Correlating crosslink formation with enzymatic activity in cysteine dioxygenase

Cysteine dioxygenase (CDO) from rat and other mammals exhibits a covalent post-translational modification between the residues C93 and Y157 that is in close proximity to the active site, and whose presence enhances the enzyme's activity. Protein with and without C93-Y157 crosslink migrates as distinct bands in SDS-PAGE, allowing quantification of the relative ratios between the two forms by densitometry of the respective bands. Expression of recombinant rat wild type CDO in Escherichia coli typically produces 40-50% with the C93-Y157 crosslink. A strategy was developed to increase the ratio of the non-crosslinked form in an enzyme preparation of reasonable quantity and purity, allowing direct assessment of the activity of non-crosslinked CDO and mechanism of formation of the crosslink. The presence of ferrous iron and oxygen is a prerequisite for C93-Y157 crosslink formation. Absence of oxygen during protein expression increased the fraction of non-crosslinked CDO, while presence of the metal chelator EDTA had little effect. Metal affinity chromatography was used to enrich non-crosslinked content. Both the enzymatic rate of cysteine oxidation and the amount of cross-linking between C93 and Y157 increased significantly upon exposure of CDO to air/oxygen and substrate cysteine in the presence of iron in a hitherto unreported two-phase process. The instantaneous activity was proportional to the amount of crosslinked enzyme present, demonstrating that the non-crosslinked form has negligible enzymatic activity. The biphasic kinetics suggest the existence of an as yet uncharacterised intermediate in crosslink formation and enzyme activation.     

Fukutin-related protein resides in the Golgi cisternae of skeletal muscle fibres and forms disulfide-linked homodimers via an N-terminal interaction

Limb-Girdle Muscular Dystrophy type 2I (LGMD2I) is an inheritable autosomal, recessive disorder caused by mutations in the FuKutin-Related Protein (FKRP) gene (FKRP) located on chromosome 19 (19q13.3). Mutations in FKRP are also associated with Congenital Muscular Dystrophy (MDC1C), Walker-Warburg Syndrome (WWS) and Muscle Eye Brain disease (MEB). These four disorders share in common an incomplete/aberrant O-glycosylation of the membrane/extracellular matrix (ECM) protein α-dystroglycan. However, further knowledge on the FKRP structure and biological function is lacking, and its intracellular location is controversial. Based on immunogold electron microscopy of human skeletal muscle sections we demonstrate that FKRP co-localises with the middle-to-trans-Golgi marker MG160, between the myofibrils in human rectus femoris muscle fibres. Chemical cross-linking experiments followed by pairwise yeast 2-hybrid experiments, and co-immune precipitation, demonstrate that FKRP can exist as homodimers as well as in large multimeric protein complexes when expressed in cell culture. The FKRP homodimer is kept together by a disulfide bridge provided by the most N-terminal cysteine, Cys6. FKRP contains N-glycan of high mannose and/or hybrid type; however, FKRP N-glycosylation is not required for FKRP homodimer or multimer formation. We propose a model for FKRP which is consistent with that of a Golgi resident type II transmembrane protein.     

A novel photosensitizer for light-controlled gene silencing

We here demonstrate for the first time that 5-carboxytetramethylrhodamine (TAMRA) covalently linked to nuclear localization signal (NLS)-conjugated peptide nucleic acids (PNAs) are photosensitizers (PSs) with the capacity to initiate photochemical damage to endocytic membranes, resulting in release of endocytosed material into cytosol. Our results show that TAMRA/PNA/NLS conjugates work as multifunctional molecules by offering cellular uptake, PNA-directed gene silencing, and the possibility for targeting in a light-controlled manner. In addition to PNA-directed gene silencing, we demonstrate that TAMRA/PNA/NLS molecules may function as a PS for light-controlled release of small interfering RNA molecules from the endocytic pathway when combined with an appropriate carrier. Using these strategies, we could silence the S100A4 gene at both protein and mRNA levels in a light-controlled manner, without any detectable reduction in cell viability. Our data demonstrate the possibility for light-controlled delivery of macromolecules entrapped within endocytic vesicles using multifunctional TAMRA/PNA/NLS molecules as PSs.     

