (unsaturated bonds) at specifically defined positions of fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. Desaturation patterns of the glycerolipids of Cylindrospermopsis raciborskii a filamentous cyanobacterium, were determined in cells grown at 35°C and 25°C. The lowering of the growth at 35°C and 25°C. The lowering of the growth temperature from 35°C to 25°C resulted in a considerable accumulation of polyunsaturated octadecanoic fatty acids in all lipid classes. The tolerance to low-temperature photo-inhibition of the C. raciborskii cells grown at 25°C and 35°C was also compared. The lower growth temperature increased the tolerance of C. raciborskii cells. These results strengthen the importance of polyunsaturated glycerolipids in the tolerance to environmental stresses and may give a physiological explanation for the determinative role of C. raciborskii in algal blooming in Lake Balaton (Hungary).
High-oleic acid Australian Brassica napus and B. juncea varieties produced by co-suppression of endogenous Δ12-desaturases
Authors: Stoutjesdijk P. A. , Hurlestone C. , Singh S. P. , Green A. G.
Published: January 2005

Genetic engineering methods have been used successfully to modify the fatty acid profile of elite Australian germplasm of Brassica napus and B. juncea. Co-suppression plasmids carrying oleate desaturase genes from each species have been constructed and transferred into Australian elite breeding lines of B. napus and B. juncea using Agrobacterium tumifaciens plant-transformation techniques. Modifications to existing Brassica transformation protocols and the use of an intron-interrupted hygromycin-resistance gene as the selectable marker have resulted in improved transformation efficiencies. Silencing of the endogenous oleate desaturase genes has resulted in substantial increases in oleic acid levels, up to 89% in B. napus and 73% in B. juncea.
Characterization of an acyl-CoA synthetase from Arabidopsis thaliana
Authors: Schnurr J. A. , Shockey J. , Browse John
Published: January 2005

One of the major goals of modern plant biotechnology is to manipulate lipid metabolism in oilseed crops to produce new and improved edible and industrial vegetable oils. Lipids constitute the structural components of cellular membranes and act as sources of energy for the germinating seed and are therefore essential to plant cell function. Both de novo synthesis and modification of existing lipids are dependent on the activity of acyl-CoA synthetases (ACSs). To date, ACSs have been recalcitrant to traditional methods of purification due to their association with membranes. In our laboratory, several isoforms of ACSs have been identified in Arabidopsis thaliana. Reverse genetics allowed us to identify a mutant containing a transfer DNA-interrupted ACS gene. Results will be presented that describe the isolation and characterization of this mutant. The elucidation of the specific roles of ACSs will lead to a greater understanding of plant lipid metabolism.
Cloning of acyl-acyl carrier protein (ACP) thioesterase gene from Brassica juncea
Authors: Mandal M. N. A. , Santha I. M. , Lodha M. L. , Mehta S. L.
Published: January 2005

Acyl-acyl carrier protein (ACP) thioesterase is the chain-length-determining enzyme in de novo biosynthesis of plant fatty acids. For cloning the gene encoding acyl-ACP thioesterase from Brassica juncea genomic DNA was used as a template to amplify a 0.7 kb thioesterase fragment in a PCR with the primers designed from the known sequences available in the GenBank. This 0.7 kb fragment was used as a probe to study the expression of the gene in developing seeds and also to screen a genomic library of B. juncea constructed in λEMBL-3 to get the full length of the gene. A 4.0 kb BamHI fragment containing the full gene was finally cloned in a plasmid vector from a recombinant phage clone λ5.12 after a series of screening, sub-cloning and Southern hybridization.
Evidence of cross-reactivity between porcine pancreatic and rapeseed (Brassica napus L.) lipases
Authors: Belguith Hatem , Jridi Taoufik , Hamida Jeannette Ben
Published: January 2005

A cross-reactivity between animal and plant lipases was determined, using immunological techniques. It was shown by ELISA and dot-blotting that these antibodies react with lipases in the rapeseed crude extract and in the different cellular fractions obtained by differential centrifugation. Pre-incubation of the antiserum with the rapeseed crude extract affects the amount of antibodies binding to the porcine pancreatic lipase. Antibodies were able to precipitate lipase activity from 3-day-old rapeseed crude extract. These epitopes seem to be located in the catalytic site, suggesting that a consensus sequence exists in oleaginous lipases and that it will be universal.
Society and the Human Genome
Authors: Sulston J. ,   the International Human Genome Sequencing Consortium
Published: January 2005

