NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB-domain 4 heterodimer formation in solution

UvrB is a central DNA damage recognition protein involved in bacterial nucleotide excision repair. Structural information has been limited by the apparent disorder of the C-terminal domain 4 in crystal structures of intact UvrB; in solution, the isolated domain 4 is found to form a helix-loop-helix dimer. In order to gain insight into the behavior of UvrB in solution, we have performed NMR studies on [methyl-13C]methionine-labeled UvrB from Bacillus caldotenax (molecular mass=75 kDa). The 13 methyl resonances were assigned on the basis of site-directed mutagenesis and domain deletion. Solvent accessibility was assessed based on the relaxation and chemical shift responses of the probe methyl resonances to the stable nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL). M632, located at the potential dimer interface of domain 4, provides an ideal probe for UvrB dimerization behavior. The M632 resonance of UvrB is very broad, consistent with some degree of monomer-dimer exchange and/or conformational instability of the exposed dimer interface. Upon addition of unlabeled domain 4 peptide, the M632 resonance of UvrB sharpens and shifts to a position consistent with a UvrB-domain 4 heterodimer. A dissociation constant (KD) value of 3.3 microM for the binding constant of UvrB with the domain 4 peptide was derived from surface plasmon resonance studies. Due to the flexibility of the domain 3-4 linker, inferred from limited proteolysis data and from the relaxation behavior of linker residue M607, the position of domain 4 is constrained not by the stiffness of the linking segment but by direct interactions with domains 1-3 in UvrB. In summary, UvrB homodimerization is disfavored, while domain 4 homodimerization and UvrB-domain 4 heterodimerization are allowed.     

NosX function connects to nitrous oxide (N2O) reduction by affecting the Cu(Z) center of NosZ and its activity in vivo

The effect of loss of the 34-kDa periplasmic NosX protein on the properties of N2O reductase was investigated with an N2O-respiration negative, double mutant of the paralogous genes nosX and nirX of Paracoccus denitrificans. In spite of absence of whole-cell N2O-reducing activity, the purified reductase was catalytically active, which attributes NosX a physiological role in sustaining the reaction cycle. N2O reductase exhibited the spectroscopic features of Cu(A) and the redox-inert, paramagnetic state, Cu(Z)*, of the catalytic center. Cu(Z)*, hitherto considered the result of spontaneous reaction of the reductase with dioxygen, attains cellular significance.     

Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats

Homocystinuria is a neurometabolic disease caused by severe deficiency of cystathionine beta-synthase activity, resulting in severe hyperhomocysteinemia. Affected patients present several symptoms including a variable degree of motor dysfunction, being that the pathomechanism is not fully understood. In the present study we investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative stress, namely 2′7′dichlorofluorescein (DCFH) oxidation, levels of thiobarbituric acid-reactive substances (TBARS), antioxidant enzyme activities (SOD, CAT and GPx), reduced glutathione (GSH), total sulfhydryl and carbonyl content, as well as nitrite levels in soleus skeletal muscle of young rats subjected to model of severe hyperhomocysteinemia. We also evaluated the effect of creatine on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of homocysteine (0.3–0.6 μmol/g body weight), and/or creatine (50 mg/kg body weight) from their 6th to the 28th days age. Controls and treated rats were decapitated at 12 h after the last injection. Chronic homocysteine administration increased 2′7′dichlorofluorescein (DCFH) oxidation, an index of production of reactive species and TBARS levels, an index of lipoperoxidation. Antioxidant enzyme activities, such as SOD and CAT were also increased, but GPx activity was not altered. The content of GSH, sulfhydril and carbonyl were decreased, as well as levels of nitrite. Creatine concurrent administration prevented some homocysteine effects probably by its antioxidant properties. Our data suggest that the oxidative insult elicited by chronic hyperhomocystenemia may provide insights into the mechanisms by which homocysteine exerts its effects on skeletal muscle function. Creatine prevents some alterations caused by homocysteine.     

