Regulation by SREBP-2 defines a potential link between isoprenoid and adenosylcobalamin metabolism

Mevalonate kinase (MVK) catalyses an early step in cholesterol biosynthesis converting mevalonate to phosphomevalonate. Cob(I)alamin adenosyltransferase (MMAB) converts cob(I)alamin to adenosylcobalamin, functionally required for mitochondrial methylmalonyl-CoA mutase activity and succinyl-CoA formation. These two synthenic genes are found in a head-to-head formation on chromosome 12 in man and chromosome 5 in mouse. The 330bp intergenic region showed several conserved NF-Y sites indicative of potential bidirectional regulatory SREBP synergism. Both MVK and MMAB appear to be regulated in a similar manner, to a large extent by SREBP-2, since their tissue expression pattern was similar and both genes were suppressed by an excess of cholesterol as well as SREBP-2 knockdown. Statin treatment in mice upregulated both Mvk and Mmab mRNA levels indicating that this treatment may be useful in inborn errors of cblB complementation associated with methylmalonic aciduria as well as hyper IgD and periodic fever syndrome (HIDS).     

Properties of spin and fluorescent labels at a receptor-ligand interface.

Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently attached to an engineered cysteine in position 140 in sTF, a position normally occupied by a Phe residue previously characterized as an important contributor to the sTF:FVIIa interaction. Two spin labels, IPSL [N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide] and MTSSL [(1-oxyl-2,2,5, 5-tetramethylpyrroline-3-methyl)methanethiosulfonate], and two fluorescent labels, IAEDANS [5-((((2-iodoacetyl)amino) ethyl)amino)naphthalene-1-sulfonic acid] and BADAN [6-bromoacetyl-2-dimethylaminonaphthalene], were used. Spectral data from electron paramagnetic resonance (EPR) and fluorescence spectroscopy showed a substantial change in the local environment of all labels when the sTF:FVIIa complex was formed. However, the interaction was probed differently by each label and these differences in spectral appearance could be attributed to differences in label properties such as size, polarity, and/or flexibility. Accordingly, molecular modeling data suggest that the most favorable orientations are unique for each label. Furthermore, line-shape simulations of EPR spectra and calculations based on fluorescence depolarization measurements provided additional details of the local environment of the labels, thereby confirming a tight protein-protein interaction between FVIIa and sTF when the complex is formed. The tightness of this local interaction is similar to that seen in the interior of globular proteins.     

Reidentification of the female sex pheromone of the Indian meal moth, Plodia interpunctella: evidence for a four-component pheromone blend

Pheromone gland extracts from calling female Plodia interpunctella contained at least seven compounds that consistently elicited electroantennographic responses from male antennae upon gas chromatographic analysis. Three of these compounds were found to be the previously identified gland constituents, i.e., (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:OAc), (Z,E)-9,12-tetradecadienal (Z9,E12-14:Ald) and (Z,E)-9,12-tetradecadienol (Z9,E12-14:OH). A fourth EAD-active compound was identified as (Z)-9-tetradecenyl acetate (Z9-14:OAc). The homologue (Z)-11-hexadecenyl acetate (Z11-16:OAc) was also identified in the extracts, but showed no EAD activity. The identity of all five compounds was confirmed by comparison of GC retention times and mass spectra with those of synthetic standards. In flight tunnel tests there were no significant differences in response of male P. interpunctella to the bait containing all four EAD-active compounds and the responses to female gland extacts. A behavioural assay of different two-compound blends in the flight tunnel showed that only addition of the corresponding aldehyde to the major pheromone component Z9,E12-14:OAc raised the male response. A subtractive assay, however, revealed that the exclusion of any of the compounds from the complete four-compound blend reduced its activity significantly. We thus conclude that the female-produced sex pheromone of P. interpunctella consists of at least four components, i.e., Z9,E12-14:OAc, Z9,E12-14:Ald, Z9,E12-14:OH and Z9-14:OAc.In a field trapping test performed in a storage facility, the four-component blend attracted significantly more males of P. interpunctella than traps baited with Z9,E12-14:OAc alone. In contrast, the highest number of Ephestia kuehniella males was found in the traps baited with this major component, suggesting that the secondary pheromone components contribute to the species specificity of the blend.     

X-ray absorption spectroscopic analysis of Fe(II) and Cu(II) forms of a herbicide-degrading α-ketoglutarate dioxygenase

 The first step in the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by Ralstonia eutropha JMP134 is catalyzed by the α-ketoglutarate (α-KG)-dependent dioxygenase TfdA. Previously, EPR and ESEEM studies on inactive Cu(II)-substituted TfdA suggested a mixture of nitrogen/oxygen coordination with two imidazole-like ligands. Differences between the spectra for Cu TfdA and α-KG- and 2,4-D-treated samples were interpreted as a rearrangement of the g–tensor principal axis system. Herein, we report the use of X-ray absorption spectroscopy (XAS) to further characterize the metal coordination environment of Cu TfdA as well as that in the active, wild-type Fe(II) enzyme. The EXAFS data are interpreted in terms of four N/O ligands (two imidazole-like) in the Cu TfdA sample and six N/O ligands (one or two imidazole-like) in the Fe TfdA sample. Addition of α-KG results in no significant structural change in coordination for Cu or Fe TfdA. However, addition of 2,4-D results in a decrease in the number of imidazole ligands in both Cu and Fe TfdA. Since this change is seen both in the Fe and Cu EXAFS, loss of one histidine ligand upon 2,4-D addition best describes the phenomenon. These XAS data clearly demonstrate that changes occur in the atomic environment of the metallocenter upon substrate binding. Received: 3 July 1998 / Accepted: 13 October 1998     

