The Light Dependent Reactions of Photosynthesis

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Glycation of low-density lipoprotein results in the time-dependent accumulation of cholesteryl esters and apolipoprotein B-100 protein in primary human monocyte-derived macrophages Bronwyn E. Brown 1 , Imran Rashid 1 , David M. van Reyk 2 and Michael J. Davies 1,3 1 Free Radical Group, The Heart Research Institute, Camperdown, Sydney, NSW, Australia 2 Department of Health Sciences, University of Technology Sydney, NSW, Australia 3 Faculty of Medicine, University of Sydney, NSW, Australia Complications associated with diabetes are the major cause of mortality and morbidity in people with this disease. These include microvascular complications that induce damage to the retina, nephrons and peripheral nerves, and macrovascular disease that is associated with accelerated atherosclerosis (deposition of lipids in Keywords aldehydes; atherosclerosis; foam cells; human monocyte-derived macrophages; low-density lipoproteins Correspondence M. J. Davies, 114 Pyrmont Bridge Road, Camperdown, Sydney, NSW 2050, Australia Fax: +61 2 95655584 Tel: +61 2 82088900 E-mail: daviesm@hri.org.au (Received 12 December 2006, accepted 15 January 2007) doi:10.1111/j.1742-4658.2007.05699.x Nonenzymatic covalent binding (glycation) of reactive aldehydes (from glu- cose or metabolic processes) to low-density lipoproteins has been previ- ously shown to result in lipid accumulation in a murine macrophage cell line. The formation of such lipid-laden cells is a hallmark of atheroscler- osis. In this study, we characterize lipid accumulation in primary human monocyte-derived macrophages, which are cells of immediate relevance to human atherosclerosis, on exposure to low-density lipoprotein glycated using methylglyoxal or glycolaldehyde. The time course of cellular uptake of low-density lipoprotein-derived lipids and protein has been character- ized, together with the subsequent turnover of the modified apolipoprotein B-100 (apoB) protein. Cholesterol and cholesteryl ester accumulation occurs within 24 h of exposure to glycated low-density lipoprotein, and increases in a time-dependent manner. Higher cellular cholesteryl ester lev- els were detected with glycolaldehyde-modified low-density lipoprotein than with methylglyoxal-modified low-density lipoprotein. Uptake was signifi- cantly decreased by fucoidin (an inhibitor of scavenger receptor SR-A) and a mAb to CD36. Human monocyte-derived macrophages endocytosed and degraded significantly more 125 I-labeled apoB from glycolaldehyde-modified than from methylglyoxal-modified, or control, low-density lipoprotein. Dif- ferences in the endocytic and degradation rates resulted in net intracellular accumulation of modified apoB from glycolaldehyde-modified low-density lipoprotein. Accumulation of lipid therefore parallels increased endocytosis and, to a lesser extent, degradation of apoB in human macrophages exposed to glycolaldehyde-modified low-density lipoprotein. This accumula- tion of cholesteryl esters and modified protein from glycated low-density lipoprotein may contribute to cellular dysfunction and the increased atherosclerosis observed in people with diabetes, and other pathologies linked to exposure to reactive carbonyls. Abbreviations AGE, advanced glycation end-products; apoB, apolipoprotein B-100; HBSS, Hank’s balanced salt solution; HMDM, human monocyte-derived macrophage; HSA, human serum The Light-Dependent Reactions of Photosynthesis The Light-Dependent Reactions of Photosynthesis Bởi: OpenStaxCollege How can light be used to make food? It is easy to think of light as something that exists and allows living organisms, such as humans, to see, but light is a form of energy Like all energy, light can travel, change form, and be harnessed to work In the case of photosynthesis, light energy is transformed into chemical energy, which autotrophs use to build carbohydrate molecules However, autotrophs only use a specific component of sunlight ([link]) Autotrophs can capture light energy from the sun, converting it into chemical energy used to build food molecules (credit: modification of work by Gerry Atwell, U.S Fish and Wildlife Service) Concept in Action 1/8 The Light-Dependent Reactions of Photosynthesis Visit this site and click through the animation to view the process of photosynthesis within a leaf What Is Light Energy? The sun emits an enormous amount of electromagnetic radiation (solar energy) Humans can see only a fraction of this energy, which is referred to as “visible light.” The manner in which