Structural analysis of three His32 mutants of DsbA: Support for an electrostatic role of His32 in DsbA stability

DsbA, a 21-kDa protein from Escherichia coli, is a potent oxidizing disulfide catalyst required for disulfide bond formation in secreted proteins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and includes a reversible disulfide bond formed from cysteines separated by two residues (Cys3O-Pro31-His32-Cys33). Unlike most protein disulfides, the active-site disulfide of DsbA is highly reactive and the oxidized form of DsbA is much less stable than the reduced form at physiological pH. His32, one of the two residues between the active-site cysteines, is critical to the oxidizing power of DsbA and to the relative instability of the protein in the oxidized form. Mutation of this single residue to tyrosine, serine, or leucine results in a significant increase in stability (of similar to 5-7 kcal/mol) of the oxidized His32 variants relative to the oxidized wild-type protein. Despite the dramatic changes in stability, the structures of all three oxidized DsbA His32 Variants are very similar to the wild-type oxidized structure, including conservation of solvent atoms near the active-site residue, Cys3O. These results show that the His32 residue does not exert a conformational effect on the structure of DsbA. The destabilizing effect of His32 on oxidized DsbA is therefore most likely electrostatic in nature.

Structural characterisation of galactoglucomannan secreted by suspension-cultured cells of Nicotiana plumbaginifolia

Galactoglucomannan (GGM) from cultures of Nicotiana plumbaginifolia has Man:Glc:Gal:Ara:Xyl in 1.0:1.1:1.0:0.1:0.04 ratio. Linkage analysis contained 4- and 4,6-Manp, 4-Glcp, terminal Galp and 2-Galp, small amounts and terminal Arap and terminal Xylp, and similar to 0.03 mol acetyl per mol of glucosyl residue. Treatment with alpha- and beta-D-galactosidases showed that the majority of the side-chains were either single Galp-alpha-(1 --> residues or the disaccharide Galp-beta-(1 --> 2)-Galp-alpha-(1 --> linked to O-6 of the 4-Manp residues of the glucomannan backbone. Analysis of the oligosaccharides generated by endo-(1 --> 4)-beta-mannanase digestion confirmed that the GGM comprises a backbone of predominantly alternating --> 4)-D-Manp-beta-(1 --> and --> Lt)-D-Glcp-beta-(1 --> branched at O-6 of 65% of the 4-Manp residues. The major oligosaccharide identified was D-Glcp-beta-(1 --> 4)-[D-Galp-beta-(1 --> 2)-D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> 4)-D-Glcp-beta-(I --> 4)-[D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> (27%), and most of the other oligosaccharides produced in significant quantities were based on this structure. (C) 1997 Elsevier Science Ltd.

On single laws for varieties of quasigroups associated with extended cycle systems

It has been previously shown by Lindner and Rodger that quasigroups associated with 2-perfect extended m-cycle systems can be equationally defined if and only if m is an element of {3, 5, 7}. In this paper we present a single identity for each such m which is equivalent to the identities given for these varieties.

Projections from the substantia nigra pars reticulata to the motor thalamus of the rat: Single axon reconstructions and immunohistochemical study

This is a study in the rat of the distribution of specific neurotransmitters in neurones projecting from the substantia nigra reticulata (SNR) to the ventrolateral (VL) and ventromedial (VM) thalamic nuclei. Individual axons projecting from the SNR to these thalamic nuclei have also been reconstructed following small injection of the anterograde tracer dextran biotin into the the SNR. Analysis of reconstructions revealed two populations of SNR neurones projecting onto the VL and VM thalamic nuclei. One group projects directly onto the VM and VL, and the other projects to the VM/VL and to the parafascicular nucleus. In another set of experiments Fluoro-Gold was injected into the VL/VM to label SNR projection neurones retrogradely, and immunohistochemistry was performed to determine the distribution of choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), gamma -aminobutyric acid (GABA), and glutamate in Fluoro-Gold-labelled SNR projection neurones. Most SNR-VL/VM thalamic projection neurones were immunoreactive to acetylcholine or glutamate, whereas only 25% of the projection neurones were found to be immunoreactive to GABA. (C) 2001 Wiley-Liss, Inc.

