Control of a redox reaction on lipid bilayer surfaces by membrane dipole potential

Nitro-2,1,3-benzoxadiazol-4-yl (NBD) group is a widely used, environment-sensitive fluorescent probe. The negatively charged dithionite rapidly reduces the accessible NBD-labeled lipids in liposomes to their corresponding nonfluorescent derivatives. In this study both the phospholipid headgroup and acyl chain NBD-labeled L-alpha-1,2-dipalmitoyl-sn-glycero-3-phospho-[N-(4-nitrobenz-2-oxa-1,3-diazole)-ethanolamine] (DPPN) and 1-acyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC), respectively, were employed. The correlation of both the rate coefficient k(1) of the redox reaction and the fluorescence properties of the two probes with the membrane dipole potential Psi in fluid dipalmitoylglycerophosphocholine (DPPC) liposomes is demonstrated. When Psi of the bilayer was varied (decreased by phloretin or increased by 6-ketocholestanol), the value for k1 decreased for both DPPN and NBD-PC with increasing Psi. For both fluorophores a positive correlation to Psi was evident for the relative fluorescence emission intensity (RFI, normalized to the emission of the fluorophore in a DPPC matrix). The relative changes in emission intensity as a function of Psi were approximately equal for both NBD derivatives. Changes similar to those caused by phloretin were seen when dihexadecylglycerophosphocholine (DHPC) was added to DPPC liposomes, in keeping with the lower dipole potential for the former lipid compound compared with DPPC. These effects of Psi on NBD fluorescence should be taken into account when interpreting data acquired using NBD-labeled lipids as fluorescent probes.     

Multiple quantitative trait locus analysis of bovine chromosome 6 in the Israeli Holstein population by a daughter design

Nine Israeli Holstein sire families with 2978 daughters were analyzed for quantitative trait loci effects on chromosome 6 for five milk production traits by a daughter design. All animals were genotyped for 2 markers. The three families with significant effects were genotyped for up to 10 additional markers spanning positions 0-122 cM of BTA6. Two sires were segregating for a locus affecting protein and fat percentage near position 55 cM with an estimated substitution effect of 0.18% protein, which is equivalent to one phenotypic standard deviation. This locus was localized to a confidence interval of 4 cM. One of these sires was also heterozygous for a locus affecting milk, fat, and protein production near the centromere. The hypothesis of two segregating loci was verified by multiple regression analysis. A third sire was heterozygous for a locus affecting milk and protein percentage near the telomeric end of the chromosome. Possible candidates for the major quantitative gene near position 55 cM were determined by comparative mapping. IBSP and SSP1 were used as anchors for the orthologous region on human chromosome 4. Twelve genes were detected within a 2-Mbp sequence. None of these genes have been previously associated with lactogenesis.     

New basicrania of Paleocene-Eocene Ignacius: re-evaluation of the Plesiadapiform-Dermopteran link

Plesiadapiformes has long been considered to be an archaic group of Primates. Discovery of a paromomyid plesiadapiform skull and independent analysis of referred postcrania have led investigators to conclude that Plesiadapiformes shares a closer relationship to extant flying lemurs (Dermoptera) than to Primates (= Euprimates of Hoffstetter [1977] Bull Mem Soc Anthropol Paris Ser 13 4:327-346). Despite challenges to this interpretation, the plesiadapiform-dermopteran relationship has gained currency in recent years. Here we show that newly discovered crania of Ignacius graybullianus, preserving previously undocumented portions of the ear, are more similar to primates than to dermopterans. New specimens confirm that paromomyids lacked the petrosal bulla of primates. However, these new specimens also demonstrate that paromomyids likely had: 1) a small promontorial branch of the internal carotid artery; 2) a lateral route for the internal carotid nerves crossing the promontorium; and 3) a ring-like ectotympanic with an annular bridge. This pattern is similar to primitive primates and fundamentally different from dermopterans, which have: 1) no internal carotid artery; 2) internal carotid nerves that take a more medial route; and 3) no annular bridge. Recognition of some primate-like traits, documented here by new evidence, indicates that Paromomyidae is likely to be more closely related to other Paleogene Plesiadapiformes and Eocene Primates than to extant Dermoptera. In view of these findings, a link between paromomyids and extant dermopterans ("Eudermoptera") is not convincingly supported by a single characteristic of the basicranium.     