solar energy travels can be described and measured as waves Scientists can determine the amount of energy of a wave by measuring its wavelength, the distance between two consecutive, similar points in a series of waves, such as from crest to crest or trough to trough ([link]) The wavelength of a single wave is the distance between two consecutive points along the wave Visible light constitutes only one of many types of electromagnetic radiation emitted from the sun The electromagnetic spectrum is the range of all possible wavelengths of radiation ([link]) Each wavelength corresponds to a different amount of energy carried 2/8 The Light-Dependent Reactions of Photosynthesis The sun emits energy in the form of electromagnetic radiation This radiation exists in different wavelengths, each of which has its own characteristic energy Visible light is one type of energy emitted from the sun Each type of electromagnetic radiation has a characteristic range of wavelengths The longer the wavelength (or the more stretched out it appears), the less energy is carried Short, tight waves carry the most energy This may seem illogical, but think of it in terms of a piece of moving rope It takes little effort by a person to move a rope in long, wide waves To make a rope move in short, tight waves, a person would need to apply significantly more energy The sun emits ([link]) a broad range of electromagnetic radiation, including X-rays and ultraviolet (UV) rays The higher-energy waves are dangerous to living things; for example, X-rays and UV rays can be harmful to humans Absorption of Light Light energy enters the process of photosynthesis when pigments absorb the light In plants, pigment molecules absorb only visible light for photosynthesis The visible light seen by humans as white light actually exists in a rainbow of colors Certain objects, such as a prism or a drop of water, disperse white light to reveal these colors to the human eye The visible light portion of the electromagnetic spectrum is perceived by the human eye as a rainbow of colors, with violet and blue having shorter wavelengths and, therefore, higher energy At the other end of the spectrum toward red, the wavelengths are longer and have lower energy Understanding Pigments Different kinds of pigments exist, and each absorbs only certain wavelengths (colors) of visible light Pigments reflect the color of the wavelengths that they cannot absorb 3/8 The Light-Dependent Reactions of Photosynthesis All photosynthetic organisms contain a pigment called chlorophyll a, which humans see as the common green color associated with plants Chlorophyll a absorbs wavelengths from either end of the visible spectrum (blue and red), but not from green Because green is reflected, chlorophyll appears green Other pigment types include chlorophyll b (which absorbs blue and red-orange light) and the carotenoids Each type of pigment can be identified by the specific pattern of wavelengths it absorbs from visible light, which is its absorption spectrum Many photosynthetic organisms have a mixture of pigments; between them, the organism can absorb energy from a wider range of visible-light wavelengths Not all photosynthetic organisms have full access to sunlight Some organisms grow underwater where light intensity decreases with depth, and certain wavelengths are absorbed by the water Other organisms grow in competition for light Plants on the rainforest floor must be able to absorb any bit of light that comes through, because the taller trees block most of the sunlight ([link]) Plants that commonly grow in the shade benefit from having a variety of light-absorbing pigments Each pigment can absorb different wavelengths of light, which allows the plant to absorb any light that passes through the taller trees (credit: Jason Hollinger) How Light-Dependent Reactions Work ...Enzymes for the NADPH-dependent reduction of dihydroxyacetone and D-glyceraldehyde and L-glyceraldehyde in the mould Hypocrea jecorina Janis Liepins 1,2 , Satu Kuorelahti 1 , Merja Penttila ¨ 1 and Peter Richard 1 1 VTT Biotechnology, Espoo, Finland 2 University of Latvia, Institute of Microbiology and Biotechnology, Riga, Latvia Dihydroxyacetone (DHA), d-glyceraldehyde and l-glyceraldehyde can be reduced using NADPH as a cofactor to form glycerol and NADP. Enzymes cataly- sing this reaction are generally called NADP:glycerol dehydrogenases. NADP:glycerol dehydrogenase activ- ity is common in moulds and filamentous fungi. Enzymes from different species of filamentous fungi have been purified and characterized. The enzymes purified from