Ordered and disordered phases coexist in plasma membrane vesicles of RBL-2H3 mast cells. An ESR study

Four chain spin labels and a spin-labeled cholestane were used to study the dynamic structure of plasma membrane vesicles (PMV) prepared from RBL-2H3 mast cells at temperatures ranging from 22 degrees C to 45 degrees C. Analysis shows that the spectra from most labels consist of two components. The abundant spectral components exhibit substantial ordering that is intermediate between that of a liquid-ordered (Lo) phase, and that of a liquid-crystalline (Lc) phase as represented by model membranes. Also, rotational diffusion rates of the spin labels are comparable to those in the Lo phase. In contrast, the ordering for the less abundant components is much lower. These results indicate that a Lo-like region or phase (the abundant component) and an Lc-like region or phase (the less abundant component) coexist in the PMV. In contrast, membranes reconstituted from extracted lipids exhibit the more ordered phase only. This suggests that membrane-associated proteins are important for the coexistence of Lo-like and Lc-like regions in the plasma membrane. In addition, binding of the myristoylated protein, ARF6 to PMV, leads to a new spectral component for a headgroup lipid spin label that indicates the formation of plasma membrane defects by this low molecular weight GTPase.     

Synthesis of 7alpha-substituted derivatives of 17beta-estradiol

Estrogen receptor (ER) pure antagonists such as ICI-182,780 (fulvestrant) are effective alternatives to tamoxifen (an ER antagonist/weak partial agonist) in the treatment of postmenopausal, receptor-positive human breast cancers. Structurally, these pure antagonists contain the basic core structure of 17beta-estradiol (E(2)) with a long side chain attached to its C-7alpha position. We explored and compared in this study various synthetic routes for preparing a number of C-7alpha-substituted derivatives of E(2), which are highly useful for the design and synthesis of high-affinity ER antagonists, ER-based imaging ligands, and other ER-based multi-functional agents. Using E(2) as the starting material and 1-iodo-6-benzyloxyhexane as a precursor for the C-7alpha side chain, a seven-step synthetic procedure afforded 3,17beta-bis(acetoxy)-7alpha-(6-hydroxyhexanyl)-estra-1,3,5(10)-triene (one of the derivatives prepared) in an overall yield of approximately 45% as compared to other known procedures that afforded substantially lower overall yield (8-27%). The synthetic steps for this representative compound include: (1) protection of the C-3 and C-17beta hydroxyls of E(2) using methoxymethyl groups; (2) hydroxylation of the C-6 position of the bismethoxymethyl ether of E(2); (3) Swern oxidation of the C-6 hydroxy to the ketone group; (4) C-7alpha alkylation of the C-6 ketone derivative of E(2); (5) deprotection of the two methoxymethyl groups; (6) reprotection of the C-3 and C-6 free hydroxyls with acetyl groups; (7) removal of the C-6 ketone and the benzyl group on the side chain by catalytic hydrogenation in acetic acid. As predicted, two of the representative C-7alpha-substituted derivatives of E(2) synthesized in the present study retained strong binding affinities (close to those of E(2) and ICI-182,780) for the human ERalpha and ERbeta subtypes as determined using the radioligand-receptor binding assays.     

Lipid chain-length dependence for incorporation of alamethicin in membranes: electron paramagnetic resonance studies on TOAC-spin labeled analogs

