Expression of the major tyrosyl phosphoprotein of 54 KDa in the integument of the medflyCeratitis capitata

Expression of a 54 kDa tyrosyl phosphorylated protein in epidermal cells during the third instar larval stage was followed. It was demonstrated that the 54 kDa protein moiety and its phosphorylated counterpart follow the same developmental profile. The system seems to be regulated only at the onset of the second moult, by an initial signal which regulates both the synthesis and phosphorylation of a 54 kDa protein. The continuous presence this protein in epidermal cells during the third instar stage, as well as during apolysis and histolysis, suggests that it might participate in cell activities taking place during this developmental period. However, the 54 kDa protein could no be involved in specific epidermal cell activities such as histolysis, melanization and sclerotization, since these activities occur only at specific times during the third instar stage.     

BLOEM: A spatially explicit model of bioenergy and carbon capture and storage,applied to Brazil

Bioenergy could play a major role in decarbonizing energy systems in the context of the Paris Agreement. Large-scale bioenergy deployment could be related to sustainability issues and requires major infrastructure investments. It, therefore, needs to be studied carefully. The Bioenergy and Land Optimization Spatially Explicit Model (BLOEM) presented here allows for assessing different bioenergy pathways while encompassing various dimensions that influence their optimal deployment. In this study, BLOEM was applied to the Brazilian context by coupling it with the Brazilian Land Use and Energy Systems (BLUES) model. This allowed investigating the most cost-effective ways of attending future bioenergy supply projections and studying the role of recovered degraded pasture lands in improving land availability in a sustainable and competitive manner. The results show optimizing for limiting deforestation and minimizing logistics costs results in different outcomes. It also indicates that recovering degraded pasture lands is attractive from both logistics and climate perspectives. The systemic approach of BLOEM provides spatial results, highlighting the trade-offs between crop allocation, land use and the logistics dynamics between production, conversion, and demand, providing valuable insights for regional and national climate policy design. This makes it a useful tool for mapping sustainable bioenergy value chain pathways.     

Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding casette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete less of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.     

Immunocytochemical and electrophoretic studies on the localization of the major haemolymph proteins (ceratitins) inCeratitis capitata during development

Summary The developmental profile of the major haemolymph proteins (ceratitins) inCeratitis capitata was studied. Ceratitin concentration in the haemolymph decreases dramatically during the last days of pupal life, while the amounts of ceratitins in whole organism extracts remain unchanged. By electrophoretic, immunological and immunofluorescence techniques it was revealed that ceratitins are reabsorbed by the fat body and a fraction of them is deposited in the cuticle. The possible role of ceratitins is discussed.     

Characterization of the subunit composition of HGPRTase from human erythrocytes and cultured fibroblasts

Hypoxanthine-guanine phosphoribosyltransferase is a ubiquitous human enzyme, the inherited deficiency of which leads to a specific metabolic-neurological syndrome. Native acrylamide isoelectric focusing revealed that the human enzyme consists of different numbers of isoenzymes depending on the tissue of origin. The erythrocytic enzyme has the most isoenzymes while the enzyme from cultured fibroblasts has only a single isoenzyme. The isoenzyme pattern of the erythrocytic enzyme changes on storage of the crude hemolysate at 4 C. Treatment of the stored crude hemolysate with 4.5 m urea and 0.35 mm -mercaptoethanol results in an isoenzyme pattern similar to that of the fresh crude extract. Thus the additional isoenzymes are generated on storage not by covalent modification of the enzyme but probably by binding of small molecules to the enzyme or to association of the enzyme molecules. Hypoxanthine-guanine phosphoribosyltransferase has been purified to 80% homogeneity in three steps, DEAE Sephadex chromatography, heat treatment at 85 C for 5 min, and hydroxylapatite chromatography. Denaturing two-dimensional gel electrophoresis of the erythrocytic enzyme revealed that the erythrocytic enzyme is composed of three major types of subunits (1–3) with the same molecular weight but different isoelectric points. In contrast, the fibroblast enzyme is composed of only a single type of subunit, which comigrates with subunit 1 of the erythrocytic enzyme. Since there is a single genetic locus in humans for HGPRTase (the enzyme is X linked) (Nyhan et al., 1967), the observed subunit modification of the erythrocyte enzyme appears to be the result of posttranslational modification. These findings provide a simple explanation for the observed electrophoretic properties of human HGPRTase. A patient with 0.5% of HGPRTase activity in his erythrocytes was found to have small amounts (> 0.5% but < 5% of normal) of the erythrocytic HGPRTase subunits.This work was supported by a grant from NIAMDD, National Institutes of Health, United States Public Health Service. L. J. G. was supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.Purine nucleoside phosphorylase has been purified at least a hundredfold from ³⁵S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of ³⁵S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. L. J. G. is supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Afforestation for climate change mitigation: Potentials,risks and trade‐offs