Aspergillus niger [1] and Aspergillus nidu- lans [2] catalyse the reversible reaction from glycerol and NADP to DHA and NADPH. For the A. niger enzyme, an equilibrium constant of 3.1–4.6 · 10 )12 m was estimated for the reaction: Glycerol þ NADP Ð DHA þ NADPH þ H þ A glycerol dehydrogenase with slightly different prop- erties was described in Neurospora crassa, where d-glyceraldehyde was the preferred substrate over DHA in the reductive reaction. This enzyme was also reversible, i.e. it showed activity with glycerol and NADP [3]. The purified glycerol dehydrogenases from A. nidulans and A. niger also showed low activity with d-glyceraldehyde; however, DHA was the preferred substrate [2]. The A. niger enzyme was commercially available as a partly purified preparation, and partial amino acid sequence s were available [4]. Keywords dihydroxyacetone; glycerol dehydrogenase; Hypocrea jecorina; L-glyceraldehyde; NADP- specific glycerol dehydrogenase Correspondence P. Richard, VTT, Tietotie 2, Espoo, PO Box 1000, 02044 VTT, Finland Fax: +358 20 722 7071 Tel: +358 20 722 7190 E-mail: Peter.Richard@vtt.fi (Received 4 May 2006, revised 7 July 2006, accepted 17 July 2006) doi:10.1111/j.1742-4658.2006.05423.x The mould Hypocrea jecorina (Trichoderma reesei) has two genes coding for enzymes with high similarity to the NADP-dependent glycerol dehy- drogenase. These genes, called gld1 and gld2, were cloned and expressed in a heterologous host. The encoded proteins were purified and their kinetic properties characterized. GLD1 catalyses the conversion of d-glyceralde- hyde and l-glyceraldehyde to glycerol, whereas GLD2 catalyses the con- version of dihydroxyacetone to glycerol. Both enzymes are specific for NADPH as a cofactor. The properties of GLD2 are similar to those of the previously described NADP-dependent glycerol-2-dehydrogenases (EC 1.1.1.156) purified from different mould species. It is a reversible enzyme active with dihydroxyacetone or glycerol as substrates. GLD1 resembles EC 1.1.1.72. It is also specific for NADPH as a cofactor but has otherwise completely different properties. GLD1 reduces d-glyceraldehyde and l-glyceraldehyde with similar affinities for the two substrates and sim- ilar maximal rates. The activity in the oxidizing reaction with glycerol as substrate was under our detection limit. Although the role of GLD2 is to facilitate glycerol formation under osmotic stress conditions, we hypothes- ize that GLD1 is active in pathways for sugar acid catabolism such as d-galacturonate catabolism. Abbreviations DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate. FEBS Journal 273 (2006) 4229–4235 ª 2006 The Authors Journal compilation ª 2006 FEBS 4229 Glycerol dehydrogenases have different functions in Light-induced reactions of Escherichia coli DNA photolyase monitored by Fourier transform infrared spectroscopy Erik Schleicher 1, *, Benedikt Heßling 2 , Viktoria Illarionova 1 , Adelbert Bacher 1 , Stefan Weber 3 , Gerald Richter 1, † and Klaus Gerwert 2 1 Lehrstuhl fu ¨ r Organische Chemie und Biochemie, Technische Universita ¨ tMu ¨ nchen, Germany 2 Lehrstuhl fu ¨ r Biophysik, Ruhr-Universita ¨ t-Bochum, Germany 3 Freie Universita ¨ t Berlin, Fachbereich Physik, Berlin, Germany Cyclobutane pyrimidine dimers (Pyr<>Pyr) and pyri- midine–pyrimidone (6–4) photoproducts are the predo- minant structural modifications resulting from exposure of DNA to ultraviolet light [1,2]. The structure of Pyr<>Pyr was elucidated by Blackburn and Davies already 40 years ago [3,4]. Both photoproducts result from 2p+2p cyclo-additions. The potentially mutagenic or lethal modifications [5] must be repaired in order to ensure cell survival and genetic stability. This can be effected by excision-repair or by photoreactivation Keywords DNA photolyase; DNA repair; FT-IR; pyrimidine dimer; stable-isotope labelling Correspondence G. Richter, School of Biological and Chemical Sciences, University of Exeter, Stocker Rd, Exeter, EX4 4QD, UK Fax: +44 1392 26 3434 Tel: +44 1392 26 3494 E-mail: g.richter@exeter.ac.uk K. Gerwert, Lehrstuhl fu ¨ r Biophysik, Ruhr-Universita ¨ t-Bochum, Universita ¨ tsstr. 