Alamethicin is a 19-residue hydrophobic peptide, which is extended by a C-terminal phenylalaninol but lacks residues that might anchor the ends of the peptide at the lipid-water interface. Voltage-dependent ion channels formed by alamethicin depend strongly in their characteristics on chain length of the host lipid membranes. EPR spectroscopy is used to investigate the dependence on lipid chain length of the incorporation of spin-labeled alamethicin in phosphatidylcholine bilayer membranes. The spin-label amino acid TOAC is substituted at residue positions n = 1, 8, or 16 in the sequence of alamethicin F50/5 [TOAC(n), Glu(OMe)(7,18,19)]. Polarity-dependent isotropic hyperfine couplings of the three TOAC derivatives indicate that alamethicin assumes approximately the same location, relative to the membrane midplane, in fluid diC(N)PtdCho bilayers with chain lengths ranging from N = 10-18. Residue TOAC(8) is situated closest to the bilayer midplane, whereas TOAC(16) is located farther from the midplane in the hydrophobic core of the opposing lipid leaflet, and TOAC(1) remains in the lipid polar headgroup region. Orientational order parameters indicate that the tilt of alamethicin relative to the membrane normal is relatively small, even at high temperatures in the fluid phase, and increases rather slowly with decreasing chain length (from 13 degrees to 23 degrees for N = 18 and 10, respectively, at 75 degrees C). This is insufficient for alamethicin to achieve hydrophobic matching. Alamethicin differs in its mode of incorporation from other helical peptides for which transmembrane orientation has been determined as a function of lipid chain length.     

A metabonomic investigation of the effects of 60 days exposure of rats to two types of pyrethroid insecticides

Type I and II pyrethroid insecticides display different neurotoxicity. To investigate the long-term (60 days exposure) metabolic effect of the two types of pyrethroid insecticides deltamethrin and permethrin, ¹H nuclear magnetic resonance (NMR) spectroscopy-based metabonomics was used to analyze the biochemical composition of urine and serum samples from rats administrated daily with deltamethrin or permethrin for 60 consecutive days, and principal component analysis used to visualize similarities and differences in the resultant biochemical profiles. Rats treated with either deltamethrin or permethrin displayed increased levels of urinary acetate, dimethylamine, dimethylglycine, trimethylamine and serum free amino acids, and decreased urinary 2-oxoglutarate, all of which are indicative of kidney lesions and nephrotoxicity. The reduced excretion of tricarboxylic acid cycle intermediates, together with increased 3-D-hydroxybutyrate, acetate, and lactate in treated rats could suggest disturbance of the energy metabolism, including an increased rate of anaerobic glycolysis, enhanced fatty acid β-oxidation and ketogenesis. These results show that these two types of insecticides have similarities in the urine and serum spectra, indicating that similar metabolic pathways are perturbed by the insecticides, which induced hepatotoxicity and nephrotoxicity. This approach may lead to the discovery of novel biomarkers of pyrethroids toxicity and thereby provide new insights into the toxicological mechanisms of pesticides pyrethroids.     

Characterization of the sodium binding sites in microcrystalline ATP by Na-solid-state NMR and ab initio calculations

Stepwise conformational transition of disodium adenosine 5′-triphosphate (Na₂ATP) crystals as a function of relative humidity (r.H.), was examined by means of high resolution ²³Na, ³¹P solid state NMR spectroscopy and quantum mechanics calculations. ²³Na-MQMAS solid state NMR spectra of hydrated disodium salts of adenosine 5,5′-triphosphate were measured to characterize the individual sodium sites. Quadrupolar coupling constants (C_Q), and the asymmetry parameters of the electric field gradient tensors (η) of sodium-23 determined by quantum mechanical calculations (gaussian03) using larger clusters than in previous studies were compared to the experimental ²³Na-MQMAS spectra.     

Hydration, structure, and molecular interactions in the headgroup region of dioleoylphosphatidylcholine bilayers: an electron spin resonance study

The relationship between bilayer hydration and the dynamic structure of headgroups and interbilayer water in multilamellar vesicles is investigated by electron spin resonance methods. Temperature variations of the order parameter of a headgroup spin label DPP-Tempo in DOPC in excess water and partially dehydrated (10 wt % water) show a cusp-like pattern around the main phase transition, Tc. This pattern is similar to those of temperature variations of the quadrupolar splitting of interbilayer D2O in PC and PE bilayers previously measured by 2H NMR, indicating that the ordering of the headgroup and the interbilayer water are correlated. The cusp-like pattern of these and other physical properties around Tc are suggestive of quasicritical fluctuations. Also, an increase (a decrease) in ordering of DPP-Tempo is correlated with water moving out of (into) interbilayer region into (from) the bulk water phase near the freezing point, Tf. Addition of cholesterol lowers Tf, which remains the point of increasing headgroup ordering. Using the small water-soluble spin probe 4-PT, it is shown that the ordering of interbilayer water increases with bilayer dehydration. It is suggested that increased ordering in the interbilayer region, implying a lowering of entropy, will itself lead to further dehydration of the interbilayer region until its lowered pressure resists further flow, i.e., an osmotic phenomenon.     