In June 2000, the draft sequence of the human genome was announced. It is, and will be for some years, incomplete, but the vast majority is now available. Currently about a third is finished (including two complete chromosomes); the rest has good coverage, but not long-range continuity. First-pass analysis indicates, among other things, fewer genes than expected: about 40000 now looks a likely number. This uncertainty illustrates the difficulty of interpretation: the sequence is not an end in itself, but a resource to be continually reanalysed as our biological understanding increases. That is the scientific reason for releasing it promptly, fully and freely. The social reasons for doing so are even more compelling.
Cytokines and cytokine receptors in fetal growth and development
Authors: Symonds M. E. , Mostyn A. , Stephenson T.
Published: January 2005

The cytokine receptors for growth hormone (GH), prolactin and leptin have a critical role in regulating embryo, placental and/or fetal development, which is dependent on stage of gestation and species. GH and prolactin receptors are detectable from conception, and alterations in the maternal hormonal environment may impact on placental growth from this early stage of gestation. Leptin is critical for conception, but its role in fetal growth remains elusive. During late gestation, when fetal growth accelerates and organ maturation occurs, prolactin and insulin-like growth factor-I may have interactive roles in regulating the growth of specific tissues, including adipose tissue. Prolactin, leptin and GH all have specific effects on fetal and neonatal energy balance, which are mediated in part through promoting lipolysis and/or enhancing the expression of uncoupling proteins. An increased understanding of these interactions is likely to have important implications for a number of potentially pathological conditions, including infection, obesity and hypertension.
Transcriptional regulation in the placenta during normal and compromised fetal growth
Authors: Anthony R. V. , Limesand S. W. , Jeckel K. M.
Published: January 2005

The placenta synthesizes a number of cytokines and growth factors that are involved in the establishment, maintenance or regulation of pregnancy. Included are interferons, placental lactogens, other members of the growth hormone/prolactin gene family, leptin, and an array of angiogenic growth factors. While their roles in pregnancy differ, in their absence pregnancy is either lost or compromised. Therefore an understanding of the cell-specific transcriptional regulation of these genes is imperative if we are ever to alter their expression to benefit pregnancy progression. Our understanding of transcriptional regulation in the placenta is still in its infancy, and there appears to be considerable divergence in the transcriptional regulation of these genes between species, as well as between the various cytokine genes being examined. For example, while there are some commonalities in the regulation of human, rodent and ruminant placental lactogens, there are differences that require the study of placental lactogen gene regulation across species. However, one common theme that is emerging with the angiogenic growth factors, such as vascular endothelial growth factor and the angio-poietins, is the transcriptional control of these genes by oxygen tension within the placenta. Examination of transcriptional regulation in normal and compromised pregnancies will provide additional insight in this area.
Prolactin receptor heterogeneity: processing and signalling of the long and short isoforms during development
Authors: Schuler L. A. , Lu J.-C. , Brockman J. L.
Published: January 2005

During development, the fetus is exposed to prolactin activity from the placenta, as well as from the developing fetal pituitary. Distinct prolactin receptor isoforms, having different cytoplasmic domains generated by alternative splicing, are expressed as development proceeds at different levels in different organs. The ‘long’ receptors are able to mediate transduction of all signals examined, in contrast with the ‘short’ isoforms, whose truncated cytoplasmic domains are able to mediate a much smaller repertoire of signals and can act as dominant negatives. Our studies demonstrate that, although these forms share internalization mechanisms, the long form is internalized faster, resulting in more rapid down-regulation of this form. In order to examine the mechanisms by which prolactin may exert trophic effects on its target tissues during development, we have examined the signalling pathways through which prolactin binding to the long receptor regulates the transcription of cyclin D1. Our studies reveal the importance of the JAK/STAT (Janus kinase/signal transduction and activators of transcription) pathway, and the complexity of prolactin signalling to this promoter.
Maturation of cytokine receptors in preparation for birth
Authors: McMillen I. C. , Houghton D. C. , Phillips I. D.
Published: January 2005

