bluuebunny/crossref_metadata_2025 huggingface dataset:
Inverted-repeat DMA: a new gene-silencing tool for seed lipid modification
Authors: Singh S. , Green A. , Stoutjesdijk P. , Liu Q.
Published: January 2005

Post-transcriptional gene silencing (PTGS) has been successfully used to modify seed lipids in oilseed crops like soybean, canola and sunflower. Conventionally, PTGS has been induced by transforming the plants with either antisense or co-suppression constructs targeted against key seed lipid biosynthesis genes. A major drawback of this approach has been the recovery of only a modest proportion of silenced individuals from large populations of transgenic plants. In this report we show that inverted-repeat DNA constructs containing an intron encoding RNA with a hairpin structure can induce PTGS with very high frequency.
Do extraction procedures affect olive oil quality and stabilility?
Authors: Miled D. D. Ben , Smaoui A. , Zarrouk M. , Chérif A.
Published: January 2005

Quality and stability of olive oil extracted using discontinuous (pressing) or continuous (centrifuging) procedures were studied. Virgin olive oils extracted from two olive varieties were stored for 17 months. Analytical parameters of oil quality (acidity, peroxide value, polyphenol content, chlorophyll amount, fatty acid composition and triacylglycerol molecular species) were followed at different time intervals. Our results showed that no differences exist between the two types of tested mill, since the studied parameters were unchanged or varied slightly in the same range during the time of oil conservation. Changes depend on olive varieties rather than extraction systems used. Moreover, oils produced by the continuous system contain comparatively higher levels of polyphenols, which may confer to them greater resistance against oxidation for long-term storage.
Two families of acyl-CoA thioesterases in Arabidopsis
Authors: Tilton G. , Shockey J. , Browse J.
Published: January 2005

We have identified two families of acyl-CoA thioesterase (ACHs) in Arabidopsis thaliana. One family, consisting of AtACH1 and AtACH2, appears to be peroxisomal, as they have type-1 peroxisomal targeting sequences. The other family, consisting of AtACH4 and AtACH5, resides in the endoplasmic reticulum, as shown by green fluorescent protein studies. AtACH2 has been overexpressed in Escherichia coli and shows high levels of acyl-CoA thioesterase activity against both 16:0-CoA and 18:1-CoA. AtACH5 has also been overexpressed in E. coli and shows thioesterase activity as well. ACHs have been characterized in other many other organisms and in various subcellular locations, but their true physiological role is not yet understood. Indeed, atach5 gene knockout mutants have no observable phenotype.
Production of hydroxy fatty acids in the seeds of Arabidopsis thaliana
Authors: Smith M. , Moon H. , Kunst L.
Published: January 2005

Seed-specific expression in Arabidopsis thaliana of oleate hydroxylase enzymes from castor bean and Lesquerella fendleri resulted in the accumulation of hydroxy fatty acids in the seed oil. By using various Arabidopsis mutant lines it was shown that the endoplasmic reticulum (ER) n–-3 desaturase (FAD3) and the FAE1 condensing enzyme are involved in the synthesis of polyunsaturated and very-long-chain hydroxy fatty acids, respectively. In Arabidopsis plants with an active ER Δ12-oleate desaturase the presence of hydroxy fatty acids corresponded to an increase in the levels of 18:1 and a decrease in 18:2 levels. Expression in yeast indicates that the castor hydroxylase also has a low level of desaturase activity.
Anaerobic lipoxygenase activity from Chlorella pyrenoidosa
Authors: Nuñez A. , Foglia T. A. , Piazza G. J.
Published: January 2005

The micro-alga Chlorella pyrenoidosa expresses an enzymatic activity that cleaves the 13hydroperoxide derivatives of linoleic acid [13-hydroperoxy-9(Z),11 (E-octadecadienoic acid, 13-HPOD] and linolenic acid [13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid, 13-HPOT] into volatile C5 and non-volatile C13 oxo-products. This enzymic activity initially was attributed to a hydroperoxide lyase enzyme; however, subsequent studies showed that this cleavage activity is the result of lipoxygenase activity under anaerobic conditions. Headspace analysis of the volatile products by GC/MS showed the formation of pentane when the substrate was 13-HPOD, whereas a more complex mixture of hydrocarbons was formed when 13-HPOT was the substrate. Analysis of the non-volatile cleavage products from 13-HPOD by liquid chromatography/MS indicated the formation of 13-oxo-9(Z),11(E)-tridecadienoic acid (13-OTA) along with the 13-keto-octadecadienoic acid derivative. When the substrate is 13-HPOT, liquid chromatography/MS analysis indicated the formation of 13-OTA as the major non-volatile product. Aldehyde dehydrogenase (AldDH) oxidizes 13-OTA to an ω-dicarboxylic acid, whereas alcohol dehydrogenase (ADH) reduces 13-OTA to an ω-hydroxy carboxylic acid. AldDH and ADH require the oxidized (NAD+) and reduced (NADH) forms of the cofactor NAD, respectively. By combining the action of AldDH and ADH into a continuous cofactor-recycling process, it is possible to simultaneously convert 13-OTA to the corresponding ω-dicarboxylic acid and ω-hydroxy carboxylic acid derivatives.
Characterization of the AMP-binding protein gene family in Arabidopsis thaliana: will the real acyl-CoA synthetases please stand up?
Authors: Shockey J. , Schnurr J. , Browse J.
Published: January 2005