Spin label electron paramagnetic resonance study in thylakoid membranes from a new herbicide-resistant D1 mutant from soybean cell cultures deficient in fatty acid desaturation

We examined the effect of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on the production of hydroxyl radical (*OH) generation via nitric oxide synthase (NOS) activation by an in vivo microdialysis technique. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and tissue was perfused with Ringer's solution through the microdialysis probe at a rate of 1 microl/min. Sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused directly through a microdialysis probe to detect the generation of *OH. Induction of [K(+)](o) (70 mM) or tyramine (1 mM), significantly increased the formation of *OH trapped as 2,3-dihydroxybenzoic acid (DHBA). The application of N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, significantly decreased the K(+) depolarization-induced *OH formation, but the effect of tyramine significantly increased the level of 2,3-DHBA. When fluvastatin (100 microM), an inhibitor of low-density lipoprotein (LDL) oxidation, was administered to L-NAME-pretreated animals, both KCl and tyramine failed to increase the level of 2,3-DHBA formation. The effect of fluvastatin may be unrelated to K(+) depolarization-induced *OH generation. To examine the effect of fluvastatin on ischemic/reperfused rat myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery (LAD). When the heart was reperfused, a marked elevation of the level of 2,3-DHBA was observed. However, in the presence of fluvastatin (100 microM), the elevation of 2,3-DHBA was not observed in ischemia/reperfused rat heart. Fluvastatin, orally at a dose of 3 mg/kg/day for 4 weeks, significantly blunted the rise of serum creatine phosphokinase and improved the electrocardiogram 2 h after coronary occlusion. These results suggest that fluvastatin is associated with a cardioprotective effect due to the suppression of noradrenaline-induced *OH generation by inhibiting LDL oxidation in the heart.     

Pathophysiological significance of in vivo ESR signal decay in brain damage caused by X-irradiation. Radiation effect on nitroxyl decay of a lipophilic spin probe in the head region

X-irradiation of mice decreased the decay rate of the in vivo ESR signal in the head region to 75% of the control when 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy (MCPROXYL), a lipophilic and blood-brain barrier-permeable spin probe, was used. We attempted to identify the specific factor responsible for the decrease in the signal decay rate caused by X-irradiation. The signal decay of MCPROXYL in the head region depends on the following three factors: (1) blood concentration of MCPROXYL, (2) reduction to the corresponding hydroxylamine in the brain tissue, and (3) effusion of MCPROXYL from the brain tissue. Irradiation at 15 Gy did not significantly change the rate of decrease of blood concentration of MCPROXYL at 1 h post-irradiation. The reducing activity of the brain homogenate was not changed by the X-irradiation (15 Gy). The contents of MCPROXYL and its hydroxylamine derivative in the brain of 15 Gy-irradiated mice remained higher than in non-irradiated mice. These findings suggest that the effect of X-irradiation observed by in vivo ESR is attributable not to the redox reaction of MCPROXYL in the brain but to the change of the efflux rate of the MCPROXYL from the brain.     

Pre-steady-state kinetic studies of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase mutants identify residues involved in catalysis

MyristoylCoA:protein N-myristoyltransferase (Nmt, EC 2.3.1.97), a member of the GCN5 acetyltransferase (GNAT) superfamily, is an essential eukaryotic enzyme that catalyzes covalent attachment of myristate (C14:0) to the N-terminal Gly of proteins involved in myriad cellular functions. The 2.5 A resolution structure of a ternary complex of Saccharomyces cerevisiae Nmt1p with a bound substrate peptide (GLYASKLA) and nonhydrolyzable myristoylCoA analogue [Farazi, T. A., et al. (2001) Biochemistry 40, 6335] was used as the basis for a series of mutagenesis experiments designed to define the enzyme's catalytic mechanism. The kinetic properties of an F170A/L171A Nmt mutant are consistent with the proposal that their main chain amides, located in a beta-bulge structure conserved among GNATs, function as an oxyanion hole to polarize the thioester carbonyl of bound myristoylCoA prior to subsequent nucleophilic attack. Removal of the two C-terminal residues (M454 and L455) produces a 300--400-fold reduction in the chemical transformation rate and converts the rate-limiting step from a step after the transformation to the transformation event itself. This finding is consistent with the main chain C-terminal carboxylate of L455 functioning as a catalytic base that abstracts a proton from the N-terminal Gly ammonium of the bound peptide to generate the nucleophilic amine. Mutating N169 and T205 in concert reduces the rate of the chemical transformation, supporting their role as components of an H-bonding network that facilitates attack of the Gly1 amine and stabilizes the tetrahedral intermediate.     

The IL-2 receptor in hemocytes of the sea mussel Mytilus galloprovincialis Lmk

In the hemolymph of the sea mussel Mytilus galloprovincialis, two different cell types have been found. Rounded (RH) cells display a nucleus that is very large in relation to the cell size; spread (SH) cells have an expanded cytoplasm and multiple granules. We determined by flow cytometry that only the SH cell types express three interleukin-2 receptor (IL-2R) subunits. Mussel IL-2R alpha and IL-2R beta subunits display a molecular weight similar to those in vertebrates tissues, whereas mussel IL-2R gamma is smaller than that in vertebrates. Both lipopolysaccharide and IL-2 induce IL-2R alpha expression, and such induction depends on the concentration of both agonists.