The elucidation of the tissue-specific profile of expression of the prolactin (PRL) and growth hormone (GH) receptors during embryonic and fetal development in a range of species has provided a new impetus for the delineation of the specific roles of the hormone ligands for these receptors in development. During late gestation, there is a requirement to shift from a phase of predominant cellular proliferation, where placental nutrient supply is a dominant influence on organ and body growth, to one of functional differentiation, which is required for independent homoeostasis after birth. In this review we discuss the interactions between the pre-partum increases in cortisol and thyroid hormones and the synthesis, secretion and actions of fetal PRL and GH. We also review the changes that occur in the tissue-specific expression of the PRL and GH receptors before birth which may play an important role in precocial species in the successful transition of the fetus to extra-uterine life.
Regional differences in protein production by human adipose tissue
Authors: Arner P.
Published: January 2005

Human adipose tissue has an important protein secretory function. Cytokines, hormones, prohormones and enzymes are secreted from fat cells and act in an endocrine or paracrine fashion. The production of several of these proteins is affected by obesity; normally there is an increase in the obese state. Protein production is, as a metabolic activity, subject to regional variations. In particular, the production of leptin, angiotensinogen, interleukin-6 and plasmin activator inhibitor-1 differs between subcutaneous and visceral adipose tissue sites, but no regional differences have been reported in the production of tumour necrosis factor α. It is possible that regional variations in protein production by adipose tissue are of importance in some of the endocrine and metabolic disturbances seen in various forms of obesity, such as visceral and upper-body obesity.
Dynamic biosynthesis of heparan sulphate sequences in developing mouse brain: a potential regulatory mechanism during development
Authors: Guimond S. , Turner K. , Kita M. , Ford-Perriss M. , Turnbull J.
Published: January 2005

Over recent years our understanding of the functions of the heparan sulphate (HS) family of complex polysaccharides has shifted dramatically. Once seen as simply structural scaffolding in the extracellular matrix, they are now viewed as critical players in the regulatory network of cells. They are strategically located at the cell surface and in the extracellular matrix, and there has been an increasing realization that specific sequences in the HS chains are designed for selective interactions with many proteins. Functionally, these interactions result in regulation of the protein activities. It is becoming clear that HS functions as a new class of multifunctional cell regulator. There is also growing evidence that cells can dynamically alter the structure of HS sequences that they express. Here we review recent developments and describe evidence for regulated changes in the synthesis and structure of HS chains expressed during early mouse brain development. The data suggest a new concept in which dynamic changes in biosynthesis of different HS sequences create distinct cellular HS repertoires, the heparanome [1]. Their expression, in specific spatio-temporal patterns, is likely to endow organisms with novel regulatory mechanisms for controlling the activity of specific HS-binding proteins.
The life and work of Dermot Hedley (‘Derek’) Williamson (1929–1998)
Authors: Evans R. D. , Stubbs M. , Gibbons G. F. , Newsholme E. A.
Published: January 2005

Derek Williamson's scientific career spanned the ‘Golden Age’ of research into metabolic regulation, to which he made an important and sustained contribution. Derek joined Hans Krebs' laboratory at Sheffield University in 1946 and moved to Krebs' MRC Unit in Oxford in 1960. He elaborated an enzymic method for the determination of acetoacetate and 3-hydroxybutyrate [Williamson, Mellanby and Krebs, Biochem. J. (1962) 82, 90–96], which opened up the field of ketone body metabolism and its regulation and became a Citation Classic. Another Citation Classic followed [Williamson, Lund and Krebs, Biochem. J. (1967) 103, 514–527]. He moved with Krebs to the Metabolic Research Laboratory at the Radcliffe Infirmary in 1967, where he blossomed, formulating his ideas about the integrated regulation of metabolic pathways, particularly with regard to fatty acid oxidation, lipid synthesis and ketone body metabolism. His success was illustrated by more than 200 publications. Derek implanted and nurtured a sense of the excitement of scientific discovery in his colleagues and students, and he worked hard to provide a friendly, supportive and encouraging environment. Many lives have been enriched by the privilege of working with him.
Travels with carnitine palmitoyltransferase I: from liver to germ cell with stops in between
Authors: McGarry J. D.
Published: January 2005