One of the most prominent and important topics in modern agricultural biotechnology is the manipulation of oilseed triacylglycerol composition. Towards this goal, we have sought to identify and characterize acyl-CoA synthetases (ACSs), which play an important role in both de novo synthesis and modification of existing lipids. We have identified and cloned 20 different genes that bear strong sequence homology to known ACSs from other organisms. Through sequence comparisons and functional characterization, we have identified several members of this group that encode ACSs, while the other genes fall into the broader category of genes for AMP-binding proteins (AMPBPs). Distinguishing ACSs from AMPBPs will simplify our efforts to understand the role of ACS in triacylglycerol metabolism.
Enhancement of seed oil content by expression of glycerol-3-phosphate acyltransferase genes
Authors: Jain R. K. , Coffey M. , Lai K. , Kumar A. , MacKenzie S. L.
Published: January 2005

Arabidopsis thaliana was transformed with a plastidial safflower glycerol-3-phosphate acyltransferase (GPAT) and an Escherichia coli GPAT. The genes were used directly and in modified forms with, as applicable, the plastidial targeting sequence removed, and with an endoplasmic reticulum targeting sequence added. Seeds of plants transformed using only the vector were indistinguishable in oil content from wild-type control plants. All other gene constructs increased seed oil content. The unmodified safflower gene (spgpat) produced oil increases ranging from 10 to 21%. On average, the greatest increase (+22%) was observed in seeds of transformants carrying the spgpat with the targeting peptide removed. The E. coli plsB gene increased seed oil content by an average of 15%.
Mutagenesis of a plastidial lysophosphatidic acid acyltransferase
Authors: Maisonneuve S. , Bessoule J.-J. , Lessire R. , Delseny M. , Roscoe T. J.
Published: January 2005

A combination of site-directed and random mutagenesis generated sequence variants of a plastidial lysophosphatidic acid acyltransferase. Alanine substitutions of residues present within two conserved motifs including the putative catalytic histidine resulted in a loss of acyltransferase activity assessed as complementation competance. Substitutions at five sites within the central core resulted in reduced or loss of activity. Truncation mutants reveal that sequences in the C-terminal moiety are essential for function.
Limnanthes douglasii erucic acid-specific lysophospatidic acid acyltransferase activity in oilseed rape: an analysis of biochemical effects
Authors: Wilmer J. A. , Brown A. P. , Forsyth K. , Carnaby S. , Barsby T. , Slabas A. R.
Published: January 2005

It has been shown previously that erucic acid accumulates in the sn-2 position of triglycerides after introduction of a Limnanthes lysophosphatidic acid acyltransferase. However, in none of the studies to date has a proper evaluation been made of the relationship between transgene expression and the erucic acid accumulation at sn-2. One of the necessary tools to study the variability is the presence of a specific antibody raised against the introduced acyltransferase. In the present study, we have looked at the correlation between erucic acid accumulation at sn-2, production of trierucin and the expression of the transgene, as detected by Western blotting. The data presented include comparisons of progeny of individual lines grown under various conditions and at various times, and an analysis of a number of lines during development. We also present calculations that could indicate from which position the erucic acid found at position sn-2 is derived.
Transgenic oil palm: production and projection
Authors: Parveez Ghulam Kadir Ahmad , Masri Masnita Mohd , Zainal Alizah , Majid Na'imatulapidah Abdul , Yunus Abdul Masani Mat , Fadilah Haliza Haris , Rasid Omar , Cheah Suan-Choo
Published: January 2005

Oil palm is an important economic crop for Malaysia. Genetic engineering could be applied to produce transgenic oil palms with high value-added fatty acids and novel products to ensure the sustainability of the palm oil industry. Establishment of a reliable transformation and regeneration system is essential for genetic engineering. Biolistic was initially chosen as the method for oil palm transformation as it has been the most successful method for monocotyledons to date. Optimization of physical and biological parameters, including testing of promoters and selective agents, was carried out as a prerequisite for stable transformation. This has resulted in the successful transfer of reporter genes into oil palm and the regeneration of transgenic oil palm, thus making it possible to improve the oil palm through genetic engineering. Besides application of the Biolistics method, studies on transformation mediated by Agrobacterium and utilization of the green fluorescent protein gene as a selectable marker gene have been initiated. Upon the development of a reliable transformation system, a number of useful targets are being projected for oil palm improvement. Among these targets are high-oleate and high-stearate oils, and the production of industrial feedstock such as biodegradable plastics. The efforts in oil palm genetic engineering are thus not targeted as commodity palm oil. Due to the long life cycle of the palm and the time taken to regenerate plants in tissue culture, it is envisaged that commercial planting of transgenic palms will not occur any earlier than the year 2020.
Ontogenesis of prolactin receptors in the human fetus: roles in fetal development
Authors: Freemark M.
Published: January 2005