Conditions affecting the re-alignment of the antimicrobial peptide PGLa in membranes as monitored by solid state H-NMR

The cationic antimicrobial peptide PGLa is electrostatically attracted to bacterial membranes, binds as an amphiphilic α-helix, and is thus able to permeabilize the lipid bilayer. Using solid state ²H-NMR of non-perturbing Ala-d₃ labels on the peptide, we have characterized the helix alignment under a range of different conditions. Even at a very high peptide-to-lipid ratio (1:20) and in the presence of negatively charged lipids, there was no indication of a toroidal wormhole structure. Instead, PGLa re-aligns from a surface-bound S-state to an obliquely tilted T-state, which is presumably dimeric. An intermediate structure half-way between the S- and T-state was observed in fully hydrated multilamellar DMPC vesicles at 1:50, suggesting a fast exchange between the two states on the time scale of >50 kHz. We demonstrate that this equilibrium is shifted from the S- towards the T-state either upon (i) increasing the peptide concentration, (ii) adding negatively charged DMPG, or (iii) decreasing the level of hydration. The threshold concentration for re-alignment in DMPC is found to be between 1:200 and 1:100 in oriented samples at 96% humidity. In fully hydrated multilamellar DMPC vesicles, it shifts to an effective peptide-to-lipid ratio of 1:50 as some peptides are able to escape into the bulk water phase.     

The effect of Ginkgo biloba extract on 3-nitropropionic acid-induced neurotoxicity in rats

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase enzyme (SDH), induces neurodegeneration similar to that observed in Huntington’s disease (HD). Reduction of prepulse inhibition (PPI) of acoustic startle response, locomotor hypoactivity, bilateral striatal lesions as well as brain oxidative stress are major features of HD. The present study was designed to investigate neuroprotective effect of Ginkgo biloba extract (EGb 761) on 3-NP induced neurobehavioral changes and striatal lesions.Rats administered 3-NP (20 mg/kg, s.c.) for five consecutive days exhibited PPI deficits and locomotor hypoactivity whereas, pretreatment of animals with EGb 761 (100 mg/kg, i.p. for 15 days) ahead of and during the induction of HD by 3-NP (20 mg/kg for 5 days starting at day 8) ameliorated 3-NP-induced neurobehavioral deficits. Administration of 3-NP increased the level of striatal malondialdehyde (MDA). This effect was prevented in animals pre-treated with EGb 761. Changes in the level of apoptotic regulatory gene expressions, following 3-NP treatment, were demonstrated as both an up-regulation and a down-regulation of the expression levels of striatal Bax and Bcl-xl genes, respectively. In addition, an up-regulation of the expression level of striatal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed. Pre-treatment with EGb 761 caused a down-regulation in striatal GAPDH and Bax together with an up-regulation of striatal Bcl-xl expression level as compared to the 3-NP treated group. Histochemical examination of striatal tissue showed that EGb 761 significantly prevented 3-NP induced inhibition of SDH activity. Histopathological examination further affirmed the neuroprotective effect of EGb 761 against 3-NP toxicity.Taken together, these results suggest that EGb 761 has a neuroprotective role in the current HD paradigm, which may be related to improvement of energy metabolism, antioxidant properties and antiapoptotic effects.     

