A fast and simplein situ method for the determination of the absolute configuration of amino acid esters using the chiroptical properties of their Eu(FOD)3 complexes

Summary The NMR shift reagent, Europium(III)-tris-(1,1,1,2,2,3,3)-heptafluoro-7,7-dimethyl-4-6-octanedione [Eu(fod)₃], complexes efficiently with-amino acid esters in chloroform. These complexes exhibit characteristic circular dichroism (CD) spectral patterns in the 350-250 nm region. A fast and simple procedure (also in microscale) has been worked out which utilizes the signs of these CD bands for the determination of the absolute configuration at the-carbon atomin situ. In the L-series, a positive CD band is observed at around 310 nm and a negative one in the 290-280 nm region. The CD spectra of the Eu complexes of the D-isomers are mirror images of those of the L-configurations. An empirical rule is proposed.Presented in part at the 2nd International Congress on Amino Acids and Analogues, Vienna, Austria, August 5–9, 1991.     

Critical importance of microsome concentration in mutagenesis assay with V79 Chinese hamster cells.

For optimum mutagensis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1-5%) concentrations of the liver 15 000 X g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the S15 concentrations of more than 10%. Similarly, aflatoxin B1 induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic aflatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased continuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the S15. In contrast with these compounds, N-nitrosodimethylamine induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the lipid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.     

Resistance to tolerance induction in B cells of autoimmune mice—Abnormal expansion of low affinity IgG-producing B-cell population

Several strains of mice are known to develop spontaneous autoimmune diseases like lupus erythematosus and they show various immunological abnormalities as well. Despite different genetic backgrounds, they manifest various immunological abnormalities in common, e.g., polyclonal B-cell activation (PBA) and resistance to tolerance induction. To elucidate mechanisms of the development of autoimmunity, tolerance inducibility was examined in autoimmune and normal mice using trinitrophenylated carboxymethyl cellulose (TNP-CMC) as tolerogen which is known to induce TNP-specific B-cell tolerance without the participation of T cells. NZB and MRL/Mp-lpr/lpr mice were used as autoimmune mice and C57BL/6, BALB/c, and MRL/Mp-+/+ mice as nonautoimmune mice. When TNP-CMC-injected mice were challenged with T-independent antigens, all of the mice tested were shown to be tolerant. In contrast, when TNP-CMC-injected mice were challenged with T-dependent antigen and secondary IgG responses were assessed, autoimmune mice showed rather hyperreactivity, while nonautoimmune mice showed hyporesponsiveness. Cyclophosphamide improved this defective tolerance inducibility. By the solid-phase radioimmunoassay it was revealed that average affinity of serum anti-TNP antibodies produced in TNP-CMC-injected mice was low. Such low affinity antibodies were produced in large amount in autoimmune mice. Hence, it was suggested that B-cell clones destined to produce low affinity IgG antibodies were responsible for the resistance to tolerance induction and such clones were expanding in autoimmune mice.     

Detecting seasonal variation of antifreeze protein distribution in Rhagium mordax using immunofluorescence and high resolution microscopy

Larvae of the blackspotted pliers support beetle, Rhagium mordax, were collected monthly, for the duration of 2012 and fixed. The larvae were embedded in paraffin wax and sectioned. Using fluorophore-coupled antibodies specific to the R. mordax antifreeze protein, RmAFP1, sections were visualised with UV reflected light microscopy. An automated software analysis method was developed in order to discard autofluorescence, and quantify fluorescence from bound antibodies. The results show that R. mordax cuticle and gut exhibit a higher degree of fluorophore-bound fluorescence during summer, than in the cold months. It is hypothesised that R. mordax stores RmAFP1 in, or near, the fat body during times when freeze avoidance is not needed.     

Interstrain crosses enhance excision of Tc1 transposable elements in Caenorhabditis elegans

Summary We have studied spontaneous and UV mutagenesis of the glyU gene in Escherichia coli trpA461 (GAG) strains carrying the pIP11 plasmid, in which the dnaQ gene encoding the 3–5 exonuclease subunit (epsilon) of DNA polymerase III is fused to the tac(trp-lac) promoter. We have used a pair of M13glyU phage in which the gene encoding the glycyl-tRNA is cloned in opposite orientations, consequently the phage present either GGG or CCC anticodon triplets for mutagenesis. The presence of IPTG, the inducer of the tac-dnaQ fusion, results in about 100-fold decrease in frequency of spontaneous Su⁺ (GAG) mutations arising in the CCC phage. The enhanced expression of tac-dnaQ reduces 10-fold the frequency of UV-induced Su⁺ (GAG) mutations in the CCC phage and nearly completely prevents generation by UV of Su⁺ (GAG) mutations in the GGG phage, in which UV-induced pyrimidine photoproducts can be formed only in the vicinity of the target triplet. These results suggest that both locally and regionally targeted mutagenesis is affected by overproduction of the epsilon subunit. By delayed photoreversal mutagenesis we have shown that UV-induced chromosomal mutagenesis of the umuC36 trpA461 strain harboring pIP11 is completely abolished in the presence of IPTG. This result seems to indicate that the misinocorporation step of DNA translesion synthesis is affected by excess of the epsilon subunit. Finally, we have introduced the pIP13 plasmid carrying the dnaQ gene into the recA1207 strain, which is deficient in the recombinase activity of RecA but constitutive in the protease activity. We demonstrate that the transformant shows much higher UV sensitivity than recA1207 carrying the vector plasmid pBR325, indicating that translesion synthesis significantly contributes to DNA repair capacity of cells deficient in recombination.     