The lactogenic hormones prolactin (PRL) and placental lactogen circulate in human fetal plasma during mid and late gestation. To explore potential roles for the lactogens in fetal development, we examined the cellular distribution and changes in expression of PRL receptors (PRLRs) during ontogeny, and the metabolic effects of PRL signalling and PRLR dysregulation. PRLRs are expressed in diverse tissues of the human fetus by 7.5 weeks of gestation. In fetal bone, adrenal gland and lung, the receptor is expressed first in mesenchymal cells and subsequently in maturing chondrocytes, adrenocortical cells and bronchiolar epithelial cells. That the lactogens play roles in fetal chondrogenesis is suggested by studies in PRLR-deficient mice, which show a delayed ossification of the calvarium. In the central nervous system, the PRLR is detected initially in periventricular neuroepithelium and later in mature neurons of the hypothalamus and olfactory bulb. Finally, in the pancreas, the PRLR is detected first in exocrine tissue and ductal epithelium. Later in gestation and in the postnatal period, PRLRs predominate in pancreatic β-cells. The lactogens regulate β-cell proliferation and insulin production in pancreatic islets, and the insulin secretory response to glucose is blunted in PRLR-deficient mice. These observations suggest roles for the lactogens in pancreatic development and function during pregnancy and postnatal life.
Prolactin receptor signal transduction pathways and actions determined in prolactin receptor knockout mice
Authors: Kelly P. A. , Binart N. , Freemark M. , Lucas B. , Goffin V. , Bouchard B.
Published: January 2005

Prolactin-receptor-deficient mice are a good model in which to study the various actions of prolactin. Female homozygous knockout mice are completely infertile and show a lack of mammary development, while hemizogotes are unable to lactate following their first pregnancy. Male and female homozygotes have markedly elevated serum prolactin levels, and in some instances pituitary hyperplasia is present. Maternal behaviour is severely affected in both hemizygous and homozygous animals. Bone formation is reduced in young animals and in adults (males and females). Finally, older males and females show a slight reduction in body weight, which seems to be due to reduced abdominal fat deposition in the knockout animals.
Leptin and its role in pregnancy and fetal development — an overview
Authors: Reitman M. L. , Bi S. , Marcus-Samuels B. , Gavrilova O.
Published: January 2005

Leptin is a hormone that is secreted by adipose cells in proportion to adipose mass, and therefore a low leptin level signifies depletion of energy stores. It has been proposed that leptin is one of the signals controlling sexual maturation. For example, humans and rodents lacking leptin fail to undergo complete puberty, while overexpression of leptin in mice causes early puberty. The placenta also produces leptin in human pregnancy, increasing the amount in the maternal circulation. The effects of the increased leptin levels during pregnancy are not clear. In contrast, the mouse placenta does not produce endocrinologically significant amounts of leptin. The mouse placenta does secrete a leptin-binding protein, the production of which correlates with a large increase in maternal leptin levels. The physiology of leptin during pregnancy and fetal development differs significantly between species, and is not well understood in any.
Fetal and neonatal adipose maturation: a primary site of cytokine and cytokine-receptor action
Authors: Stephenson T. , Budge H. , Mostyn A. , Pearce S. , Webb R. , Symonds M. E.
Published: January 2005

During late gestation, the maturation of fetal adipose tissue is geared towards the synthesis of high levels of uncoupling protein 1 (UCP1), which is unique to brown adipose tissue. At birth, rapid activation of UCP1 ensures a large increase in heat production. These adaptations are nutritionally sensitive, and may be mediated in part by rapid changes in prolactin and leptin secretion after birth. Restriction of maternal nutrition reduces adipose tissue deposition, with no effect on UCP1. Increased maternal food intake results in increases in levels of UCP1 and the short form of the prolactin receptor, but in a decrease in adipose tissue content per kg of fetus. The ontogeny of the long and short forms of the prolactin receptor follows that of UCP1, to peak at birth. Then, during postnatal life, UCP1 disappears in parallel with the loss of prolactin receptors. Treatment of neonatal lambs with prolactin increases body temperature and the thermogenic potential of brown adipose tissue. In contrast, acute leptin treatment results in maintenance of colonic temperature, but chronic leptin treatment accelerates UCP1 loss. Increasing our understanding of the interaction between prolactin and leptin during perinatal development may enable the establishment of strategies aimed at maximizing adipose tissue development in order to promote metabolic adaptation to the extra-uterine environment.
Plant peroxidases: substrate complexes with mechanistic implications
Authors: Gajhede M.
Published: January 2005

Plant peroxidases are capable of binding phenolic substrates, and it has been possible to crystallize complexes between horseradish peroxidase C (HRP C) and benzhydroxamic acid. The X-ray structures of the binary HRP C: ferulic acid complex and the ternary HRP C: CN− :ferulic acid complex to 2.0 and 1.45 Å resolution, respectively, have also been solved recently. Ferulic acid is a naturally occurring phenolic compound found in the plant cell wall and it is an in vivo substrate for plant peroxidases. The X-ray structures demonstrate the flexibility of the aromatic-donor-binding site in plant peroxidases and highlight the role of the distal arginine in substrate oxidation and ligand binding. A general mechanism of peroxidase substrate oxidation (compound I → compound II and compound II → resting state) can be proposed on the basis of the complexes and a large body of biochemical evidence.
Engineering the active site of ascorbate peroxidase
Authors: Raven E. Lloyd , Celik A. , Cullis P. M. , Sangar R. , Sutcliffe M. J.
Published: January 2005