Although malonyl-CoA was first recognized as a central component in the regulation of hepatic ketogenesis, its inhibitory effect on mitochondrial carnitine palmitoyltransferase I (CPT I) has come to be viewed as a key element in fuel sensing in a variety of body tissues. This broadening role of the malonyl-CoA/CPT I partnership in the control of glucose and fatty acid metabolism, as well as current understanding of structure-function relationships surrounding the CPT proteins, are outlined. The intriguing question of whether CPT I has an important function in sperm development is also addressed briefly.
Carnitine palmitoyl transferase I and the control of myocardial β-oxidation flux
Authors: Eaton S. , Fukumoto K. , Duran N. Paladio , Pierro A. , Spitz L. , Quant P. A. , Bartlett K.
Published: January 2005

Carnitine palmitoyltransferase I is assumed to be rate limiting for β-oxidation in all tissues. However, the concentration of malonyl-CoA in heart and muscle is high and is enough to completely inhibit β-oxidation if this assumption is correct. In this review, we consider whether: (i) there is a malonyl-CoA-insensitive carnitine palmitoyltransferase I activity; (ii) the measured malonyl-CoA concentration in the heart is physiologically meaningful; and (iii) carnitine palmitoyltransferase I is rate-limiting for β-oxidation in the heart.
Role of ketone bodies in perinatal myocardial energy metabolism
Authors: Bartelds B. , van der Leij F. R. , Kuipers J. R. G.
Published: January 2005

Metabolic changes at around the time of birth are crucial for life. Here we review the energy utilization in the myocardium, emphasizing ketone body metabolism. Before birth, glucose and lactate are the major energy substrates for the myocardium. Long-chain fatty acids (LCFA) are normally not available as an energy substrate for the fetal heart; however, when LCFA are supplied artificially in near-term fetal lambs, they are readily oxidized. Hence the myocardium has no limitation to its ability to use LCFA before birth. After birth, lactate remains an important energy source for the myocardium, whereas the contribution of glucose to myocardial energy production decreases despite an increase in the supply of glucose. The oxidation of ketone bodies increases after birth in relation to an increase in supply. However, ketone bodies account for only 7% of left ventricular oxygen consumption. The supply and contribution of LCFA to the myocardium increases after birth; the oxidation of LCFA accounts for most of the left ventricular oxygen consumption. Hence the role of ketone bodies in myocardial metabolism is limited. However, there are interesting observations on interference between the uptake of different substrates and the release of ketone bodies, which might have consequences for our interpretation of ketone body utilization.
Oxidative DNA damage, antioxidants and DNA repair: applications of the comet assay
Authors: Collins A. R. , Horváthová E.
Published: January 2005

Estimates of background levels of oxidative base damage in human white blood cells vary enormously, from 300 down to 0.4 molecules of 8-oxoguanine per 106 guanines. An EC-funded Concerted Action, the European Standards Committee on Oxidative DNA Damage, is currently attempting to resolve the discrepancy and to agree a realistic estimate of basal endogenous oxidation. Oxidation of lymphocyte DNA is a useful marker of oxidative stress, and this can be decreased by supplementation with pure antioxidants or with foods rich in antioxidants. The steady-state level of DNA oxidation is ultimately controlled by the process of DNA repair; the extent to which this varies between individuals has yet to be established.
Conjugated linoleic acid and oxidative behaviour in cancer cells
Authors: Devery R. , Miller A. , Stanton C.
Published: January 2005

Convincing evidence from rodent models of carcinogenesis indicates that cis-9,trans-11 (c9t11) conjugated linoleic acid (CLA) is a potent naturally occurring anti-carcinogen in the human diet. CLA has been reported to alter the fatty acid composition of biological tissues in a manner that increases their oxidative stability. However, recent information suggests that an antioxidant role for CLA does not seem plausible. Given the knowledge that c9t11 CLA is present in a wide range of meat and dairy food products, our studies have begun to investigate mechanisms by which CLA-enriched milk fat exerts its anti-carcinogenic effects. An oxidative mechanism appears to be involved in its growth-suppressive effects, since supplementation of growth culture medium with CLA (17–71.5 μM) made breast cancer cells more susceptible to lipid peroxidation. Studies have indicated that cancer cells may become enriched in CLA during growth in culture. This may make intracellular lipids more susceptible to ordinary levels of oxidative stress, to the point of producing a cytotoxic effect.
Oxidative stress in cells exposed to low levels of ionizing radiation
Authors: Lyng F. M. , Seymour C. B. , Mothersill C.
Published: January 2005

