Kinetics and mechanism of exogenous anion exchange in FeFbpA–NTA: significance of periplasmic anion lability and anion binding activity of ferric binding protein A

The bacterial transferrin ferric binding protein A (FbpA) requires an exogenous anion to facilitate iron sequestration, and subsequently to shuttle the metal across the periplasm to the cytoplasmic membrane. In the diverse conditions of the periplasm, numerous anions are known to be present. Prior in vitro experiments have demonstrated the ability of multiple anions to fulfill the synergistic iron-binding requirement, and the identity of the bound anion has been shown to modulate important physicochemical properties of iron-bound FbpA (FeFbpA). Here we address the kinetics and mechanism of anion exchange for the FeFbpA–nitrilotriacetate (NTA) assembly with several biologically relevant anions (citrate, oxalate, phosphate, and pyrophosphate), with nonphysiologic NTA serving as a representative synergistic anion/chelator. The kinetic data are consistent with an anion-exchange process that occurs in multiple steps, dependent on the identity of both the entering anion and the leaving anion. The exchange mechanism may proceed either as a direct substitution or through an intermediate FeFbpA–X* assembly based on anion (X) identity. Our kinetic results further develop an understanding of exogenous anion lability in the periplasm, as well as address the final step of the iron-free FbpA (apo-FbpA)/Fe³⁺ sequestration mechanism. Our results highlight the kinetic significance of the FbpA anion binding site, demonstrating a correlation between apo-FbpA/anion affinity and the FeFbpA rate of anion exchange, further supporting the requirement of an exogenous anion to complete tight sequestration of iron by FbpA, and developing a mechanism for anion exchange within FeFbpA that is dependent on the identity of both the entering anion and the leaving anion.     

Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding

In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment; (4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate in a resin; and (5) preservation of antigens for sensitive immunogold labeling.     

ILCD Handbook Public Consultation Workshop

Introduction

The European Commission is supporting the development of the International Reference Life Cycle Data System (ILCD). This consists primarily of the ILCD Handbook and the ILCD Data Network. This paper gives an insight into the scientific positions of business, governments, consultants, academics, and others that were expressed at this public consultation workshop.

Workshop focus

The workshop focused on four of the topics of the main guidance documents of the ILCD Handbook: (1) general guidance on life cycle assessment (LCA); (2) guidance for generic and average life cycle inventory (LCI) data sets; (3) requirements for environmental impact assessment methods, models and indicators for LCA; and (4) review schemes for LCA.

Workshop participation

This consultation workshop was attended by more than 120 participants during the 4 days of the workshop. Representatives came from 23 countries, from both within and outside the European Union.

Workshop structure

Approximately half of the participants were from business associations or individual companies. Another 20% were governmental representatives. Others came predominantly from consultancies and academia.

Results

This public consultation workshop provided valuable inputs into the overall ILCD Handbook developments as well as for further development. This paper focuses on some of the main scientific issues that were raised.     

A review on the potential of triploid tench for aquaculture

Performance and physiological traits and health of spontaneous and induced triploid tench are reviewed. Triploidy is best induced with cold shock; with triploids exhibiting 13.5–51.5% better weight gain, 2.69–3.94% higher slaughtering value, 20–60% lower gonadosomatic index, 0.9–4.5% higher dry matter in flesh and up to 107% more flesh fat than diploids, if farmed untill post sexual maturity. Triploids exhibit more abdominal fat and less polyunsaturated fatty acids of the n-3 and n-6 groups in the flesh. Triploid females are sterile, while triploid males may produce aneuploid spermatozoa with varying DNA content (1–1.9n) which may initiate development of embryos. Triploids have milder seasonal dynamics in their erythrocyte profile than the diploids. Thinner diffusion distance in gills of triploids than in diploids is interpreted as adaptation to lower aerobic capacity. Triploids show neither stronger tendencies to anatomic malformations, nor have bigger affinity to parasitic diseases than the diploids. Production of triploid tench could be an economically interesting method of farming to higher marketable weight, bringing a relatively high product quality.     

