Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins : Detection probes and affinity standards

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four CuI ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their CuI affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with CuI to yield distinct 1:2 chromatophoric complexes [CuIL2] 3- with formation constants β2 = 1017.2 and 1019.8 M-2, respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu+ concentrations from 10-12 to 10-19 M. Dtt binds CuI with KD∼10-15 M at pH 7, but it is air-sensitive, and its CuI affinity varies with pH. The CuI binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity CuI binding and the individual quantitative affinities (KD values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind CuI with sub-femtomolar affinities, consistent with tight control of labile Cu+ concentrations in living cells.

Concomitant reactivity of the m-Terphenylindium dihydroxide [2,6-Mes2C6H3In(OH)2]4 toward carbon dioxide and ethylene glycol

The stepwise reaction of [2,6-Mes₂C₆H₃In(OH)₂]₄ with carbon dioxide and ethylene glycol proceeded with the formation of (2,6-Mes₂C₆H₃In)₄(CO₃)₂(OH)₄(H₂O)₂ (1) and (2,6-Mes₂C₆H₃In)₄(OCH₂CH₂O)₂(OH)₄ (2), respectively, and eventually produced (2,6-Mes₂C₆H₃In)₄(CO₃)₂(OCH₂CH₂OH)₂(OH)₂ (3). Attempts to liberate ethylene carbonate upon heating of 3 were unsuccessful.

Depolymerization of aryltellurinic anhydrides with sodium hydroxide. Synthesis and structure of the hydrated sodium aryltellurinates [Na(H2O)4](RTeO2) (R = 4-MeOC6H4, 8-Me2NC10H6)

Depolymerization of the aryltellurinic anhydrides [(RTe)₂O₃]_n (9, R = 4-MeOC₆H₄; 6, R = 8-Me₂NC₁₀H₆) with aqueous NaOH afforded the hydrated sodium aryltellurinates [Na(H₂O)₄](4-MeOC₆H₄TeO₂) (10) and [Na(H₂O)₄](8-Me₂NC₁₀H₆TeO₂)·H₂O (11·H₂O) as highly crystalline materials.

New series of intramolecularly coordinated diaryltellurium compounds. Rational synthesis of the diarylhydroxytelluronium triflate [(8-Me 2NC 10H 6) 2Te(OH)](O 3SCF 3)

The reaction of 8-dimethylaminonaphthyllithium etherate with the tellurium(II) bis(dithiocarbamate) Te(S₂CNEt₂)₂ provided the diaryltelluride (8-Me₂NC₁₀H₆)₂Te (1). The oxidation of 1 with an excess of H₂O₂ did not afford the expected diaryltellurium(IV) oxide (8-Me₂NC₁₀H₆)₂TeO (2), but the diaryltellurium(VI) dioxide (8-Me₂NC₁₀H₆)₂TeO₂ (3). The preparation of 2 was achieved by the comproportionation reaction of 1 and 3. The protonation of 2 using triflic acid gave rise to the formation of diarylhydroxytelluronium triflate [(8-Me₂NC₁₀H₆)₂Te(OH)](O₃SCF₃) (4), which features the protonated diaryltellurium oxide [(8-Me₂NC₁₀H₆)₂Te(OH)]+ (4a). Compounds 1, 3·H₂O·H₂O₂, 3·2H₂O, and 4 were characterized by X-ray crystallography. The experimentally obtained molecular structures were compared to those calculated for 1–3, 4a, and (8-Me₂NC₁₀H₆)₂Te(OH)₂ (5) as well as the related diphenyltellurium compounds Ph₂Te (6), Ph₂TeO (7), Ph₂TeO₂ (8), [Ph₂Te(OH)]+ (9a), and Ph₂Te(OH)₂ (10) at the DFT/B3PW91 level of theory.