The ability of medium from γ-irradiated cells to induce early events in the apoptotic cascade, such as the mobilization of intracellular calcium, loss of mitochondrial membrane potential and increased levels of reactive oxygen species, in unirradiated cells was investigated. Medium from irradiated human keratinocytes was harvested and transferred to unirradiated keratinocytes. Intracellular calcium levels, mitochondrial membrane potential and the level of reactive oxygen species were all monitored for a period of 24 h following medium transfer. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential and increases in reactive oxygen species (from 6 h after medium transfer) were observed. There was no significant difference between the effects of medium generated by cells irradiated at 0.5 Gy or 5 Gy. The data suggest that a signal that leads to apoptosis is released from cells undergoing radiation-induced oxidative stress.
Trace elements and DNA damage
Authors: O'Connor J. M.
Published: January 2005

A number of studies (mainly in vitro and in vivo animal models) have examined the interaction of trace elements with DNA. Normal dietary levels of various trace elements are required to prevent the occurrence of oxidative damage, and deficiency may increase susceptibility. Conversely, overload of some trace elements, including copper and iron, has been demonstrated to result in adverse effects. However, under normal physiological conditions, such overloads are unlikely to occur.
Degradation of unassembled and damaged thylakoid proteins
Authors: Adam Z. , Ostersetzer O.
Published: January 2005

To study protein degradation in thylakoid membranes we identified, characterized and cloned thylakoid proteases, and then linked them to known proteolytic processes. Several families of chloroplast proteases were identified and characterized to different extents. FtsH, an ATP-dependent metalloprotease that belongs to the AAA-protein family, was found to be integral to the thylakoid membrane, facing the stroma. It is involved in both the degradation of unassembled subunits of membrane complexes, such as the Rieske Fe-S protein of the cytochrome complex, and the degradation of oxidatively damaged proteins such as the D1 protein of the photosystem II (PS II) reaction centre. Plant genomes contain multiple isomers of this protease but the functional significance of this multiplication is not clear yet. A second protease, the serine ATP-independent DegP, was found to be strongly associated with the luminal side of the thylakoid membrane. Although a specific role has not yet assigned for it, its location suggests that it can degrade luminal soluble proteins as well as luminally exposed regions of thylakoid membrane proteins.
The ubiquitin-proteasome pathway regulates lysosomal degradation of the growth hormone receptor and its ligand
Authors: van Kerkhof P. , Strous G. J.
Published: January 2005

The growth hormone (GH) receptor (GHR) is a mammalian plasma membrane protein whose internalization is mediated by the ubiquitin-proteasome pathway. GH internalization and degradation are inhibited when cells are treated with proteasome inhibitors. Here we show that a GHR truncated at residue 369 can enter the cells in the presence of a proteasome inhibitor, but that the subsequent lysosomal degradation of GH is blocked. Lysosomal inhibitors prolong the half-life of both receptor and ligand. Experiments with antibodies against different receptor tail sections show that degradation of the GHR cytosolic domain precedes degradation of the extracellular GH-binding domain. A possible role for the ubiquitin-proteasome pathway in the degradation of the receptor and ligand is discussed.
Multiple pathways for the dynamin-regulated internalization of muscarinic acetylcholine receptors
Authors: van Koppen C. J.
Published: January 2005

An important regulatory pathway of G-protein-coupled receptors (GPCRs) is the internalization of receptors into the cell interior. To unravel the molecular mechanisms by which GPCRs are internalized, we have studied the internalization of various members of the family of muscarinic acetylcholine receptors (mAChRs). Using the transient expression system of HEK-293 cells, we showed that the M1, M3 and M4 mAChRs are internalized into clathrin-coated vesicles and recycle back to the plasma membrane. This internalization pathway is dependent on the concerted action of β-arrestin, c-Src and the GTPase dynamin, which ‘catalyses’ the budding of clathrin-coated vesicles (and other vesicles) from the plasma membrane. Internalization of the M2 mAChR (which is highly structurally and functionally related to the M4 receptor subtype) also requires dynamin, but proceeds in an apparent β-arrestin-, c-Src- and clathrin-independent manner. Internalized M2 mAChRs also show virtually no receptor recycling, but are down-regulated. This demonstrates that GPCRs can be internalized by multiple dynamin-dependent pathways in a highly regulated manner.
Interplay between insulin and nutrients in the regulation of translation factors
Authors: Proud C. G. , Wang X. , Patel J. V. , Campbell L. E. , Kleijn M. , Li W. , Browne G. J.
Published: January 2005