Mepyramine-JNJ7777120-hybrid compounds show high affinity to hH(1)R, but low affinity to hH(4)R

In literature, a synergism between histamine H(1) and H(4) receptor is discussed. Furthermore, it was shown, that the combined application of mepyramine, a H(1) antagonist and JNJ7777120, a H(4) receptor ligand leads to a synergistic effect in the acute murine asthma model. Thus, the aim of this study was to develop new hybrid ligands, containing one H(1) and one H(4) pharmacophor, connected by an appropriate spacer, in order to address both, H(1)R and H(4)R. Within this study, we synthesized nine hybrid compounds, which were pharmacologically characterized at hH(1)R and hH(4)R. The new compounds revealed (high) affinity to hH(1)R, but showed only low affinity to hH(4)R. Additionally, we performed molecular dynamic studies for some selected compounds at hH(1)R, in order to obtain information about the binding mode of these compounds on molecular level.     

Identification of the Ubiquitin-like Domain of Midnolin as a New Glucokinase Interaction Partner

Glucokinase acts as a glucose sensor in pancreatic beta cells. Its posttranslational regulation is important but not yet fully understood. Therefore, a pancreatic islet yeast two-hybrid library was produced and searched for glucokinase-binding proteins. A protein sequence containing a full-length ubiquitin-like domain was identified to interact with glucokinase. Mammalian two-hybrid and fluorescence resonance energy transfer analyses confirmed the interaction between glucokinase and the ubiquitin-like domain in insulin-secreting MIN6 cells and revealed the highest binding affinity at low glucose. Overexpression of parkin, an ubiquitin E3 ligase exhibiting an ubiquitin-like domain with high homology to the identified, diminished insulin secretion in MIN6 cells but had only some effect on glucokinase activity. Overexpression of the elucidated ubiquitin-like domain or midnolin, containing exactly this ubiquitin-like domain, significantly reduced both intrinsic glucokinase activity and glucose-induced insulin secretion. Midnolin has been to date classified as a nucleolar protein regulating mouse development. However, we could not confirm localization of midnolin in nucleoli. Fluorescence microscopy analyses revealed localization of midnolin in nucleus and cytoplasm and co-localization with glucokinase in pancreatic beta cells. In addition we could show that midnolin gene expression in pancreatic islets is up-regulated at low glucose and that the midnolin protein is highly expressed in pancreatic beta cells and also in liver, muscle, and brain of the adult mouse and cell lines of human and rat origin. Thus, the results of our study suggest that midnolin plays a role in cellular signaling of adult tissues and regulates glucokinase enzyme activity in pancreatic beta cells.     

Global Material Flows and Resource Productivity: Forty Years of Evidence

The international industrial ecology (IE) research community and United Nations (UN) Environment have, for the first time, agreed on an authoritative and comprehensive data set for global material extraction and trade covering 40 years of global economic activity and natural resource use. This new data set is becoming the standard information source for decision making at the UN in the context of the post‐2015 development agenda, which acknowledges the strong links between sustainable natural resource management, economic prosperity, and human well‐being. Only if economic growth and human development can become substantially decoupled from accelerating material use, waste, and emissions can the tensions inherent in the Sustainable Development Goals be resolved and inclusive human development be achieved. In this paper, we summarize the key findings of the assessment study to make the IE research community aware of this new global research resource. The global results show a massive increase in materials extraction from 22 billion tonnes (Bt) in 1970 to 70 Bt in 2010, and an acceleration in material extraction since 2000. This acceleration has occurred at a time when global population growth has slowed and global economic growth has stalled. The global surge in material extraction has been driven by growing wealth and consumption and accelerating trade. A material footprint perspective shows that demand for materials has grown even in the wealthiest parts of the world. Low‐income countries have benefited least from growing global resource availability and have continued to deliver primary materials to high‐income countries while experiencing few improvements in their domestic material living standards. Material efficiency, the amount of primary materials required per unit of economic activity, has declined since around 2000 because of a shift of global production from very material‐efficient economies to less‐efficient ones. This global trend of recoupling economic activity with material use, driven by industrialization and urbanization in the global South, most notably Asia, has negative impacts on a suite of environmental and social issues, including natural resource depletion, climate change, loss of biodiversity, and uneven economic development. This research is a good example of the IE research community providing information for evidence‐based policy making on the global stage and testament to the growing importance of IE research in achieving global sustainable development.     