Understanding the catalytic versatility of haem enzymes, and in particular the relationships that exist between different classes of haem-containing proteins and the mechanisms by which the apo-protein structure controls chemical reactivity, presents a major experimental and theoretical challenge. These issues are discussed in the general context of peroxidase and cytochrome P450 chemistry, and specific issues relating to the catalytic chemistry of ascorbate peroxidase are highlighted.
An old activity in the cytochrome P450 superfamily (CYP51) and a new story of drugs and resistance
Authors: Kelly S. L. , Lamb D. C. , Cannieux M. , Greetham D. , Jackson C. J. , Marczylo T. , Ugochukwu C. , Kelly D. E.
Published: January 2005

Cytochrome P450 51 (CYP51) is sterol 14α-demethylase, known also as Ergllp in yeast. First studied in yeast, where it is one of three CYPs in the genome, it has subsequently gained attention as the only CYP found so far in different kingdoms of life. As such it is central to considerations of CYP evolution. Recent use of CYP51-inhibiting antifungal drugs, such as fluconazole, has also been associated with dramatic CYP51 evolution to numerous resistant forms in fungal pathogens. CYP51 has also been discovered in mycobacteria where antifungal azoles have effect and might be of value against tuberculosis. Evolutionary and therapeutic aspects of CYP51 studies are discussed.
Predicting drug pharmacokinetics in humans from in vitro metabolism studies
Authors: McGinnity D. F. , Riley R. J.
Published: January 2005

The pharmaceutical industry is committed to market safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. There is an increasing need to develop robust, enhanced-throughput in vitro assays, which accurately extrapolate to humans. The major drug metabolizing human hepatic cytochrome P450s (CYPs; CYP1A2, 2C9, 2C19, 2D6 and 3A4) have been co-expressed functionally in Escherichia coli with human NADPH-cytochrome P450 reductase and validated as surrogates to their counterparts in human liver microsomes (HLM) with respect to their kinetic and inhibition properties. Using these recombinant enzymes, fully automated in vitro assays to assess CYP inhibition and determine the enzymology of drug oxidation have been developed and validated. IC50 values determined for a series of test compounds in HLM and recombinant CYPs were similar (r2 = 0.9, P < 0.001). There was a good correlation between the sum of individual CYP intrinsic clearance (Clint) and HLM CIint (r2 = 0.8, P< 0.001) for ten prototypic substrates for which clearance was CYP-dependent. Several in vitro incubation milieu (e.g. CYPs, HLM, human hepatocytes) are routinely used and the level of non-specific binding was investigated with respect to effects on Km and Ki determinations. There were clear correlations between binding and lipophilicity (logD7.4) for a selection of bases (r2 = 0.98, P < 0.001) and acids (r2 = 0.79, P < 0.001) that may allow prediction of this property. Our laboratory has shown that recombinant enzymes are suitable for ‘frontline’ predictive human metabolism studies in early drug discovery.
MUCI, glycans and the cell-surface barrier to embryo implantation
Authors: Aplin J. D. , Meseguer M. , Simon C. , Ortíz M. E. , Croxatto H. , Jones C. J. P.
Published: January 2005

As it approaches the maternal surface, the attaching embryo encounters the epithelial glycocalyx, which contains the mucin, MUC1. A high density of MUC1 at the cell surface can inhibit cell adhesion. This raises the possibility of the existence of a uterine barrier to implantation that might allow maternal rejection of poorer quality embryos. To investigate the mechanism of implantation, human embryos were incubated with endometrial epithelial monolayers. Hatched blastocysts were found to attach readily to the epithelial surface. MUC1 was lost from epithelial cells beneath and near to the attached embryo, while normal expression persisted in neighbouring cells.
Glycosylation changes during differentiation of the murine uterine epithelium
Authors: Kimber S. J. , Stones R. E. , Sidhu S. S.
Published: January 2005

In mouse uterine luminal epithelium (LE) several terminal carbohydrate structures are regulated by ovarian steroids and show stage-specific expression during early pregnancy. We have demonstrated that expression of H-type-1 antigen (Fucα1-2Galβ1-3GlcNAcβ1-) is regulated by oestrogenic stimulation of α-2fucosyltransferase (fut1) mRNA levels in LE. H-type-1 expression is high after ovulation but becomes negligible after implantation. In contrast, NeuNAcα2-3Galβ1-, and specifically sialyl Le-x (NeuAcα2,3Galβ1,4-[Fucα1-3]GlcNAcβ1-), is stimulated by progesterone. It is not expressed on LE after ovulation but is expressed maximally on apical LE at the time of and after implantation. However, mRNA levels for 4 out of 5 known Gal/GalNAc α2-3sialyltransferases appear not to change in LE during early pregnancy, suggesting an alternative level of control.
Glycosaminoglycans in the study of mammalian organ development
Authors: Davies J. A. , Fisher C. E. , Barnett M. W.
Published: January 2005