Protein synthesis in mammalian cells is regulated through alterations in the states of phosphorylation of eukaryotic initiation factors and elongation factors (eIFs and eEFs respectively) and of other regulatory proteins. This modulates their activities or their abilities to interact with one another. Insulin activates several of these proteins including the following: the guanine-nucleotide exchange factor eIF2B; the eIF4F complex, which (through eIF4E) interacts with the cap of the mRNA; p70 S6 kinase; and elongation factor eEF2, which mediates the translocation step of elongation. Control of the last three of these is linked to mTOR (mammalian target of rapamycin). In Chinese hamster ovary cells, regulation of all these proteins by insulin is modulated by the presence of amino acids and/or glucose in the medium. For example, p70 S6 kinase activity declines in the absence of amino acids and cannot be stimulated by insulin under this condition. The readdition of amino acids, especially leucine, restores activity and sensitivity to insulin. With eIF2B and eEF2, both amino acids and glucose must be provided for insulin to regulate their activities. In contrast, insulin-stimulation of the formation of eIF4F complexes requires glucose but not amino acids. Glucose metabolism is required for this permissive effect. Our recent studies have also identified the mechanism by which mTOR signalling regulates the phosphorylation of eEF2. eEF2 kinase is phosphorylated by p70 S6 kinase at Ser-366; this results in the inactivation of eEF2 kinase, especially at low (micromolar) Ca concentrations.
De novo design, synthesis and characterization of membrane-active peptides
Authors: Lear J. D. , Gratkowski H. , DeGrado W. F.
Published: January 2005

Our current level of understanding of membrane-protein folding is primitive, but it is beginning to advance. Previously [Choma, Gratkowski, Lear and DeGrado (2000) Nat. Struct. Biol. 7, 161–166], we described studies of the association in detergent micelles of short, simple-sequence hydrophobic peptides modified from the sequence of the water-soluble, homodimeric coiled-coil GCN4-P1 peptide using the principle that the interiors of membrane proteins are similar to those of water-soluble proteins. Here, we discuss more quantitative aspects of the association equilibrium and compare the free energies of association of a number of mutant peptides designed to explore specific features responsible for the association.
Application of electron spin resonance for investigating peptide-lipid interactions, and correlation with thermodynamics
Authors: Marsh D.
Published: January 2005

Peptide-lipid interactions can be investigated with spin-labelled lipid probes by using electron spin resonance (ESR) methods that have been developed for studying lipid-protein interactions with both integral and peripheral membrane proteins and also with surface-binding proteins that additionally penetrate the membrane. This approach has the advantage that a direct comparison can be made with the databank of ESR results from the various types of membrane protein. The appropriateness of the peptides as models for membrane proteins, or for their specific segments, can then be assessed. Further, differences in behaviour can be readily identified, as for example in the case of surface-active cytolytic or fusogenic peptides. Comparison with thermodynamic predictions for membrane insertion provides a useful adjunct to the spin-label method.
Uncovering BRCAI-regulated signalling pathways by microarray-based expression profiling
Authors: Mullan P. B. , McWilliams S. , Quinn J. , Andrews H. , Gilmore P. , McCabe N. , McKenna S. , Harkin D. P.
Published: January 2005

The introduction of microarray technology to the scientific and medical communities has dramatically changed the way in which we now address basic biomedical questions. Expression profiling using microarrays facilitates an experimental approach where alterations in the transcript level of entire transcriptomes can be simultaneously assayed in response to defined stimuli. We have used microarray analysis to identify downstream transcriptional targets of the BRCA1 (Breast Cancer 1) tumour-suppressor gene as a means of defining its function. BRCA1 has been implicated in the predisposition to early onset breast and ovarian cancer and while its exact function remains to be defined, roles in DNA repair, cell-cycle control and transcriptional regulation have been implied. In the current study we have generated cell lines with tetracycline-regulated, inducible expression of BRCA1 as a tool to identify genes, which might represent important effectors of BRCA1 function. Oligonucleotide array-based expression profiling identified a number of genes that were upregulated at various times following inducible expression of BRCA1 including the DNA damage-responsive gene GADD45 (Growth Arrest after DNA Damage). Identified targets were confirmed by Northern blot analysis and their functional significance as BRCA1 targets examined.
UV stimulation induces nuclear factor κB (NF-κB) DNA-binding activity but not transcriptional activation
Authors: Campbell K. J. , Chapman N. R. , Perkins N. D.
Published: January 2005

