1,3-Dipolar cycloaddition to the FeSC fragment 20. Preparation and properties of carbonyliron complexes of di-thiooxamide. Reactivity of the mononuclear (di-thiooxamide)Fe(CO)3 towards dimethyl acetylenedicarboxylate

Reaction of Fe₂(CO)₉ at room temperature in THF with the di-thiooxamides (L), SC{N(R,R′)}C{(R,R′)N}S [R=Me, R′-R′=(CH₂)₂ (a); R=H, R′=ⁱPr (b); R=H, R′=iPr (c), R=H, R′=benzyl (d); R=H, R′=H (e)], results for ligands a-d initially in the formation of the mononuclear σ-S, σ-S′ chelate complexes Fe(CO)₃(L) (7a-d), which could be isolated in case of 7a and 7d. Under the reaction conditions, complexes 7a-d react further with [Fe(CO)₄] fragments to give three types of Fe₂(CO)₆(L) complexes (8a-d) in high yields, depending on the di-thiooxamide ligand used together with traces of the known complex S₂Fe₃(CO)₉ (14). The molecular structures of these complexes have been established by the single crystal X-ray diffraction determinations of 8a, 8b and 8d. In the reaction with ligand e the corresponding complex 7e was not detected and the well-known complexes 14 and S₂Fe₃(CO)₉ (15) were isolated in low yield. In situ prepared 7a reacts in a slow reaction with 1 equiv. of dimethyl acetylene dicarboxylate in a 1,3-dipolar cycloaddition reaction to give the stable initial ferra [2.2.1] bicyclic complex 10a in 60% yield. In complex 10a an additional Fe(CO)₄ fragment is coordinated to the sulfido sulfur atom of the cycloadded FeSC fragment. When a toluene solution of 10a is heated to 50 °C it loses two terminal CO ligands to give the binuclear FeFe bonded complex 11a in almost quantitative yield. The molecular structures of 10a and 11a have been confirmed by single crystal X-ray diffraction. Reaction of 7d at room temperature with 2 equiv. of dimethyl acetylene dicarboxylate results in the mononuclear complex 12d in 5% yield. The molecular structure of 12b has been established by single crystal X-ray diffraction and comprises a tetra dentate ligand with two ferra-sulpha cyclobutene, and a ferra-disulpha cyclopentene moiety. When the reaction is performed at 60 °C a low yield of 2,3,4,5-thiophene tetramethyl tertracarboxylate is obtained besides complex 12d.     

Analysis of phytochrome-deficient yellow-green-2 and aurea mutants of tomato

Two alleles of the yellow-green-2 ( yg-2) and eight different alleles of the aurea ( au ) locus of tomato ( Lycopersicon esculentum Mill.) were compared. All are characterized by a paler green colour compared with wild-type (WT), an elongated hypocotyl in red light, and low or below detection limits of spectrophotometrically active phytochrome. Hypocotyl length was variable in white light, ranging from that of WT to more elongated. Immunochemical analysis revealed that etiolated seedlings of the yg-2 mutant have approximately 25% of the WT level of phytochrome A protein (PHYA), whereas that of phytochrome B protein (PHYB) is normal. In this it resembles the au mutant. The au,yg-2 double mutant has a more extreme chlorophyll deficiency than either parent. Since the yg-2 and au mutants have a less severe phenotype at the adult stage, that is, are leaky, the additive effect can be explained by assuming that the mutants control two steps in the chromophore biosynthesis pathway. Combination, by crossing, of the yg-2 and au mutants with a transgenic tomato line that overexpresses oat phytochrome A3 (PhyA-3) essentially failed to restore the WT phenotype under white fluorescent light conditions, although under greenhouse conditions some evidence for increased sensitivity to light was observed. Immunochemically, oat PHYA-3 protein is detectable in both the yg-2,PhyA-3 and au,PhyA-3 'double' mutants. Spectrophotometrical analysis, however, revealed that holophytochrome was undetectable in the yg-2,PhyA-3 and au,PhyA-3 'double' mutants. These results are compatible with both mutants being disturbed in phytochrome chromophore biosynthesis.     

Quirks of dye nomenclature. 1. Evans blue

The history, origin, identity, chemistry and use of Evans blue dye are described along with the first application to staining by Herbert McLean Evans in 1914. In the 1930s, the dye was marketed under the name, Evans blue dye, which was profoundly more acceptable than the ponderous chemical name.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

A novel functional class I lineage in zebrafish (Danio rerio), carp (Cyprinus carpio), and large barbus (Barbus intermedius) showing an unusual conservation of the peptide binding domains

Species from all major jawed vertebrate taxa possess linked polymorphic class I and II genes located in an MHC. The bony fish are exceptional with class I and II genes located on different linkage groups. Zebrafish (Danio rerio), common carp (Cyprinus carpio), and barbus (Barbus intermedius) represent highly divergent cyprinid genera. The genera Danio and Cyprinus diverged 50 million years ago, while Cyprinus and Barbus separated 30 million years ago. In this study, we report the first complete protein-coding class I ZE lineage cDNA sequences with high similarity between the three cyprinid species. Two unique complete protein-coding cDNA sequences were isolated in zebrafish, Dare-ZE*0101 and Dare-ZE*0102, one in common carp, Cyca-ZE*0101, and six in barbus, Bain-ZE*0101, Bain-ZE*0102, Bain-ZE*0201, Bain-ZE*0301, Bain-ZE*0401, and Bain-ZE*0402. Deduced amino acid sequences indicate that these sequences encode bonafide class I proteins. In addition, the presence of conserved potential peptide anchoring residues, exon-intron organization, ubiquitous expression, and polymorphism generated by positive selection on putative peptide binding residues support a classical nature of class I ZE lineage genes. Phylogenetic analyses revealed clustering of the ZE lineage clade with nonclassical cyprinid class I Z lineage clade away from classical cyprinid class I genes, suggesting a common ancestor of these nonclassical genes as observed for mammalian class I genes. Data strongly support the classical nature of these ZE lineage genes that evolved in a trans-species fashion with lineages being maintained for up to 100 million years as estimated by divergence time calculations.     