150, 44780 Bochum, Germany Fax: +49 2343 21 4238 Tel: +49 2343 22 4461 E-mail: gerwert@bph.ruhr-uni-bochum *Present address Freie Universita ¨ t Berlin, Fachbereich Physik, Arnimallee 14, 14195 Berlin, Germany †Present address School of Biological and Chemical Sciences, University of Exeter, UK (Received 9 December 2004, revised 10 February 2005, accepted 16 February 2005) doi:10.1111/j.1742-4658.2005.04617.x Cyclobutane-type pyrimidine dimers generated by ultraviolet irradiation of DNA can be cleaved by DNA photolyase. The enzyme-catalysed reaction is believed to be initiated by the light-induced transfer of an electron from the anionic FADH ) chromophore of the enzyme to the pyrimidine dimer. In this contribution, first infrared experiments using a novel E109A mutant of Escherichia coli DNA photolyase, which is catalytically active but unable to bind the second cofactor methenyltetrahydrofolate, are described. A stable blue-coloured form of the enzyme carrying a neutral FADH radical cofactor can be interpreted as an intermediate analogue of the light-driven DNA repair reaction and can be reduced to the enzymatically active FADH ) form by red-light irradiation. Difference Fourier transform infra- red (FT-IR) spectroscopy was used to monitor vibronic bands of the blue radical form and of the fully reduced FADH ) form of the enzyme. Preliminary band assignments are based on experiments with 15 N-labelled enzyme and on experiments with D 2 O as solvent. Difference FT-IR mea- surements were also used to observe the formation of thymidine dimers by ultraviolet irradiation and their repair by light-driven photolyase catalysis. This study provides the basis for future time-resolved FT-IR studies which are aimed at an elucidation of a detailed molecular picture of the light- driven DNA repair process. Abbreviations DTT, dithiothreitol; FT-IR, Fourier transform infrared; MTHF, 5,10-methenyltetrahydrofolylpolyglutamate; Pyr<>Pyr, cyclobutane pyrimidine dimmer. FEBS Journal 272 (2005) 1855–1866 ª 2005 FEBS 1855 mediated by DNA photolyases. Specifically, photolyases catalyse the light-driven cleavage of the cyclobutane ring of tricyclic pyrimidine dimers, and (6–4) photolyases cleave the pyrimidine (6–4) pyrimidone Analysis of the CK2-dependent phosphorylation of serine 13 in Cdc37 using a phospho-specific antibody and phospho-affinity gel electrophoresis Yoshihiko Miyata and Eisuke Nishida Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Japan Protein kinases play pivotal roles in cellular signal transduction systems. Reversible protein phosphoryla- tion is one of the major mechanisms used to control the function, localization and stability of proteins inside cells [1]. Therefore, the analysis of protein kinase activity and the phosphorylation level of their sub- strates are important for understanding signal trans- duction pathways at a molecular level. Many methods have been described for determining protein kinase activity and protein phosphorylation levels. In vitro, phosphorylation reactions can be monitored by incu- bating a protein kinase and a substrate in the presence of radioactive ATP ([ 32 P]ATP[cP]), followed by SDS ⁄ PAGE and autoradiography to quantify radio- activity in the substrate. When a peptide substrate is used, the amount of radioactivity incorporated into Keywords Cdc37; CK2; gel electrophoresis; Hsp90; protein kinase Correspondence Y. Miyata, Department of Cell & Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan Fax: +81 75 753 4235 Tel: +81 75 753 4231 E-mail: ymiyata@lif.kyoto-u.ac.jp (Received 22 March 2007, revised 9 August 2007, accepted 4 September 2007) doi:10.1111/j.1742-4658.2007.06090.x The CK2-dependent phosphorylation of Ser13 in cell division cycle pro- tein 37 (Cdc37), a kinase-specific heat shock protein 90 (Hsp90) cochaper- one, has previously been reported to be essential for the association of Cdc37 with signaling protein kinases [Bandhakavi S, McCann RO, Hanna DE & Glover CVC (2003) J Biol Chem 278, 2829–2836; Shao J, Prince T, Hartson SD & Matts RL (2003) J Biol Chem 278, 38117–38220; Miyata Y & Nishida E (2004) Mol Cell Biol 24, 4065–4074]. Here we describe a new phospho-specific antibody against Cdc37 that recognizes recombinant puri- fied Cdc37 only when incubated with CK2 in the presence of Mg 2+ and ATP. The replacement of Ser13 in Cdc37 by nonphosphorylatable amino acids abolished binding to this antibody. The antibody was specific for phosphorylated Cdc37 and did not crossreact with other CK2 substrates such as Hsp90 and FK506-binding protein 52. Using this antibody, we showed that complexes of Hsp90 with its client signaling kinases, Cdk4, MOK, v-Src, and Raf1, contained the CK2-phosphorylated form of Cdc37 in vivo. Immunofluorescent staining showed that Hsp90 and the phosphory- lated form of Cdc37 accumulated in epidermal growth factor-induced mem- brane ruffles. We further characterized the phosphorylation of Cdc37 using phospho-affinity gel electrophoresis. Our analyses demonstrated that the CK2-dependent phosphorylation of Cdc37 