The cellular response to DNA-damaging agents is partly mediated by DNA-binding transcription factors such as p53 and nuclear factor κB (NF-κB). Typically NF-κB activation is associated with resistance to apoptosis. Following stimulation with UV light however, NF-κB activation has been shown to be required for programmed cell death. To study this effect further and to determine the relationship between NF-κB and p53 function, we have examined the effect of UV light on U2OS cells. UV stimulation resulted in the activation of NF-κB DNA-binding and the induction of p53. Surprisingly, and in contrast with tumour necrosis factor α stimulation, this UV-induced NF-κB was transcriptionally inert. These observations suggest a model in which the NF-κB switch from an anti-apoptotic to a pro-apoptotic role within the cell results from modulation of its ability to stimulate gene expression, possibly as a result of the ability of p53 to sequester transcriptional co-activator proteins such as p300/CREB (cAMP-response-element-binding protein)-binding protein.
Impact of oligomerization on the function of the human serotonin transporter
Authors: Schmid J. A. , Just H. , Sitte H. H.
Published: January 2005

The formation of oligomeric structures has been proposed for a large number of membrane proteins, including G-protein-coupled receptors and ion channels. Biochemical studies employing gel filtration, cross-linking or co-immunoprecipitation techniques showed that the serotonin [5-hydroxytryptamine (5-HT)] transporter is also capable of forming oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. To test this working hypothesis, we generated fusion proteins of hSERT and spectral variants of green fluorescent protein [cyan and yellow fluorescent proteins (CFP and YFP, respectively)]. When expressed in HeLa or HEK-293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were inserted into the plasma membrane and were indistinguishable from wild-type hSERT on functional testing (5-HT uptake assays, inhibition of 5-HT uptake by blockers such as imipramine). Oligomers were visualized by fluorescence resonance energy transfer (FRET) microscopy in living cells using complementary methods. Interestingly, oligomerization was not confined to hSERT; FRET was also observed between CFP-and YFP-labelled rat γ-aminobutyric acid transporter. Gel filtration experiments showed that most of the protein was recovered as higher molecular weight complexes; almost no monomeric form was detected. This indicates that the homo-oligomeric form is the favoured state of hSERT in living cells. The formation of oligomers was not significantly affected by co-incubation with transporter substrates or blockers. Based on our observations, oligomer formation might not be essential for the physiological function of the transporter protein, the re-uptake of substrates. Furthermore, we conclude that constitutive oligomer formation might be a general property of Na+/Cl−-dependent neurotransmitter transporters.
Regulation of glycine transporters
Authors: López-Corcuera B. , Aragón C. , Geerlings A.
Published: January 2005

The regulation of neurotransmitter transporters is a central aspect of their physiology. Recent studies that focused on syntaxin-1 transporter interactions led to the postulation that syntaxin-1 is somehow implicated in protein trafficking. Because syntax – in-1 is involved in the exocytosis of neurotransmitters and it interacts with glycine transporter 2 (GLYT2), we stimulated exocytosis in synaptosomes and examined its effect on GLYT2 surface-expression and transport activity. We found that GLYT2 is rapidly trafficked first towards the plasma membrane and then internalized under conditions that stimulate vesicular glycine release. However, when syntaxin-1 was inactivated by pre-treatment of synaptosomes with the botulinum neurotoxin C, GLYT2 was unable to reach the plasma membrane but still was able to leave it. These results indicate the existence of a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated regulatory mechanism that controls the surface expression of GLYT2. Syntaxin-1 is involved in the transport of GLYT2 to, but not its retrieval from, the plasma membrane. Immunogold-labelling on purified vesicular preparations from synaptosomes showed that GLYT2 is present in small synaptic-like vesicles. This may represent neurotransmitter transporter that is being trafficked. The subcellular distribution of the glycine transporters was further examined in PC12 cells that were stably transfected with the fusions of GLYT1 and GLYT2 with green fluorescent protein. There was a clear difference in their intracellular distribution, GLYT1 being present mainly on the plasma membrane and GLYT2 being localized mainly on large, dense-core vesicles. We are trying to find signal sequences responsible for this differential localization.
A history of UCPI
Authors: Nicholls D. G.
Published: January 2005