Serum Antibodies to Bovine Coronavirus in Swedish Sheep

Altogether 218 sheep sera from 40 flocks in different parts of Sweden were screened for antibodies to bovine Coronavirus (BCV). Nineteen per cent of the sera were positive and there was a significantly higher frequency (p<0.05) of at least one positive sample in flocks with more than 100 adult sheep than in smaller flocks. There was also a significantly higher frequency (p<0.001) of positive samples from sheep older than 4 years than from younger ones. Only a weak relationship between BCV positivity (2 or more positive samples, p<0.05) and cattle contact was demonstrated in this study. Possible transmission routes and other factors that could have affected the result are discussed. In light of our finding that all 5 sheep experimentally exposed to BCV through contact with infectious cow faeces seroconverted, we conclude that the antibodies found in Swedish sheep are probably the result of BCV infections directly or indirectly transmitted from cattle.     

Improved quantitation of DNA curvature using ligation ladders.

It is often desirable to estimate accurately the local shape of DNA molecules. Such measurements are useful in understanding the intrinsic contribution of DNA sequence to curvature, as well as in assessing the effects of chemical modifications. We have been investigating the effects of asymmetric phosphate neutralization on DNA shape using the well-characterized ligation ladder approach developed by Crothers and co-workers [D.M.Crothers and J.Drak (1992) Meth. Enzymol.,212, 46-71]. This technique is remarkably sensitive to differences in DNA shape. We now report a general quantitative assay of DNA curvature that we have validated using a set of phased A(5)tract standards. This approach allows simultaneous estimation of helix axis deflection magnitude and direction when a test sequence is monitored in at least three phasings relative to a reference A(5-6)tract in short DNA duplexes. Analysis using this improved approach confirms our published data on DNA curvature due to electrostatic effects.     

The Impact of Genetic Removal of GFAP and/or Vimentin on Glutamine Levels and Transport of Glucose and Ascorbate in Astrocytes

The importance of the intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP) and vimentin for astrocyte function was studied by investigating astrocytes prepared from GFAP-/-and/or vimentin-/- mice. The rate of glucose uptake through facilitative hexose transporters was not affected by depletion of GFAP or vimentin. Similarly, the absence of these IF proteins did not affect ascorbate uptake, under control or cyclic AMP-stimulated conditions, or ascorbate efflux through volume-sensitive organic anion channels. However, compared with wild-type astrocytes, glutamine concentrations were increased up to 200% in GFAP-/- astrocytes and up to 150% in GFAP+/-astrocytes and this increase was not dependent on the presence of vimentin. GFAP-/- astrocytes in culture still contain IFs (made of vimentin and nestin), whereas GFAP-/-vim-/- cultured astrocytes lack IFs. Thus, glutamine levels appear to correlate inversely with GFAP, rather than depend on the presence of IFs per se. Furthermore, the effect of GFAP is dose-dependent since the glutamine concentration in GFAP+/- astrocytes falls between those in wild-type and GFAP-/-astrocytes.     

The metal site of Pseudomonas aeruginosa azurin,revealed by a crystal structure determination of the co(II) derivative and co-EPR spectroscopy

The crystal structure of cobalt-substituted azurin from Pseudomonas aeruginosa has been determined to final crystallographic R value of 0.175 at 1.9 Å resolution. There are four molecules in the asymmetric unit in the structure, and these four molecules are packed as a dimer of dimers. The dimer packing is very similar to that of the wild-type Pseudomonas aeruginosa azurin dimer. Replacement of the native copper by the cobalt ion has only small effects on the metal binding site presumably because of the existence of an extensive network of hydrogen bonds in its immediate neighborhood. Some differences are obvious, however. In wild-type azurin the copper atom occupies a distorted trigonal bipyramidal site, while cobalt similar to zinc and nickel occupy a distorted tetrahedral site, in which the distance to the Met121,S^δ atom is increased to 3.3–3.5 Å and the distance to the carbonyl oxygen of Gly45 has decreased to 2.1–2.4 Å. The X-band EPR spectrum of the high-spin Co(II) in azurin is well resolved (apparent g values g_x′ = 5.23; g_y′ = 3.83; g_z′ = 1.995, and hyperfine splittings A_x′ = 31; A_y′ = 20–30; A_z′ = 53 G) and indicates that the ligand field is close to axial. Proteins 27:385–394, 1997. © 1997 Wiley-Liss, Inc.