Synthesis and structure of an intramolecularly coordinated diaryltelluronic acid and its dimethyl ester

The oxidation of the telluroxane cluster (8Me₂NC₁₀H₆Te)₆O₈(OH)₂ (4) or the diaryl ditelluride (8-Me₂NC₁₀H₆Te)(₂) (7) using H₂O₂ provided the diarylditelluronic acid [8-Me₂NC₁₀H₆Te(O)(OH)₂]₂(O) (6), which is the second member of this compound class and the first one to contain an intramolecularly coordinated substituent. Attempts at recrystallizing 6 from Methanol provided the partial ester [8-Me₂NC₁₀H₆Te(O)(OH)(OMe)]₂(O) (8). In addition structural motifs of known diaryltelluronic acids were compared using DFT calculations.

Mesityltellurenyl cations stabilized by triphenylpnictogens [MesTe(EPh₃)] + (E = P, As, Sb)

The homoleptic 1:1 Lewis pair (LP) complex [MesTe(TeMes₂)]O₃SCF₃ (1) featuring the cation [MesTe(TeMes₂)]+ (1a) was obtained by the reaction of Mes₂Te with HO₃SCF₃. The reaction of 1 with Ph₃E (E = P, As, Sb, Bi) proceeded with substitution of Mes₂Te and provided the heteroleptic 1:1 LP complexes [MesTe(EPh₃)]O₃SCF₃ (2, E = P; 3, E = As) and [MesTe(SbPh₃)][Ph₂Sb(O₃SCF₃)₂] (4) featuring the cations [MesTe(EPh₃)]+ (2a, E = P; 3a, E = As; 4a, E = Sb) and the anion [Ph₂Sb(O₃SCF₃)₂]− (4b). In the reaction with Ph₃Bi, the crude product contained the cation [MesTe(BiPh₃)]+ (5a) and the anion [Ph₂Bi(O₃SCF₃)₂]− (5b); however, the heteroleptic 1:1 LP complex [MesTe(BiPh₃)][Ph₂Bi(O₃SCF₃)₂] (5) could not be isolated because of its limited stability. Instead, fractional crystallization furnished a large amount of Ph₂BiO₃SCF₃ (6), which was also obtained by the reaction of Ph₃Bi with HO₃SCF₃. The formation of the anions 4b and 5b involves a phenyl group migration from Ph₃E (E = Sb, Bi) to the MesTe+ cation and afforded MesTePh as the byproduct, which was identified in the mother liquor. The heteroleptic 1:1 LP complexes 2–4 were also obtained by the one-pot reaction of Mes₂Te, Ph₃E (E = P, As, Sb) and HO₃SCF₃. Compounds 1–4 and 6 were investigated by single-crystal X-ray diffraction. The molecular structures of 1a–4a were used for density functional theory calculations at the B3PW91/TZ level of theory and studied using natural bond order (NBO) analyses as well as real-space bonding descriptors derived from an atoms-in-molecules (AIM) analysis of the theoretically obtained electron density. Additionally, the electron localizability indicator (ELI-D) and the delocalization index are derived from the corresponding pair density.

Determinants of green supply chain implementation in the food and beverage sector

Using a grounded theory approach, this paper extracts emerging concepts in the implementation of green supply chain management from case data of New Zealand food and beverage (F&B) companies. In search of factors that may lead to theory-building, the study relates case studies in fruit, juice, and dairy product companies through in-depth interviews with ranking general and line managers. We uncover the outline of a theoretical framework focusing on determinants of GSCM behaviour. These include strategic and operational planning; management structure, systems, and decision-making; management of people and company culture; and relationships with supply-chain members.