Interest in the enormous thermogenic capacity of brown adipose tissue (BAT) began in the 1960s and focused on BAT mitochondria (BATM), which when prepared by conventional techniques respired rapidly but displayed no respiratory control. Two apparently distinct treatments, fatty acid removal and purine nucleotide addition, induced respiratory control. In 1972, we found that BATM were highly permeant to halides and protons, and that albumin decreased the proton conductance while purine nucleotides decreased both. Devising techniques to quantify the proton leak in respiring mitochondria we found a nucleotide-sensitive conductance pathway whose ‘break-point’, the protonmotive force at which conductance suddenly increased, could be subtly modulated by free fatty acids. The nucleotide-binding site on the outer face of the inner membrane was characterized and identified by photoaffinity labelling as a 32 kDa ‘uncoupling protein’, now UCP1. Studies with intact brown adipocytes generated the currently accepted model, namely that fatty acids liberated by β3-adrenergic receptor activation act as both self-regulating second messengers for UCP1 and substrates for fatty acid activation and oxidation. Fatty acid concentration increases at the outset of thermogenesis, binding to UCP1 lowers the protonmotive force below that giving respiratory control and rapid thermogenesis proceeds. At the termination of receptor activation oxidation of residual fatty acid ‘recouples’ the mitochondria. The challenge with the novel UCPs is to demonstrate a similar coherent mechanism.
Uncoupling protein 3 biological activity
Authors: Giacobino J. P.
Published: January 2005

The hypothesis that uncoupling protein 3 (UCP3) is an uncoupling protein involved in heat dissipation is not unequivocally supported. An update of in vitro, ex vivo and in vivo studies testing this hypothesis is presented. Data are provided showing that exercise induces a fatty acid-dependent increase in muscle UCP3 mRNA in humans. The proposed positive correlation between glycolytic capacity and UCP3 level in various muscle-fibre types in the mouse is reassessed. Finally, an association between an intronic polymorphism of UCP3 and adiposity is reported.
Uncoupling protein, H+ transport and regulation
Authors: Klingenberg M. , Winkler E. , Echtay K.
Published: January 2005

The biochemical functions of uncoupling proteins (UCPs) are discussed with the view of UCP1 as a paradigm. In contrast with UCP1, the heterologous expression of UCP3 in yeast is found to result primarily in extra-mitochondrial deposits and thus is unsuitable for studying UCP3 function. On expression in Escherichia coli inclusion bodies, UCPs extracted and incorporated into vesicles showed no H+ transport, only Cl– transport. Only after addition of coenzyme Q was fully nucleotide-sensitive high-H+ transport reconstituted, with UCP1 as well as with UCP2 and UCP3. The newly discovered cofactor role of coenzyme Q in H+ transport is proposed to imply co-operation with fatty acids for the injection of H+ into the UCP channel.
Regulation of de novo sphingolipid biosynthesis and the toxic consequences of its disruption
Authors: Linn S. C. , Kim H. S. , Keane E. M. , Andras L. M. , Wang E. , Merrill A. H.
Published: January 2005

Complex sphingolipids are ‘built’ on highly bio-active backbones (sphingoid bases and ceramides) that can cause cell death when the amounts are elevated by turnover of complex sphingolipids, disruption of normal sphingolipid metabolism, or over-induction of sphingolipid biosynthesis de novo. Under normal conditions, it appears that the bioactive intermediates of this pathway (3-keto-sphinganine, sphinganine and ceramides) are kept at relatively low levels. Both the intrinsic activity of serine palmitoyltransferase (SPT) and the availability of its substrates (especially palmitoyl-CoA) can have toxic consequences for cells by increasing the production of cytotoxic intermediates. Recent work has also revealed that diverse agonists and stresses (cytokines, UV light, glucocorticoids, heat shock and toxic compounds) modulate SPT activity by induction of SPTLC2 gene transcription and/or post-translational modification. Mutation of the SPTLC1 component of SPT has also been shown to cause hereditary sensory neuropathy type I, possibly via aberrant oversynthesis of sphingolipids. Another key step of the pathway is the acylation of sphinganine (and sphingosine in the recycling pathway) by ceramide synthase, and up-regulation of this enzyme (or its inhibition to cause accumulation of sphinganine) can also be toxic for cells. Since it appears that most, if not all, tissues synthesize sphingolipids de novo, it may not be surprising that disruption of this pathway has been implicated in a wide spectrum of disease.