Spin distribution of the H-cluster in the Hox–CO state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans: HYSCORE and ENDOR study of 14N and 13C nuclear interactions

Hydrogenases are enzymes which catalyze the reversible cleavage of molecular hydrogen into protons and electrons. In [FeFe] hydrogenases the active center is a 6Fe6S cluster, referred to as the “H-cluster.” It consists of the redox-active binuclear subcluster ([2Fe]_H) coordinated by CN⁻ and CO ligands and the cubane-like [4Fe–4S]_H subcluster which is connected to the protein via Cys ligands. One of these Cys ligands bridges to the [2Fe]_H subcluster. The CO-inhibited form of [FeFe] hydrogenase isolated from Desulfovibrio desulfuricans was studied using advanced EPR methods. In the H_ox–CO state the open coordination site at the [2Fe]_H subcluster is blocked by extrinsic CO, giving rise to an EPR-active S = 1/2 species. The CO inhibited state was prepared with ¹³CO and illuminated under white light at 273 K. In this case scrambling of the CO ligands occurs. Three ¹³C hyperfine couplings of 17.1, 7.4, and 3.8 MHz (isotropic part) were observed and assigned to ¹³CO at the extrinsic, the bridging, and the terminal CO-ligand positions of the distal iron, respectively. No ¹³CO exchange of the CO ligand to the proximal iron was observed. The hyperfine interactions detected indicate a rather large distribution of the spin density over the terminal and bridging CO ligands attached to the distal iron. Furthermore, ¹⁴N nuclear spin interactions were measured. On the basis of the observed ¹⁴N hyperfine couplings, which result from the CN⁻ ligands of the [2Fe]_H subcluster, it has been concluded that there is very little unpaired spin density on the cyanides of the binuclear subcluster.

Characterization of Substrate Preference for Slc1p and Cst26p in Saccharomyces cerevisiae Using Lipidomic Approaches and an LPAAT Activity Assay

Background

Phosphatidic acid (PA) is a key regulated intermediate and precursor for de novo biosynthesis of all glycerophospholipids. PA can be synthesized through the acylation of lysophosphatidic acid (LPA) by 1-acyl-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase, LPAAT). Recent findings have substantiated the essential roles of acyltransferases in various biological functions.

Methodologies/Principal Findings

We used a flow-injection-based lipidomic approach with ∼200 multiple reaction monitoring (MRM) transitions to pre-screen fatty acyl composition of phospholipids in the yeast Saccharomyces cerevisiae mutants. Dramatic changes were observed in fatty acyl composition in some yeast mutants including Slc1p, a well-characterized LPAAT, and Cst26p, a recently characterized phosphatidylinositol stearoyl incorporating 1 protein and putative LPAAT in S. cerevisiae. A comprehensive high-performance liquid chromatography–based multi-stage MRM approach (more than 500 MRM transitions) was developed and further applied to quantify individual phospholipids in both strains to confirm these changes. Our data suggest potential fatty acyl substrates as well as fatty acyls that compensate for defects in both Cst26p and Slc1p mutants. These results were consistent with those from a non-radioactive LPAAT enzymatic assay using C17-LPA and acyl-CoA donors as substrates.

Conclusions

We found that Slc1p utilized fatty acid (FA) 18:1 and FA 14:0 as substrates to synthesize corresponding PAs; moreover, it was probably the only acyltransferase responsible for acylation of saturated short-chain fatty acyls (12:0 and 10:0) in S. cerevisiae. We also identified FA 18:0, FA 16:0, FA 14:0 and exogenous FA 17:0 as preferred substrates for Cst26p because transformation with a GFP-tagged CST26 restored the phospholipid profile of a CST26 mutant. Our current findings expand the enzymes and existing scope of acyl-CoA donors for glycerophospholipid biosynthesis.     

Fast and reliable determination of the antifungal drug voriconazole in plasma using monolithic silica rod liquid chromatography

In the present study, we developed a fast and reliable HPLC assay for the determination of the new triazole antifungal agent voriconazole in plasma, using a Chromolith RP 18e (100 mm x 4.6 mm) monolithic silica rod HPLC column. After liquid-liquid extraction, plasma samples were separated with a mobile phase consisting of ammoniumdihydrogencarbonate buffer (pH 5.8)-acetonitrile-tetrahydrofuran (72:25:3) at a flow-rate of 3.5 mL/min and UV detection at 255 nm. The retention times for voriconazole and internal standard (UK-115794) were 2.3 and 2.7 min, respectively, and total run time was 4 min. The calibration curves were linear between 0.05 and 10 microg/mL, and within-assay and between-assay coefficients of variation were <4%. The proposed assay for voriconazole in plasma is fast, sensitive and reliable, and, thus, well suited for routine therapeutic monitoring of patients and for pharmacokinetic studies. It can be predicted that the use of monolithic silica rod chromatography will substantially shorten the turn-around time in the therapeutic drug monitoring laboratory.     