Tyrosine residue 271 of the norepinephrine transporter is an important determinant of its pharmacology

The aim was to examine the functional importance in the norepinephrine transporter (NET) of (i) the phenylalanine residue at position 531 in transmembrane domain (TMD) 11 by mutating it to tyrosine in the rat (rF531Y) and human (hF531Y) NETs and (ii) the highly conserved tyrosine residues at positions 249 in TMD 4 of human NET (hNET) (mutated to alanine: hY249A) and 271 in TMD 5, by mutating to alanine (hY271A), phenylalanine (hY271F) and histidine (hY271H). The effects of the mutations on NET function were for uptake of the substrates, examined by expressing the mutant and wildtype NETs in COS-7 cells and measuring the K-m and V-max for uptake of the substrates, [H-3]norepinephrine, [H-3]MPP+ and [H-3]dopamine, the K-D and B-max for [H-3]nisoxetine binding and the K-i of the inhibitors, nisoxetine, desipramine and cocaine, for inhibition of [H-3]norepinephrine uptake. The K-m values of the substrates were lower for the mutants at amino acid 271 than hNET and unaffected for the other mutants, and each mutant had a significantly lower than NET for substrate uptake. The mutations at position 271 caused an increase in the K-i or K-D values of nisoxetine, desipramine and cocaine, but there were no effects for the other mutations. Hence, the 271 tyrosine residue in TMD 5 is an important determinant of NET function, with the mutants showing an increase in the apparent affinities of substrates and a decrease in the apparent affinities of inhibitors, but the 249 tyrosine and 531 phenylalanine residues do not have a major role in determining NET function. (C) 2001 Elsevier Science B.V. All rights reserved.

A single nucleotide polymorphism in the 5' untranslated region of RAD51 and risk of cancer among BRCA1/2 mutation carriers

RAD51 colocalizes with both BRCA1 and BRCA2, and genetic variants in RAD51 would be candidate BRCA1/2 modifiers. We searched for RAD51 polymorphisms by sequencing 20 individuals. We compared the polymorphism allele frequencies between female BRCA1/2 mutation carriers with and without breast or ovarian cancer and between population-based ovarian cancer cases with BRCA1/2 mutations to cases and controls without mutations. We discovered two single nucleotide polymorphisms (SNPs) at positions 135 g-->c and 172 g-->t of the 5' untranslated region. In an initial group of BRCA1/2 mutation carriers, 14 (21%) of 67 breast cancer cases carried a c allele at RAD51:135 g-->c, whereas 8 (7%) of 119 women without breast cancer carried this allele. In a second set of 466 mutation carriers from three centers, the association of RAD51:135 g-->c with breast cancer risk was not confirmed. Analyses restricted to the 216 BRCA2 mutation carriers, however, showed a statistically significant association of the 135 c allele with the risk of breast cancer (adjusted odds ratio, 3.2; 95% confidence limit, 1.4-40). BRCA1/2 mutation carriers with ovarian cancer were only about one half as likely to carry the RAD51:135 g-->c SNP. Analysis of the RAD51:135 g-->c SNP in 738 subjects from an Israeli ovarian cancer case-control study was consistent with a lower risk of ovarian cancer among BRCA1/2 mutation carriers with the c allele. We have identified a RAD51 5' untranslated region SNP that may be associated with an increased risk of breast cancer and a lower risk of ovarian cancer among BRCA2 mutation carriers. The biochemical basis of this risk modifier is currently unknown.

Single-electron measurements with a micromechanical resonator

A mechanical electroscope based on a change in the resonant frequency of a cantilever one micron in size in the presence of charge has recently been fabricated. We derive the decoherence rate of a charge superposition during measurement with such a device using a master equation theory adapted from quantum optics. We also investigate the information produced by such a measurement, using a quantum trajectory approach. Such instruments could be used in mesoscopic electronic systems, and future solid-state quantum computers, so it is useful to know how they behave when used to measure quantum superpositions of charge.