Advancing science and policy through a coordinated international study of physical activity and built environments: IPEN Adult methods

Background: National and international strategies to increase physical activity emphasize environmental and policy changes that can have widespread and long-lasting impact. Evidence from multiple countries using comparable methods is required to strengthen the evidence base for such initiatives. Because some environment and policy changes could have generalizable effects and others may depend on each country’s context, only international studies using comparable methods can identify the relevant differences. Methods: Currently 12 countries are participating in the International Physical Activity and the Environment Network (IPEN) study. The IPEN Adult study design involves recruiting adult participants from neighborhoods with wide variations in environmental walkability attributes and socioeconomic status (SES). Results: Eleven of twelve countries are providing accelerometer data and 11 are providing GIS data. Current projections indicate that 14,119 participants will provide survey data on built environments and physical activity and 7145 are likely to provide objective data on both the independent and dependent variables. Though studies are highly comparable, some adaptations are required based on the local context. Conclusions: This study was designed to inform evidence-based international and country-specific physical activity policies and interventions to help prevent obesity and other chronic diseases that are high in developed countries and growing rapidly in developing countries.

Estimating nephron number in the developing kidney using the physical disector/fractionator combination

Over the last decade the development of new molecular biology tools, advanced microscopy, live imaging and systems biology approaches have revolutionized our conception of how embryonic development proceeds. One fundamental aspect of development biology is the concept of morphogenesis: understanding how a group of multipotent cells organize and differentiate into a complex organ. In Kidney Development: Methods and Protocols, expert researchers in the field detail different approaches to tackle kidney development. These approaches include culture and live imaging aspects of kidney development, analyzing the 3-dimensional aspects of branching morphogenesis as well as nephrogenesis, manipulation of the gene/protein expression during kidney development as well as in the adult kidney, and how to assess kidney malformation and disease. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Kidney Development: Methods and Protocols seeks to aid scientists in the further study of the process of morphogenesis which is fundamental important not only for studying developmental biology but also for regenerative medicine.

Isolation breeds naivety : island living robs Australian varanid lizards of toad-toxin immunity via four-base-pair mutation

Since their introduction to the toad-free Australian continent cane toads (Bufo marinus) have caused a dramatic increase in naïve varanid mortality when these large lizards attempt to feed on this toxic amphibian. In contrast Asian–African varanids, which have coevolved with toads, are resistant to toad toxin. Toad toxins, such as Bufalin target the H1-H2 domain of the α1 subunit of the sodium-potassium-ATPase enzyme. Sequencing of this domain revealed identical nucleotide sequences in four Asian as well as in three African varanids, and identical sequences in all 11 Australian varanids. However, compared to the Asian–African varanids, the Australian varanids showed four-base-pair substitutions, resulting in the alteration in three of the 12 amino acids representing the H1-H2 domain. The phenotypic effect of the substitutions was investigated in human embryonic kidney (HEK) 293 cells stably transfected with the Australian and the Asian–African H1-H2 domains. The transfections resulted in an approximate 3000-fold reduction in resistance to Bufalin in the Australian HEK293 cells compared to the Asian–African HEK293 cells, demonstrating the critical role of this minor mutation in providing Bufalin resistance. Our study hence presents a clear link between genotype and phenotype, a critical step in understanding the evolution of phenotypic diversity.

A design-based method for estimating glomerular number in the developing kidney

Low glomerular (nephron) endowment has been associated with an increased risk of cardiovascular and renal disease in adulthood. Nephron endowment in humans is determined by 36 wk of gestation, while in rats and mice nephrogenesis ends several days after birth. Specific genes and environmental perturbations have been shown to regulate nephron endowment. Until now, design-based method for estimating nephron number in developing kidneys was unavailable. This was due in part to the difficulty associated with unambiguously identifying developing glomeruli in histological sections. Here, we describe a method that uses lectin histochemistry to identify developing glomeruli and the physical disector/fractionator principle to provide unbiased estimates of total glomerular number (Nglom). We have characterized Nglom throughout development in kidneys from 76 rats and model this development with a 5-parameter logistic equation to predict Nglom from embryonic day 17.25 to adulthood (r2 = 0.98). This approach represents the first design-based method with which to estimate Nglom in the developing kidney.