The Corruption Footprints of Nations

In this study, we innovatively apply multiregional input‐output analysis to calculate corruption footprints of nations and show the details of commodities that use the most employment affected by corruption (EAC), as they flow between countries. Every country's corruption footprint includes its domestic corruption and the corruption imported by global supply chains to meet final demand. Our results show that, generally, the net corruption exporters are developing countries, with the exception of Italy where corruption is likely to be more affected by political and cultural factors than economic factors. China is the largest gross corruption exporter, and India follows close behind, with clothing as one of the industries in which the most people are affected by corruption. This is because: (1) China and India are major clothing exporters, thus many workers are employed in the clothing industry within the country as well as in countries providing intermediate commodities by supply chains, and (2) corruption is high in China and India. Our results can be useful to identify where regulations to combat corruption can have the greatest impact. More important, the method we use can be applied to link corruption to other economic and social aspects of trade, such as working conditions, thus making it possible to find avenues for tackling the problem that are not usually considered in anticorruption strategies.     

An empirical test for a zone of canalization in thermal reaction norms

Theoretical models on the evolution of phenotypic plasticity predict a zone of canalization where reaction norms cross, and genetic variation is minimized in the environment a population most frequently encounter. Empirical tests of this prediction are largely missing, in particular for life‐history traits. We addressed this prediction by quantifying thermal reaction norms of three life‐history traits (somatic growth rate, age and size at maturation) of a Norwegian population of Daphnia magna and testing for the occurrence of an intermediate temperature (T_m) at which genetic variance in the traits is minimized. Size at maturation changed relatively little with temperature compared to the other traits, and there was no genetic variance in the shape of the reaction norm. Consequently, age at maturation and somatic growth rate were strongly negatively correlated. Both traits showed a strong genotype–environment interaction, and the estimated T_m was 14 °C for both age at maturation and growth rate. This value of T_m corresponds well with mean summer temperatures experienced by the population and suggests that the population has evolved under stabilizing selection in temperatures that fluctuate around this mean temperature. These results suggest local adaptation to temperature in the studied population and allow predicting evolutionary trajectories of thermal reaction norms under changing thermal regimes.     

Influence of exogenous thyroxine on plasma melatonin in juvenile Atlantic salmon (Salmo salar)

One of the most clearly defined endocrine changes during the parr-smolt transformation of anadromous salmonids is an increase in plasma levels of thyroid hormones. The role of pineal hormone melatonin in timing and synchronisation of smoltification is widely discussed. The effect of administration of exogenous thyroxine (T4) on plasma melatonin was investigated in juvenile Atlantic salmon (Salmo salar) at the early stages of parr-smolt transformation. Fish were kept in fresh water under simulated-natural photoperiod and exposed to exogenous T4. Fish were sampled at 12.00 and 24.00 h from treatment and control tanks, 2 and 14 days after treatment started. Plasma melatonin and L-thyroxine were measured using RIA and competitive enzyme immunoassay, respectively. After 2 days of T4 treatment, marked difference in plasma melatonin concentration measured at 12.00 and 24.00 h was still observed in both groups. However, 2-week exposure to T4 caused a reduction in night-time plasma melatonin level and thus, probably, inhibited melatonin related time-keeping system in juvenile salmon. Additional studies are needed to clarify the mechanism of the described phenomenon.     

Double‐Counting in Life Cycle Calculations

Many popular frameworks apply life cycle calculations to examine environmental burdens occurring throughout the life cycle of products and services that are either purchased by final consumers or demanded as inputs by producers. Accounting for the full supply chain of producer items can lead to double‐counting effects when results of separate studies are added up and referenced or compared to totals. If, for instance, energy life cycle inventories were prepared for all consumer and producer items in an economy and added up, the resulting total amount of energy would be greater than national energy consumption. Although this double‐counting is inconsequential if analyses are appraised in isolation without reference to national totals, it leads to serious errors when large interconnected systems are analyzed or when results are placed into wider (e.g., regional, national, or global) contexts. The article lists a number of prominent policy and decision‐making frameworks that make use of life cycle techniques, where this double‐counting error is highly undesirable. It proposes a solution to the double‐counting problem in which supply chains in the product life cycle are split and burdens shared between the supplying and demanding sides of every transaction in the economy.