Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress

The effect of supplementation with substances having antioxidant properties on the adaptive responses of human skin fibroblasts to UV-induced oxidative stress was studied in vitro. UVR was found to induce a substantial oxidative stress in fibroblasts, resulting in an increased release of superoxide anions and an increase in lipid peroxidation (shown by an elevated malonaldehyde content). Sub-lethal doses of UVR were also found to induce adaptive responses in the fibroblast antioxidant defences, with a transient rise in catalase and superoxide dismutase activities followed by a slower, large increase in cellular glutathione content. Supplementation of the fibroblasts with the antioxidants, Trolox (a water soluble analogue of alpha-tocopherol), ascorbic acid or beta-carotene, had differential effects on these responses. Trolox supplementation reduced the UVR-induced cellular oxidative stress and adaptive response in a predictable concentration-dependent manner. This was in contrast to ascorbic acid which increased superoxide release from fibroblasts. At low doses, ascorbate supplements also reduced the magnitude of the adaptive increases in catalase and superoxide dismutase activities and increase in glutathione content. Beta-carotene had a similar effect to ascorbic acid, reducing the extent of the adaptations to UVR at lower doses while simultaneously increasing superoxide release and malonaldehyde content. These in vitro data indicate that only the vitamin E analogue suppressed UVR-induced oxidative stress in a predictable manner and suggest that common dietary antioxidants may not be equally effective in reducing the potential deleterious effects of UVR-induced oxidative stress in skin.     

Synthesis and biological activities of leukotriene F4 and leukotriene F4 sulfone

Leukotriene F₄ (LTF₄ and LTF₄ sulfone have been synthesized and their biological activities determined in the guinea pig. LFT₄ displayed comparable activity to LTD₄ on guinea pig trachea and parenchyma but was less active on the ileum. When injected intravenously into the guinea pig, LTF₄ induced a bronchoconstriction (ED₅₀ 16 μg Kg⁻¹) which was blocked by indomethacin and FPL-55712 and was 50–100 X less potent than LTD₄ in this assay. LTF₄ sulfone was approximately 2–5 times less active than LTF₄ and . When injected into guinea pig skin with PGE₂ (100 ng); LTF₄ and LTF₄ sulfone (10–1000 ng) induced changes in vascular permeability. The order of potency in this assay was LTE₄ sulfone = LTD₄ = LTD₄ sulfone > LTE₄ > LTF₄ = LTF₄ sulfone.     

Nocturnal loss of muscle protein from House sparrows (Passer domesticus)

An overall loss of protein was considered to be the explanation for at least some of the observed loss of lean dry material from muscle of wild House sparrows overnight.     

Characterization of lipoprotein produced by the perfused rhesus monkey liver

Isolated livers from rhesus monkeys (Macaca mulatta) were perfused in order to asses the nature of newly synthesized hepatic lipoprotein. Perfusate containing [3H]leucine was recirculated for 1.5 hr, followed by an additional 2.5-hr perfusion with fresh perfusate. Equilibrium density gradient ultracentrifugation clearly separated VLDL from LDL. The apoprotein composition of VLDL secreted by the liver was similar to that of serum VLDL. The perfusate LDL contained some poorly radiolabeled, apoB-rich material, which appeared to be contaminating serum LDL. There was also some material of an LDL-like density, which was rich in radiolabeled apoE. Rate zonal density gradient ultracentrifugation fractionated HDL. All perfusate HDL fractions had a decreased cholesteryl ester/unesterified cholesterol ratio, compared to serum HDL. Serum HDL distributed in one symmetric peak near the middle of the gradient, with coincident peaks of apoA-I and apoA-II. The least dense fractions of the perfusate gradient were rich in radiolabeled apoE. The middle of the perfusate gradient contained particles rich in radiolabeled apoA-I and apoA-II. The peak of apoA-I was offset from the apoA-II peak towards the denser end of the gradient. The dense end of the HDL gradient contained lipoprotein-free apoA-I, apoE, and small amounts of apoA-II, probably resulting from the relative instability of nascent lipoprotein compared to serum lipoprotein. Perfusate HDL apoA-I isoforms were more basic than serum apoA-I isoforms. Preliminary experiments, using noncentrifugal methods, suggest that some hepatic apoA-I is secreted in a lipoprotein-free form. In conclusion, the isolated rhesus monkey liver produces VLDL similar to serum VLDL, but produces LDL and HDL which differ in several important aspects from serum LDL and HDL.     

Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection

The cell''s endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system.Membrane compartmentalization is an essential mechanism for eukaryotic life, by which cells separate and control biological processes. Plant growth, development, and adaptation to biotic and abiotic stress all rely on the highly dynamic endomembrane system, yet we know comparatively little about the proteins regulating these dynamic trafficking events. The plasma membrane (PM) provides the main interface between the cell and its environment, mediating the transfer of material to and from the cell and is a primary site for perception of external signals. Transmembrane proteins are synthesized in the endoplasmic reticulum (ER) and trafficked to the PM via the Golgi, although there are other secretory routes for soluble cargo (discussed in (1–4)). Post-Golgi trafficking is the main route by which newly synthesized transmembrane proteins and cell wall glycans are delivered to the PM. In plants, secretory and endocytic traffic converge at the trans-Golgi network (TGN), which also functions as an early endosome (EE). Multivesicular bodies (MVBs) are the other main endosomal compartment in plants and serve as prevacuolar compartments (PVCs) or late endosomes (LE) destined for vacuolar degradation (reviewed (1, 5, 6)).Recycling and sorting of plasma membrane proteins is essential for generating the polar localization of auxin efflux transporters (discussed in (7)), formation of the cell plate during cell division (8–11), and in defense such as localized deposition of papilla reviewed in (12, 13). Furthermore, the subcellular localization of transporters and receptors is dynamically regulated. For example, the boron transporter (BOR1) exhibits polar localization and is internalized and degraded under conditions of high boron to reduce toxicity (14, 15). Similarly the receptor-like kinases (RLKs) flagellin-sensing 2 (FLS2) and brassinosteroid insensitive 1 (BRI1), important transmembrane receptors in antibacterial immunity and plant development, respectively, are constitutively endocytosed and recycled to the PM (16–18). Both receptors and transporters are also cargoes of the LE/MVB trafficking route (16) and are probably sorted to the vacuole for degradation (19, 20). Importantly, FLS2 trafficking via the recycling endocytic or the late endocytic route depends on its activation status; inactive receptors are recycled while ligand-activated receptors are sorted to the late endosomal pathway (16). Similarly, the polar sorting of auxin efflux transporters depends on their phosphorylation status (21). These observations illustrate that membrane compartmentalization underpins important aspects of plant cell biology and has initiated a quest toward a better understanding of the endomembrane compartments and the routes and mechanisms by which cargo is trafficked and sorted within the cell.Membrane trafficking within the cell requires complex machinery consisting of a plethora of coat and adaptor proteins, small GTPases, targeting, tethering, and scission factors (reviewed in (22, 23)). Homologues of some animal and yeast and endomembrane regulators have been identified in plants, but the localization and function of many of these remain to be characterized. For example, members of the RAB GTPase family have been shown to have markedly different roles and localizations in plants compared with their animal and yeast homologs (24). Therefore, acquiring localization data for tethering complexes and other regulators in plant systems is essential. In Arabidopsis thaliana, some of these proteins have been developed as useful probes to visualize the different endomembrane compartments by fusion with fluorescent reporters (9, 25–27). These include regulators of trafficking events such as RAB GTPases that are molecular switches responsible for the assembly of tethering and docking complexes and compartment identity. RAB proteins are widely used markers of endomembrane compartments, for example RABD2a/ARA5 labels the Golgi and TGN/EE as well as post-Golgi vesicles (4, 24, 26, 28), RABF2b/ARA7 localizes to TGN/EE and LE (25), RABF1/ARA6 is a marker of the LE/MVB vesicles (25, 29), and RABG3f localizes to MVBs and the tonoplast (26, 30).Fluorescent-tagged marker lines for the live-cell imaging of plant cells have been invaluable in defining the location of proteins within and between organelles and endomembrane compartments (26). However, microscopic investigation of membrane trafficking is limited by throughput, as only few proteins can be studied simultaneously. A powerful approach to large-scale identification of proteins in endomembrane compartments is through subcellular fractionation based on physical properties to directly isolate or enrich for the subcellular compartment of interest. Subcellular fractionation-based proteomics have been successfully used to decipher the steady state and cargo proteomes of, including but not limited to, the ER, the vacuole, PM, mitochondria and chloroplasts, and smaller vesicle-like compartments such as peroxisomes and Golgi (31–41). However, the smaller, transitory vesicles of the secretory and endocytic pathways have proved challenging to purify for reliable proteomic analysis. To overcome this, affinity purification of vesicles was established in animal cells (42, 43) and recently successfully applied in plants in combination with subcellular fractionation. Affinity purification and mass spectrometry (MS) of syntaxin of plants 61 (SYP61)-positive TGN/EE compartments identified 145 proteins specifically enriched in (44), while affinity isolation of VHA-a1-GFP (vacuolar H⁺ ATPase A1) identified 105 proteins associated with the TGN/EE (45). The VHA-A1 affinity purification data were then further refined using density gradient centrifugation to differentiate cargo and steady-state proteins (45).We have further explored affinity purification of fluorescent-tagged markers localizing to defined compartments to identify proteins associated with trafficking. Our motivation was to dissect the trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with small GTPases in the RAB family. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for Golgi/TGN/EE/secretory vesicles, LE/MVB compartments, LE/MVB compartments and LE/MVB/tonoplast, respectively. Additionally, we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles (CCVs) and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast (26, 27, 29, 46, 47) as comparisons. Our objective was to identify transient cargo proteins, tethers, and docking factors associated with dynamic subdomains of the endomembrane system, to supplement better-characterized “steady-state” components, and to identify components of recycling and vacuolar trafficking pathways.