Glycosaminoglycans (GAGs) are linear polymers of amino sugar uronic acid disaccharides, and are generally attached to protein cores to form proteoglycans. GAGs interact with a large number of proteins and can participate in matrix organization, cell adhesion, differentiation, growth and apoptosis. Proteoglycans are expressed in tightly regulated spatio-temporal patterns during organ development, and changes in expression frequently correlate with developmental events. Here we review the evidence that GAGs play important roles in the development of mouse kidneys, which are organs that will undergo organotypic development in simple culture conditions and that are therefore highly accessible to experimentation. Depleting kidneys of GAGs, either biochemically or genetically, blocks the development of the urinary collecting-duct system, probably because critical signalling molecules require GAGs to form stable associations with their receptors. The in-sensitivity of GAG-deprived organ rudiments to physiological concentrations of growth factors can be used to screen candidate signalling molecules for morphoregulatory activity; candidate growth factors are applied at supraphysiological levels to GAG-deprived kidneys and assessed for their ability to rescue normal development. This approach has assisted the identification of four collecting-duct morphogens: hepatocyte growth factor, glial cell line-derived neurotrophic factor, nerturin and persephin.
DNA polymerases II and V mediate respectively mutagenic (-2 frameshift) and error-free bypass of a single N-2-acetylaminofluorene adduct
Authors: Fuchs R. P. P. , Koffel-Schwartz N. , Pelet S. , Janel-Bintz R. , Napolitano R. , Becherel O. J. , Broschard T. H. , Burnouf D. Y. , Wagner J.
Published: January 2005

The Nar I sequence represents a strong mutation hot spot for - 2 frameshift mutations induced by N-2-acetylaminofluorene (AAF), a strong chemical carcinogen. Only when bound to the third (underlined) guanine (5′-GGCGCC → GGCC) can AAF trigger frameshift mutations, suggesting the involvement of a slipped replication intermediate with a two-nucleotide bulge. While base substitutions induced by UV light or abasic sites require DNA polymerase V (Pol V; umuDC), the AAF-induced - 2 frameshift pathway requires DNA polymerase II, the polB gene product. Interestingly, error-free bypass of the G-AAF adduct requires Pol V. The genes encoding both Pol II and Pol V are induced by the SOS regulon, a co-ordinated cellular response to environmental stress. A given lesion, G-AAF, can thus be bypassed by two SOS-controlled DNA polymerases (II and V), generating mutagenic (-2 frameshifts) and error-free replication products respectively. Therefore both Pol II and Pol V can compete for the blocked replication intermediate in the vicinity of the lesion and engage in replication by transiently replacing the replicative DNA Pol III. Our data suggest that, in order to cope with the large diversity of existing DNA lesions, cells use a single or a combination of translesional DNA polymerases to achieve translesion synthesis.
Double-strand-break-induced homologous recombination in mammalian cells
Authors: Johnson R. D. , Jasin M.
Published: January 2005

In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.
Role of the Bloom's syndrome helicase in maintenance of genome stability
Authors: Hickson I. D. , Davies S. L. , Li J.-L. , Levitt N. C. , Mohaghegh P. , North P. S. , Wu L.
Published: January 2005

The RecQ family of DNA helicases has members in all organisms analysed. In humans, defects in three family members are associated with disease conditions: BLM is defective in Bloom's syndrome, WRN in Werner's syndrome and RTS in Rothmund-Thomson syndrome. In each case, cells from affected individuals show inherent genomic instability. The focus of our work is the Bloom's syndrome gene and its product, BLM. Here, we review the latest information concerning the roles of BLM in the maintenance of genome integrity.
Do dietary phytoestrogens influence susceptibility to hormone-dependent cancer by disrupting the metabolism of endogenous oestrogens?
Authors: Kirk C. J. , Harris R. M. , Wood D. M. , Waring R. H. , Hughes P. J.
Published: January 2005

Phytoestrogens are natural constituents of our diets that have been suggested to protect against hormone-dependent breast cancer. Some of the diverse effects of these compounds may be attributed to ligand-dependent differences in their interaction with oestrogen receptor sub-classes. However, phytoestrogens can also inhibit enzymes that are involved in the generation and removal of endogenous steroid hormones. Among the most potent effects of dietary phytoestrogens is their ability to inhibit the sulphotransferases that sulphate both oestrogenic steroids and a variety of environmental chemicals, including dietary pro-carcinogens. Circulating steroid sulphates are thought to be the major source of oestradiol in post-menopausal breast tumours and sulphation is a key step in the activation of some dietary procarcinogens. Hence the inhibition of sulpho-transferases by dietary phytoestrogens may have complex effects upon human susceptibility to breast cancer.
Actions of the soy phytoestrogen genistein in models of human chronic disease: potential involvement of transforming growth factor β
Authors: Kim H. , Xu J. , Su Y. , Xia H. , Li L. , Peterson P. , Murphy-Ullrich J. , Barnes S.
Published: January 2005

The structural similarity, but non-identity, between 17β-oestradiol and the soy phytoestrogen genistein suggests that the two compounds will have actions that may be identical in some target biological systems, but different in others. Epidermal growth factor (EGF)-stimulated proliferation of human mammary epithelial cells (that do not express the oestrogen receptor) was significantly suppressed at genistein concentrations (5–10μM) that are attainable physiologically. Others have shown previously that transforming growth factor β (TGFβ) has similar growth-inhibitory effects on human cells. Analysis of the conditioned medium of human mammary epithelial cells exposed to genistein plus EGF showed increased levels of TGFβ relative to those in the medium of cells exposed to EGF or genistein alone. Related experiments in a primate model of menopause demonstrated that ingestion of soy containing isoflavones was correlated with the suppression of neurodegeneration-relevant phosphorylation of the microtubule-associated protein tau, while intake of Premarin (a hormone replacement therapy that is commonly prescribed for women) was not correlated. The results discussed here indicate that genistein, and probably other related phytoestrogens, have pleiotropic actions, some of which may involve TGFβ activity.
Resistance to thyroid hormone, and peroxisome-proliferator-activated receptor γ resistance
Authors: Chatterjee V. K. K.
Published: January 2005