Structural diversity of nucleoside phosphonic acids as a key factor in the discovery of potent inhibitors of rat T-cell lymphoma thymidine phosphorylase

Structurally diverse, sugar-modified, thymine-containing nucleoside phosphonic acids were evaluated for their ability to inhibit thymidine phosphorylase (TP, EC 2.4.2.4) purified from spontaneous T-cell lymphomas of an inbred Sprague-Dawley rat strain. From a large set of tested compounds, among them a number of pyrrolidine-based derivatives, 10 nucleotide analogues with IC₅₀ values below 1 μM were selected. Out of them, four compounds strongly inhibited the enzyme with IC₅₀ values lying in a range of 11–45 nM. These most potent compounds might be bi-substrate analogues.     

Structure of the catalytic domain of the human mitochondrial Lon protease: Proposed relation of oligomer formation and activity

ATP‐dependent proteases are crucial for cellular homeostasis. By degrading short‐lived regulatory proteins, they play an important role in the control of many cellular pathways and, through the degradation of abnormally misfolded proteins, protect the cell from a buildup of aggregates. Disruption or disregulation of mammalian mitochondrial Lon protease leads to severe changes in the cell, linked with carcinogenesis, apoptosis, and necrosis. Here we present the structure of the proteolytic domain of human mitochondrial Lon at 2 Å resolution. The fold resembles those of the three previously determined Lon proteolytic domains from Escherichia coli, Methanococcus jannaschii, and Archaeoglobus fulgidus. There are six protomers in the asymmetric unit, four arranged as two dimers. The intersubunit interactions within the two dimers are similar to those between adjacent subunits of the hexameric ring of E. coli Lon, suggesting that the human Lon proteolytic domain also forms hexamers. The active site contains a 3₁₀ helix attached to the N‐terminal end of α‐helix 2, which leads to the insertion of Asp852 into the active site, as seen in M. jannaschii. Structural considerations make it likely that this conformation is proteolytically inactive. When comparing the intersubunit interactions of human with those of E. coli Lon taken with biochemical data leads us to propose a mechanism relating the formation of Lon oligomers with a conformational shift in the active site region coupled to a movement of a loop in the oligomer interface, converting the proteolytically inactive form seen here to the active one in the E. coli hexamer.     

A critical role of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase in the control of plant metabolism and development

Glycolysis is a central metabolic pathway that provides energy and generates precursors for the synthesis of primary metabolites such as amino acids and fatty acids.^1–3 In plants, glycolysis occurs in the cytosol and plastids, which complicates the understanding of this essential process.¹ As a result, the contribution of each glycolytic pathway to the specific primary metabolite production and the degree of integration of both pathways is still unresolved. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described biochemically. But, up to now, little attention had been paid to GAPCps, probably because they have been considered as “minor isoforms” that catalyze a reversible reaction in plastids where it has been assumed that key glycolytic intermediates are in equilibrium with the cytosol. In the associated study,⁴ we have elucidated the crucial role of Arabidopsis GAPCps in the control of primary metabolism in plants. GAPCps deficiency affects amino acid and sugar metabolism and impairs plant development. Specifically, GAPCp deficiency affects the serine supply to roots, provoking a drastic phenotype of arrested root development. Also, we show that the phosphorylated serine biosynthesis pathway is critical to supply serine to non-photosynthetic organs such as roots. These studies provide new insights of the contribution of plastidial glycolysis to plant metabolism and evidence the complex interactions existing between metabolism and development.Key words: GAPDH, glycolysis, serine biosynthesis, Arabidopsis, plastid     

Genetic diversity and its effect on fitness in an endangered plant species, <Emphasis Type="Italic">Dracocephalum austriacum</Emphasis> L.

The aim of this study was to estimate genetic diversity and assess its importance for plant fitness in a species belonging to the most endangered species in Europe, Dracocephalum austriacum L., and to select the most valuable populations for conservation of genetic diversity within the species in the studied regions. We analyzed allozyme variation of 12 populations in three distinct regions (Czech Karst, Moravia and Slovak Karst) in Central Europe. The results showed high genetic diversity within populations (80.14%) and relatively low differentiation among populations within regions (9.42%) and between regions (10.45%). Seed production was significantly higher in larger, genetically more diverse and less inbred populations. The results suggest that genetic diversity has important effect on seed production in this species and thus can be expected to have strong direct consequences for plant fitness and vitality of the whole populations. They also show large variation in genetic diversity between populations and indicate which populations should get a priority in attempts to conserve all the genetic diversity within the region.