Afforestation is considered a cost‐effective and readily available climate change mitigation option. In recent studies afforestation is presented as a major solution to limit climate change. However, estimates of afforestation potential vary widely. Moreover, the risks in global mitigation policy and the negative trade‐offs with food security are often not considered. Here we present a new approach to assess the economic potential of afforestation with the IMAGE 3.0 integrated assessment model framework. In addition, we discuss the role of afforestation in mitigation pathways and the effects of afforestation on the food system under increasingly ambitious climate targets. We show that afforestation has a mitigation potential of 4.9 GtCO₂/year at 200 US$/tCO₂ in 2050 leading to large‐scale application in an SSP2 scenario aiming for 2°C (410 GtCO₂ cumulative up to 2100). Afforestation reduces the overall costs of mitigation policy. However, it may lead to lower mitigation ambition and lock‐in situations in other sectors. Moreover, it bears risks to implementation and permanence as the negative emissions are increasingly located in regions with high investment risks and weak governance, for example in Sub‐Saharan Africa. Afforestation also requires large amounts of land (up to 1,100 Mha) leading to large reductions in agricultural land. The increased competition for land could lead to higher food prices and an increased population at risk of hunger. Our results confirm that afforestation has substantial potential for mitigation. At the same time, we highlight that major risks and trade‐offs are involved. Pathways aiming to limit climate change to 2°C or even 1.5°C need to minimize these risks and trade‐offs in order to achieve mitigation sustainably.     

Proteasome dysfunction in Drosophila signals to an Nrf2‐dependent regulatory circuit aiming to restore proteostasis and prevent premature aging

The ubiquitin–proteasome system is central to the regulation of cellular proteostasis. Nevertheless, the impact of in vivo proteasome dysfunction on the proteostasis networks and the aging processes remains poorly understood. We found that RNAi‐mediated knockdown of 20S proteasome subunits in Drosophila melanogaster resulted in larval lethality. We therefore studied the molecular effects of proteasome dysfunction in adult flies by developing a model of dose‐dependent pharmacological proteasome inhibition. Impaired proteasome function promoted several ‘old‐age’ phenotypes and markedly reduced flies' lifespan. In young somatic tissues and in gonads of all ages, loss of proteasome activity induced higher expression levels and assembly rates of proteasome subunits. Proteasome dysfunction was signaled to the proteostasis network by reactive oxygen species that originated from malfunctioning mitochondria and triggered an Nrf2‐dependent upregulation of the proteasome subunits. RNAi‐mediated Nrf2 knockdown reduced proteasome activities, flies' resistance to stress, as well as longevity. Conversely, inducible activation of Nrf2 in transgenic flies upregulated basal proteasome expression and activity independently of age and conferred resistance to proteotoxic stress. Interestingly, prolonged Nrf2 overexpression reduced longevity, indicating that excessive activation of the proteostasis pathways can be detrimental. Our in vivo studies add new knowledge on the proteotoxic stress‐related regulation of the proteostasis networks in higher metazoans. Proteasome dysfunction triggers the activation of an Nrf2‐dependent tissue‐ and age‐specific regulatory circuit aiming to adjust the cellular proteasome activity according to temporal and/or spatial proteolytic demands. Prolonged deregulation of this proteostasis circuit accelerates aging.     

Lysoglycerophospholipids in chronic inflammatory disorders: The PLA2/LPC and ATX/LPA axes

Lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA), the most prominent lysoglycerophospholipids, are emerging as a novel class of inflammatory lipids, joining thromboxanes, leukotrienes and prostaglandins with which they share metabolic pathways and regulatory mechanisms. Enzymes that participate in LPC and LPA metabolism, such as the phospholipase A₂ superfamily (PLA₂) and autotaxin (ATX, ENPP2), play central roles in regulating LPC and LPA levels and consequently their actions. LPC/LPA biosynthetic pathways will be briefly presented and LPC/LPA signaling properties and their possible functions in the regulation of the immune system and chronic inflammation will be reviewed. Furthermore, implications of exacerbated LPC and/or LPA signaling in the context of chronic inflammatory diseases, namely rheumatoid arthritis, multiple sclerosis, pulmonary fibrosis and hepatitis, will be discussed. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Significance of the hydrophobic residues 225–230 of apoA-I for the biogenesis of HDL

We studied the significance of four hydrophobic residues within the 225–230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I^−/− mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I^−/− × apoE^−/− mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-β- and α4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal αHDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225–230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225–230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-β- and α4-HDL particles, a phenotype that could be corrected by administration of LCAT.