Resistance to thyroid hormone (RTH) is usually inherited in a dominant fashion, and is characterized by elevated serum thyroid hormone levels and failure to suppress pituitary secretion of thyroid-stimulating hormone, with variable refractoriness to hormone action in peripheral tissues. Two major forms of the disorder are recognized: asymptomatic individuals with generalized resistance (GRTH) and patients with thyrotoxic features suggesting predominant pituitary resistance (PRTH). In over 100 families with GRTH or PRTH, we have identified heterozygous mutations in the thyroid hormone receptor β isoform (TRβ), which localize to three regions (amino acids 234–282, 310–353 and 429–461) of the hormone-binding domain of the receptor. The mutant receptors are transcriptionally impaired, due either to reduced ligand binding or to attenuated interaction with co-activators, and inhibit wild-type TR action in a dominant-negative manner. In the TRβ crystal structure, most RTH mutations cluster around the hormone-binding pocket, with receptor regions that mediate functions (DNA binding, dimerization, corepressor recruitment) required for dominant-negative activity being devoid of natural mutations. The pathogenesis of variable tissue resistance is not fully understood, but may be related to the differing tissue distributions of TRα and TRβ, and to variable dominant-negative activity of mutant receptors on different target genes. The nuclear receptor peroxisome-proliferator-activated receptor γ (PPARγ) regulates adipogenesis and mediates the action of thiazolidinediones - novel antidiabetic agents which enhance tissue insulin sensitivity. The PPARγ gene was screened in 85 subjects with severe insulin resistance, and two different heterozygous receptor mutations (P467L and V290M) were identified in three affected individuals. The PPARγ mutants are markedly transcriptionally impaired due to altered ligand binding and co-activator recruitment. Analogous to RTH, they inhibit the function of wild-type PPARγ when co-expressed, and such dominant-negative inhibition is linked to their ability to silence basal gene transcription via aberrant interaction with co-repressors. In addition to insulin resistance, all three affected subjects developed Type II diabetes mellitus and hypertension at an unusually early age. Our findings provide compelling evidence that PPARγ is important in the control of insulin sensitivity, glucose homoeostasis and blood pressure in humans. Future studies aim to elucidate the mechanism by which this receptor regulates insulin action and vascular tone.
Peroxisomal fatty acid α- and β-oxidation in humans: enzymology, peroxisomal metabolite transporters and peroxisomal diseases
Authors: Wanders R. J. A. , Vreken P. , Ferdinandusse S. , Jansen G. A. , Waterham H. R. , van Roermund C. W. T. , Van Grunsven E. G.
Published: January 2005

Peroxisomes are subcellular organelles with an indispensable role in cellular metabolism. The importance of peroxisomes for humans is stressed by the existence of a group of genetic diseases in humans in which there is an impairment in one or more peroxisomal functions. Most of these functions have to do with lipid metabolism including the α and β-oxidation of fatty acids. Here we describe the current state of knowledge about peroxisomal fatty acid α- and β-oxidation with particular emphasis on the following: (1) the substrates β-oxidized in peroxisomes; (2) the enzymology of the α- and β-oxidation systems; (3) the permeability properties of the peroxisomal membrane and the role of the different transporters therein; (4) the interaction with other subcellular compartments, including the mitochondria, which are the ultimate site of NADH reoxidation and full degradation of acetyl-CoA to CO2 and water; and (5) the different disorders of peroxisomal α- and β-oxidation.
Microsomal malonyl-CoA-sensitive carnitine acyltransferase
Authors: Broadway N. M. , Pease R.J. , Birdsey G. , Turner N. A. , Shayeghi M. , Saggerson E. D.
Published: January 2005

Liver microsomes contain two carnitine acyltransferase activities. One of these has properties closely corresponding to those of 88 kDa mitochondrial carnitine palmitoyltransferase-1 (CPT-1). Antisera against CPT-1 cross-react with an 88 kDa microsomal protein, suggesting that CPT-1 may be targeted to both microsomal and mitochondrial membranes. However, no experiments using cDNAs corresponding to CPT-1 involving in vitro translation with microsomes or involving in vivo COS-1 cell transfection provided any evidence to support this hypothesis.
Fuel-sensing mechanisms integrating lipid and carbohydrate utilization
Authors: Sugden M. C. , Bulmer K. , Holness M.J.
Published: January 2005