on Ser13 caused a specific gel mobility shift, and that Cdc37 in the complexes between Hsp90 and its cli- ent signaling protein kinases was in the phosphorylated form. Our results show the physiological importance of CK2-dependent Cdc37 phosphoryla- tion and the usefulness of phospho-affinity gel electrophoresis Glycation of low-density lipoprotein results in the time-dependent accumulation of cholesteryl esters and apolipoprotein B-100 protein in primary human monocyte-derived macrophages Bronwyn E. Brown 1 , Imran Rashid 1 , David M. van Reyk 2 and Michael J. Davies 1,3 1 Free Radical Group, The Heart Research Institute, Camperdown, Sydney, NSW, Australia 2 Department of Health Sciences, University of Technology Sydney, NSW, Australia 3 Faculty of Medicine, University of Sydney, NSW, Australia Complications associated with diabetes are the major cause of mortality and morbidity in people with this disease. These include microvascular complications that induce damage to the retina, nephrons and peripheral nerves, and macrovascular disease that is associated with accelerated atherosclerosis (deposition of lipids in Keywords aldehydes; atherosclerosis; foam cells; human monocyte-derived macrophages; low-density lipoproteins Correspondence M. J. Davies, 114 Pyrmont Bridge Road, Camperdown, Sydney, NSW 2050, Australia Fax: +61 2 95655584 Tel: +61 2 82088900 E-mail: daviesm@hri.org.au (Received 12 December 2006, accepted 15 January 2007) doi:10.1111/j.1742-4658.2007.05699.x Nonenzymatic covalent binding (glycation) of reactive aldehydes (from glu- cose or metabolic processes) to low-density lipoproteins has been previ- ously shown to result in lipid accumulation in a murine macrophage cell line. The formation of such lipid-laden cells is a hallmark of atheroscler- osis. In this study, we characterize lipid accumulation in primary human monocyte-derived macrophages, which are cells of immediate relevance to human atherosclerosis, on exposure to low-density lipoprotein glycated using methylglyoxal or glycolaldehyde. The time course of cellular uptake of low-density lipoprotein-derived lipids and protein has been character- ized, together with the subsequent turnover of the modified apolipoprotein B-100 (apoB) protein. Cholesterol and cholesteryl ester accumulation occurs within 24 h of exposure to glycated low-density lipoprotein, and increases in a time-dependent manner. Higher cellular cholesteryl ester lev- els were detected with glycolaldehyde-modified low-density lipoprotein than with methylglyoxal-modified low-density lipoprotein. Uptake was signifi- cantly decreased by fucoidin (an inhibitor of scavenger receptor SR-A) and a mAb to CD36. Human monocyte-derived macrophages endocytosed and degraded significantly more 125 I-labeled apoB from glycolaldehyde-modified than from methylglyoxal-modified, or control, low-density lipoprotein. Dif- ferences in the endocytic and degradation rates resulted in net intracellular accumulation of modified apoB from glycolaldehyde-modified low-density lipoprotein. Accumulation of lipid therefore parallels increased endocytosis and, to a lesser extent, degradation of apoB in human macrophages exposed to glycolaldehyde-modified low-density lipoprotein. This accumula- tion of cholesteryl esters and modified protein from glycated low-density lipoprotein may contribute to cellular dysfunction and the increased atherosclerosis observed in people with diabetes, and other pathologies linked to exposure to reactive carbonyls. Abbreviations AGE, advanced glycation end-products; apoB, apolipoprotein B-100; HBSS, Hank’s balanced salt solution; HMDM, human monocyte-derived macrophage; HSA, human serum The Light-Dependent Reactions of Photosynthesis The Light-Dependent Reactions of Photosynthesis Bởi: OpenStaxCollege How can light be used to make food? When a person turns on a lamp, electrical energy becomes light energy Like all other forms of kinetic energy, light can travel, change form, and be harnessed to work In the case of ... find their way to the surrounding environment The hydrogen ions play critical roles in the remainder of the lightdependent reactions Keep in mind that the purpose of the light- dependent reactions. .. Absorption of Light Light energy enters the process of photosynthesis when pigments absorb the light In plants, pigment molecules absorb only visible light for photosynthesis The visible light seen.. .The Light- Dependent Reactions of Photosynthesis Visit this site and click through the animation to view the process of photosynthesis within a leaf What Is Light Energy? The sun emits