Understanding the chemistry of the artificial electron acceptors PES,PMS, DCPIP and Wurster’s Blue in methanol dehydrogenase assays

JBIC Journal of Biological Inorganic Chemistry - Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize...     

Enhanced Performance of the Eurostat Method for Comprehensive Assessment of Urban Metabolism: A Material Flow Analysis of Amsterdam

Sustainable urban resource management depends essentially on a sound understanding of a city's resource flows. One established method for analyzing the urban metabolism (UM) is the Eurostat material flow analysis (MFA). However, for a comprehensive assessment of the UM, this method has its limitations. It does not account for all relevant resource flows, such as locally sourced resources, and it does not differentiate between flows that are associated with the city's resource consumption and resources that only pass through the city. This research sought to gain insights into the UM of Amsterdam by performing an MFA employing the Eurostat method. Modifications to that method were made to enhance its performance for comprehensive UM analyses. A case study of Amsterdam for the year 2012 was conducted and the results of the Eurostat and the modified Eurostat method were compared. The results show that Amsterdam's metabolism is dominated by water flows and by port‐related throughput of fossil fuels. The modified Eurostat method provides a deeper understanding of the UM than the urban Eurostat MFA attributed to three major benefits of the proposed modifications. First, the MFA presents a more complete image of the flows in the UM. Second, the modified resource classification presents findings in more detail. Third, explicating throughput flows yields a much‐improved insight into the nature of a city's imports, exports, and stock. Overall, these advancements provide a deeper understanding of the UM and make the MFA method more useful for sustainable urban resource management.     

ALS/FTD‐associated FUS activates GSK‐3β to disrupt the VAPB–PTPIP51 interaction and ER–mitochondria associations

Defective FUS metabolism is strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), but the mechanisms linking FUS to disease are not properly understood. However, many of the functions disrupted in ALS/FTD are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling is facilitated by close physical associations between the two organelles that are mediated by binding of the integral ER protein VAPB to the outer mitochondrial membrane protein PTPIP51, which act as molecular scaffolds to tether the two organelles. Here, we show that FUS disrupts the VAPB–PTPIP51 interaction and ER–mitochondria associations. These disruptions are accompanied by perturbation of Ca²⁺ uptake by mitochondria following its release from ER stores, which is a physiological read‐out of ER–mitochondria contacts. We also demonstrate that mitochondrial ATP production is impaired in FUS‐expressing cells; mitochondrial ATP production is linked to Ca²⁺ levels. Finally, we demonstrate that the FUS‐induced reductions to ER–mitochondria associations and are linked to activation of glycogen synthase kinase‐3β (GSK‐3β), a kinase already strongly associated with ALS/FTD.     

Lidocaine block of cardiac sodium channels

Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 μM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 μM, at a negative holding potential where inactivation was completely removed, to approximately 10 μM, at a depolarized holding potential where inactivation was nearly complete. Lidocaine block showed prominent use dependence with trains of depolarizing pulses from a negative holding potential. During the interval between pulses, repriming of I (Na) displayed two exponential components, a normally recovering component (τless than 0.2 s), and a lidocaine-induced, slowly recovering fraction (τ approximately 1-2 s at pH 7.0). Raising the lidocaine concentration magnified the slowly recovering fraction without changing its time course; after a long depolarization, this fraction was one-half at approximately 10 μM lidocaine, just as expected if it corresponded to drug-bound, inactivated channels. At less than or equal to 20 μM lidocaine, the slowly recovering fraction grew exponentially to a steady level as the preceding depolarization was prolonged; the time course was the same for strong or weak depolarizations, that is, with or without significant activation of I(Na). This argues that use dependence at therapeutic levels reflects block of inactivated channels, rather than block of open channels. Overall, these results provide direct evidence for the “modulated-receptor hypothesis” of Hille (1977) and Hondeghem and Katzung (1977). Unlike tetrodotoxin, lidocaine shows similar interactions with Na channels of heart, nerve, and skeletal muscle.     

Glycogenosis type II: protein and DNA analysis in five South African families from various ethnic origins.

The molecular nature of lysosomal alpha-glucosidase deficiency was studied in five South African families with glycogenosis type II. Distinct ethnic origins were represented. Two new mutant acid alpha-glucosidase alleles were discovered. In two infantile patients from a consanguineous Indian family we found for the first time an acid alpha-glucosidase precursor of reduced size. The mutant precursor appeared normally glycosylated and phosphorylated but was not processed to mature enzyme. Abnormalities of the mRNA were not obvious, but digestion of genomic DNA with HindIII, BglII, and StuI revealed for each enzyme a fragment of increased length. Heterozygosity was demonstrated in the parents. Complete lack of acid alpha-glucosidase mRNA, as well as deficiency of precursor synthesis, was observed in two black baby girls from unrelated families. In these cases the length of all restriction-enzyme fragments was normal. Reduced enzyme synthesis but normal processing was registered in juvenile and young adult Cape colored patients. The extensive heterogeneity of glycogenosis type II is emphasized in these studies on various ethnic groups. The newly discovered mutants are valuable for the understanding of clinical diversity as a result of allelic variation.