Fuel metabolism is highly regulated to ensure adequate energy for cellular function. The contribution of the major metabolic fuels - glucose, lactate and fatty acids (FAs) - often reflects their circulating levels. In addition, regulatory crosstalk and fuel-induced hormone secretion ensures appropriate and co-ordinate fuel utilization. Because its activity can either determine or reflect fuel preference (carbohydrate versus fat), the pyruvate dehydrogenase complex (PDC) occupies a pivotal position in fuel cross-talk. Active PDC permits glucose oxidation and allows the formation of mitochondrially derived intermediates (e.g. malonyl-CoA and citrate) that reflect fuel abundance. FA oxidation suppresses PDC activity. PDC inactivation by phosphorylation is catalysed by pyruvate dehydrogenase kinases (PDKs) 1–4, which are regulated differentially by metabolite effectors. Most tissues contain at least two and often three of the PDK isoforms. We develop the hypothesis that PDK4 is a ‘lipid status’-responsive PDK isoform facilitating FA oxidation and signalling through citrate formation. Substrate interactions at the level of gene transcription extend glucose-FA interactions to the longer term. We discuss potential targets for substrate-mediated transcriptional regulation in relation to selective PDK isoform expression and the influence of altered PDK isoform expression in fuel sensing, selection and utilization.
Impact of the intramitochondrial enzyme organization on fatty acid oxidation
Authors: Liang X. , Le W. , Zhang D. , Schulz H.
Published: January 2005

The enzymes of mitochondrial β-oxidation are thought to be organized in at least two functional complexes, a membrane-bound, long-chain-specific β-oxidation system and a matrix system consisting of soluble enzymes with preferences for medium-chain and short-chain substrates. This hypothesis is supported by the observation that the inactivation of long-chain 3-ketoacyl-CoA thiolase by 4-bromotiglic acid (4-bromo-2-methylbut-2-enoic acid) causes the complete inhibition of palmitate β-oxidation even though 3-ketoacyl-CoA thiolase, which acts on 3-ketopalmitoyl-CoA, remains partly active. The observed substrate specificities of long-chain acyl-CoA dehydrogenase (LCAD) and very-long-chain acyl-CoA dehydrogenase prompt the suggestion that LCAD is a functional component of the long-chain-specific β-oxidation system. Altogether, a view is emerging of the organization of β-oxidation enzymes in mitochondria that supports the idea of intermediate channelling and explains the apparent absence of true intermediates of β-oxidation from mitochondria.
Structure-function relationships of the liver and muscle isoforms of carnitine palmitoyltransferase I
Authors: Zammit V. A. , Price N. T. , Fraser F. , Jackson V. N.
Published: January 2005

Elucidation of the membrane topology of carnitine palmitoyltransferase (CPT) I showed that the extreme N-terminus is involved in determining the sensitivity of the liver (L) isoform to malonyl-CoA and suggested that interaction between the two cytosolic segments of the CPT I molecule determines the kinetic characteristics of the enzyme. Work with chimaeric liver/muscle-isoform (L/M) proteins constructed from all six possible combinations of three domains [N-terminus plus transmembrane domain 1 (TM1), loop plus TM2 and C-domain] expressed in Pichia pastoris showed that the precise N-C and TM1-TM2 pairings determine the overall kinetic parameters of the protein. Discrete short sequences within the respective N-terminal regions have negative or positive effects on malonyl-CoA sensitivity (L-isoform) or the Km for carnitine (M-isoform) in the full-length proteins, thus imparting to them their distinctive kinetic characteristics. Interactions within N-terminal domains also seem to be important in the targeting of the protein to microsomes in the P. pastoris expression system.
Further insights into peroxisomal lipid breakdown via α- and β-oxidation
Authors: Van Veldhoven P. P. , Casteels M. , Mannaerts G. P. , Baes M.
Published: January 2005

Mammalian peroxisomes degrade fatty carboxylates via two pathways, β-oxidation and, as shown more recently, α-oxidation. The latter process consists of an activation step, followed by a hydroxylation at position 2 and cleavage of the 2-hydroxyacyl-CoA, generating formyl-CoA (precursor of formate/CO2) and, in case of phytanic acid as substrate, pristanal (precursor of pristanic acid). The stereochemistry of the overall pathway, cofactor requirements and substrate specificity of the hydroxylase and the cleavage enzyme, which is homologous with bacterial oxalyl-CoA decarboxylases, will be discussed. With regard to β-oxidation, peroxisomes contain different acyl-CoA oxidases, multifunctional proteins and thiolases. Based on substrate spectra and stereospecificities of these enzymes, a model was proposed whereby straight chain and branched compounds are degraded by separate pathways. The biochemical findings in mice lacking the D-specific multifunctional protein, however, do not fully support this model. These animals, together with the Pex5−/− mice, might be useful to pinpoint the pathological factors contributing to the brain abnormalities in Zellweger patients. Apparently, the deficit in docosahexaenoic acid, presumably formed via peroxisomal β-oxidation, is not the major cause.
Clinical consequences of defects in peroxisomal β-oxidation
Authors: Clayton P. T.
Published: January 2005