Organ-specific difference in the sugar chains of gamma-glutamyltranspeptidase

Sugar chains of gamma-glutamyltranspeptidase purified from neonatal mouse liver and adult mouse kidney were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. A comparative study of the structures of the oligosaccharides has revealed that the GlcNAc beta 1 leads to 4Man beta 1 leads to group is found in the sugar chains of kidney enzyme but not in those of liver enzyme. This is considered as an organ-specific difference common to mammals because the same phenomenon was found in bovine and rat enzymes.     

Characterization of a novel mutant KCNQ1 channel subunit lacking a large part of the C-terminal domain

A mutation of KCNQ1 gene encoding the alpha subunit of the channel mediating the slow delayed rectifier K⁺ current in cardiomyocytes may cause severe arrhythmic disorders. We identified KCNQ1(Y461X), a novel mutant gene encoding KCNQ1 subunit whose C-terminal domain is truncated at tyrosine 461 from a man with a mild QT interval prolongation. We made whole-cell voltage-clamp recordings from HEK-293T cells transfected with either of wild-type KCNQ1 [KCNQ1(WT)], KCNQ1(Y461X), or their mixture plus KCNE1 auxiliary subunit gene. The KCNQ1(Y461X)-transfected cells showed no delayed rectifying current. The cells transfected with both KCNQ1(WT) and KCNQ1(Y461X) showed the delayed rectifying current that is thought to be mediated largely by homomeric channel consisting of KCNQ1(WT) subunit because its voltage-dependence of activation, activation rate, and deactivation rate were similar to the current in the KCNQ1(WT)-transfected cells. The immunoblots of HEK-293T cell-derived lysates showed that KCNQ1(Y461X) subunit cannot form channel tetramers by itself or with KCNQ1(WT) subunit. Moreover, immunocytochemical analysis in HEK-293T cells showed that the surface expression level of KCNQ1(Y461X) subunit was very low with or without KCNQ1(WT) subunit. These findings suggest that the massive loss of the C-terminal domain of KCNQ1 subunit impairs the assembly, trafficking, and function of the mutant subunit-containing channels, whereas the mutant subunit does not interfere with the functional expression of the homomeric wild-type channel. Therefore, the homozygous but not heterozygous inheritance of KCNQ1(Y461X) might cause major arrhythmic disorders. This study provides a new insight into the structure–function relation of KCNQ1 channel and treatments of cardiac channelopathies.     

Alkalization of larval mosquito midgut and the role of carbonic anhydrase in different species of mosquitoes

We have previously demonstrated the involvement of carbonic anhydrase (CA) in the alkalization mechanism of the Aedes aegypti larval midgut. In this study, we used Hansson's histochemistry to examine the distribution of the enzyme in the midgut of six different species of mosquito larvae (Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex nigripalpus, Ochlerotatus taeniorhynchus, Anopheles quadrimaculatus). Additionally, we quantitated CA content in the gastric caeca, anterior and posterior midgut of fourth instar larvae from these species using the 18O isotope exchange method coupled to mass spectrometry. We also tested the effect of CA inhibitors such as methazolamide and acetazolamide in the alkalization of the midgut for these species. Our results indicate that CA is present in the larval midgut of the species studied and that it appears to be associated with the posterior midgut and gastric caeca in some species and with the anterior midgut in others. CA inhibitors appear to have a profound effect on the alkalization mechanism of the midgut with lethal consequences for most of the species tested.     

Crystal structure of the temperature-sensitive and allosteric-defective chaperonin GroELE461K

The chaperonin GroEL adopts a double-ring structure with various modes of allosteric communication. The simultaneous positive intra-ring and negative inter-ring co-operativities alternate the functionality of the folding cavities in both protein rings. Negative inter-ring co-operativity is maintained through different inter-ring interactions, including a salt bridge involving Glu 461. Replacement of this residue by Lys modifies the temperature sensitivity of the substrate-folding activity of this protein, most likely as a result of the loss of inter-ring co-operativity. The crystal structure of the mutant chaperonin GroELE461K has been determined at 3.3A and compared with other structures: the wild-type GroEL, an allosteric defective GroEL double mutant and the GroEL-GroES-(ADP)7 complex. The inter-ring region of the mutant exhibits the following characteristics: (i) no salt-bridge stabilizes the inter-ring interface; (ii) the mutated residue plays a central role in defining the relative ring rotation (of about 22 degrees) around the 7-fold axis; (iii) an increase in the inter-ring distance and solvent accessibility of the inter-ring interface; and (iv) a 2-fold reduction in the stabilization energy of the inter-ring interface, due to the modification of inter-ring interactions. These characteristics explain how the thermal sensitivity of the protein's fundamental properties permits GroEL to distinguish physiological (37 degrees C) from stress (42 degrees C) temperatures.