An “energy-auxotroph” Escherichia coli provides an in vivo platform for assessing NADH regeneration systems

An efficient in vivo regeneration of the primary cellular resources NADH and ATP is vital for optimizing the production of value-added chemicals and enabling the activity of synthetic pathways. Currently, such regeneration routes are tested and characterized mainly in vitro before being introduced into the cell. However, in vitro measurements could be misleading as they do not reflect enzyme activity under physiological conditions. Here, we construct an in vivo platform to test and compare NADH regeneration systems. By deleting dihydrolipoyl dehydrogenase in Escherichia coli, we abolish the activity of pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. When cultivated on acetate, the resulting strain is auxotrophic to NADH and ATP: acetate can be assimilated via the glyoxylate shunt but cannot be oxidized to provide the cell with reducing power and energy. This strain can, therefore, serve to select for and test different NADH regeneration routes. We exemplify this by comparing several NAD-dependent formate dehydrogenases and methanol dehydrogenases. We identify the most efficient enzyme variants under in vivo conditions and pinpoint optimal feedstock concentrations that maximize NADH biosynthesis while avoiding cellular toxicity. Our strain thus provides a useful platform for comparing and optimizing enzymatic systems for cofactor regeneration under physiological conditions.     

Force-dependent recruitment from myosin OFF-state increases end-systolic pressure–volume relationship in left ventricle

Biomechanics and Modeling in Mechanobiology - Finite element (FE) modeling is becoming increasingly prevalent in the world of cardiac mechanics; however, many existing FE models are...     

Rapid atrazine mineralisation in soil slurry and moist soil by inoculation of an atrazine-degrading Pseudomonas sp. strain

The evaluation of pesticide-mineralising microorganisms to clean-up contaminated soils was studied with the widely applied and easily detectable compound atrazine, which is rapidly mineralised by several microorganisms including the Pseudomonas sp. strain Yaya 6. The rate of atrazine removal was proportional to the water content of the soil and the amount of bacteria added to the soil. In soil slurry, 6 mg atrazine kg soil⁻¹ was eliminated within 1 day after application of 0.3 g dry weight inoculant biomass kg soil⁻¹ and within 5 days when 0.003 g kg soil⁻¹ was used. In partially saturated soil (60% of the maximal water-holding capacity) 15 mg atrazine kg soil⁻¹ was eliminated within 2 days by 1 g biomass kg soil⁻¹ and within 25 days when 0.01 g biomass kg soil⁻¹ was used. In unsaturated soil, about 60% [U-ring-¹⁴C]atrazine was converted to ¹⁴CO₂ within 14 days. Atrazine was very efficiently removed by the inoculant biomass, not only in soil that was freshly contaminated but also in soil aged with atrazine for up to 260 days. The bacteria exposed to atrazine in unsaturated sterile soil were still active after a starvation period of 240 days: 15 mg newly added atrazine kg soil⁻¹ was eliminated within 5 days. Received: 31 October 1997 / Received revision: 16 January 1998 / Accepted: 18 January 1998     

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 contribution to the generalization of the Gompertz function

SCHARF (1976) discusses various growth models. For the Gompertz function the differential equation (Formula: see text) is used. In words: the difference between relative growth rate and relative growth acceleration is constant. On the other hand, according to WENK (1973), the differential equation (Formula: see text) applies to the Gompertz function. It can be shown mathematically that (Formula: see text) applies in general. From Eq. (2) one obtains without trouble (Formula: see text). Therefore, the property leading to the Gompertz function may be defined as follows; the logarithmic derivation of the relative growth rate is constant. Eq. (2) is applicable only in special cases. It can be extended by assuming that c is not constant, but a function of time. In this way, a great number of growth functions can be found, which have to be regarded as model-based extensions of the Gompertz function.     

The effect of salts on the kinetics of iron release from N-terminal and C-terminal monoferrictransferrins

The kinetics of iron release from N-terminal and C-terminal monoferric human transferrins has been studied using EDTA as the accepting chelate. In the absence of added salts iron release from the N-terminal site is more facile but the relative lability can be reversed by the addition of NaClO₄, NaCl and LiCl. The results indicate that both anions and cations can affect the lability of the two sites. Since the relative lability of the two monoferrictransferrins is affected by fairly moderate concentrations of NaCl and NaClO₄ we suggest that the ionic composition serum may play an important role in determining the observed distribution of iron among the sites. A new method for the preparation of N-terminal monoferrictransferrin is described.     

The effect of copper supplementation on the concentration of copper in the brain of the brindled mouse.

The brindled mutant mouse is a useful model to study Menkes kinky-hair syndrome. The metabolic dysfunctions in both human and rodent are related to insufficient levels of bioavailable copper. Recently, copper supplementation therapy has been able both to prevent the appearance of various neuropathological changes and to prolong the life of these mutant mice. The optimum conditions for supplementation have been shown to be two intraperitoneal injections on postnatal days 7 and 10. The present study reports on the brain copper concentrations before, during and after the intraperitoneal copper therapy. The results demonstrate that postnatal days 7 and 10 correspond to two important epochs in copper homoeostasis. The supplementation therapy seems to provide sufficient bioavailable copper to respond to the needs of the animal at these crucial time points. The results are discussed in terms of their importance to the human copper disorder.