The disorders of peroxisomal β-oxidation, which have been well characterised at the molecular level, include defects of acyl-CoA oxidase, defects of the D-bifunctional protein (D-BP) (including specific defects of its enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase components), defects of the very-long-chain fatty acid (VLCFA)-CoA importer [X-linked adrenoleukodystrophy (ALD)] and α-methylacyl-CoA racemase deficiency. A survey of the clinical consequences of these defects indicates that defects in the acyl-CoA oxidase and D-BP can produce neonatal hypotonia, seizures in early infancy, retinopathy and progressive neurological dysfunction with leukodystrophy on imaging. Defects in the VLCFA-CoA importer and in the racemase do not produce disease until a long time after the neonatal period. However, again the clinical picture is dominated by neurological disease: impaired cognitive function with leukodystrophy in childhood X-linked ALD and retinopathy and neuropathy in racemase deficiency. It is difficult to escape the conclusion that defective peroxisomal β-oxidation has effects (such as impaired neuronal migration in the developing brain), which are more serious than those produced by the accumulation of substrates (VLCFAs, pristanic acid) alone.
Peroxisome-proliferator-activated receptors as physiological sensors of fatty acid metabolism: molecular regulation in peroxisomes
Authors: Latruffe N. , Malki M. Cherkaoui , Nicolas-Frances V. , Jannin B. , Clemencet M.-C. , Hansmannel F. , Passilly-Degrace P. , Berlot J.-P.
Published: January 2005

The enzymes required for the β-oxidation of fatty acyl-CoA are present in peroxisomes and mitochondria. Administration of hypolipidaemic compounds such as clofibrate to rodents leads to an increase in the volume and density of peroxisomes in liver cells. These proliferators also induce simultaneously the expression of genes encoding acyl-CoA oxidase, enoyl-CoA hydratase-hydroxy-acyl-CoA dehydrogenase (multifunctional enzyme) and thiolase (3-ketoacyl-CoA thiolase). All these enzymes are responsible for long-chain and very-long-chain fatty acid β-oxidation in peroxisomes. Similar results were observed when rat hepatocytes, or liver-derived cell lines, were cultured with a peroxisome proliferator. The increased expression of these genes is due to the stimulation of their transcription rate. These results show that the peroxisome proliferators act on the hepatic cells and regulate the transcription through various cellular components and pathways, including peroxisome-proliferatoractivated receptor α (PPARα). After activation by specific ligands, either fibrates or fatty acid derivatives, PPARα binds to a DNA response element: peroxisome-proliferator-responsive element (PPRE), which is a direct repeat of the following consensus sequence: TGACCTXTGACCT, found in the promoter region of the target genes. PPARα is expressed mainly in liver, intestine and kidney. PPARα is a transcriptional factor, which requires other nuclear proteins for function including retinoic acid X receptor (RXRα) and other regulatory proteins. From our results and others we suggest the role of PPARα in the regulation of the peroxisomal fatty acid β-oxidation. In this regard, we showed that although PPARα binds to thiolase B gene promoter at - 681 to - 669, a better response is observed with hepatic nuclear factor 4 (‘HNf-4’). Moreover, rat liver PPARα regulatory activity is dependent on its phosphorylated state. In contrast, a proteinkinase-C-mediated signal transduction pathway seems to be modified by peroxisome proliferators, leading to an increase in the phosphorylation level of specific proteins, some of which have been shown to be involved in the phosphoinositide metabolism.
Post-transcriptional regulation of rat carnitine octanoyltransferase
Authors: Hegardt F. G. , Bach M. , Asins G. , Caudevilla C. , Morillas M. , Codony C. , Serra D.
Published: January 2005

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which can lead to the synthesis of two proteins. The occurrence of the three COT transcripts in rat has been found in all tissues examined and does not depend on sex, fat feeding, peroxisome proliferators or hyperinsulinaemia. Rat COT exon 2 contains a putative exonic splicing enhancer (ESE) sequence. Mutation of this ESE (GAAGAAG) to AAAAAAA decreased trans-splicing in vitro, from which it is deduced that this ESE sequence is partly responsible for the formation of the three transcripts. The protein encoded by cis-spliced mRNA of rat COT is inhibited by malonyl-CoA and etomoxir. cDNA species encoding full-length wild-type COT and one double mutant COT were expressed in Saccharomyces cerevisiae. The recombinant enzymes showed full activity towards both substrates, carnitine and decanoyl-CoA. The activity of the doubly mutated H131A/H340A enzyme was similar to that of the rat peroxisomal enzyme but was completely insensitive to malonyl-CoA and etomoxir. These results indicate that the histidine residues His-131 and His-340 are the sites responsible for the interaction of these two inhibitors, which inhibit COT by interacting with the same sites.
Enzymes: Nature's Nanomachines
Authors: Mantle T.J.
Published: January 2005

The development of enzyme kinetics, protein crystallography and NMR studies allows enzymecatalysed reactions to be described in terms of mechanistic chemistry, albeit applied to relatively enormous molecules. These nanomachines, which so inspired Drexler's Engines of Creation, have been working in biological systems for over three billion years and represent a useful knowledge base for our further understanding of mechanistic biology. They also provide a tantalizing glimpse into what may be the basis for novel technologies with industrial applications for the twenty-first century.
Lipoprotein oxidation and atherosclerosis
Authors: Young I. S. , McEneny J.
Published: January 2005

Lipoprotein oxidation is a key early stage in the development of atherosclerosis. Oxidation of lowdensity lipoprotein (LDL) is initiated by both enzyme-mediated and non-enzymic mechanisms in vivo, and oxidized LDL has many atherogenic properties. Oxidation of LDL in vivo is likely to be influenced by local environmental factors, such as pH. The composition of LDL is also important, including such factors as antioxidant content, fatty acid composition and particle size.