Stepwise Transition of the Tetra-Manganese Complex of Photosystem II to a Binuclear Mn2(μ-O)2 Complex in Response to a Temperature Jump: A Time-Resolved Structural Investigation Employing X-Ray Absorption Spectroscopy

In oxygenic photosynthesis, water is oxidized at a protein-cofactor complex comprising four Mn atoms and, presumably, one calcium. Using multilayers of Photosystem II membrane particles, we investigated the time course of the disassembly of the Mn complex initiated by a temperature jump from 25°C to 47°C and terminated by rapid cooling after distinct heating periods. We monitored polarographically the oxygen-evolution activity, the amount of the Y_D^ox radical and of released Mn²⁺ by EPR spectroscopy, and the structure of the Mn complex by x-ray absorption spectroscopy (XAS, EXAFS). Using a novel approach to analyze time-resolved EXAFS data, we identify three distinct phases of the disassembly process: (1) Loss of the oxygen-evolution activity and reduction of Y_D^ox occur simultaneously (k₁ = 1.0 min⁻¹). EXAFS spectra reveal the concomitant loss of an absorber-backscatterer interaction between heavy atoms separated by ~3.3 Å, possibly related to Ca release. (2) Subsequently, two Mn(III) or Mn(IV) ions seemingly separated by ~2.7 Å in the native complex are reduced to Mn(II) and released (k₂ = 0.18 min⁻¹). The x-ray absorption spectroscopy data is highly suggestive that the two unreleased Mn ions form a di-μ-oxo bridged Mn(III)₂ complex. (3) Finally, the tightly-bound Mn₂(μ-O)₂ unit is slowly reduced and released (k₃ = 0.014 min⁻¹).     

Membrane cholesterol contents influence the protective effects of quercetin and rutin in erythrocytes damaged by oxidative stress

Flavonoids are potent scavengers of reactive oxygen species (ROS) that effectively prevent erythrocyte oxidation. Their antioxidant activities are governed by their structural characteristics and their ability to interact with and penetrate lipid bilayers. In order to gain a better understanding of the relationship between cholesterol contents and the antioxidant effectiveness of flavonoids against oxidative damage induced by ROS in cells, here we analyzed the integrity and structural stability of cholesterol-modified (enriched or depleted) and control erythrocytes exposed to tert-butyl hydroperoxide in the presence of quercetin or rutin. In control and cholesterol-enriched erythrocytes, quercetin provided greater protection against lipid peroxidation, ROS formation, and it preserved better cellular integrity than rutin. Both antioxidants suppressed the alterations in membrane fluidity and lipid losses with similar efficiency, reducing hemoglobin oxidation by 30% and GSH losses by 60% in the above-mentioned erythrocytes. Cholesterol depletion reduced the efficiency of the antioxidant power of both flavonoids against oxidative damage induced in the erythrocyte membrane, while a stronger degree of protection of GSH and hemoglobin contents was observed, mainly in the presence of rutin. These findings suggest a preferential incorporation of the antioxidants into the membranes from erythrocytes with normal and high cholesterol contents, whereas they would mainly be located in the cytoplasm of cholesterol-depleted erythrocytes.     

Coexistence of two liquid crystalline phases in dihydrosphingomyelin and dioleoylphosphatidylcholine binary mixtures

Recently, DHSM, a minor constituent in naturally occurring SMs, was indicated to form a raft-like ordered phase more effectively than a naturally occurring form of SM because DHSM has greater potential to induce the intermolecular hydrogen bond. In order to examine the influence of the DHSM-induced hydrogen bond on the phase segregation, the thermal phase behavior of stearoyl-DHSM/DOPC binary bilayers was examined using calorimetry and fluorescence observation and compared with that of SSM/DOPC binary bilayers. Results revealed that the DHSM/DOPC bilayers undergo phase segregation between two L_α phases within a limited compositional range. On the other hand, apparent phase separation was not observed above main transition temperature in SSM/DOPC mixtures. Our monolayer measurements showed that the lipid packing of DHSM is less perturbed than that of SSM by the addition of small amount of DOPC, indicating a stronger hydrogen bond between DHSM molecules. Therefore, in DHSM/DOPC binary bilayers, DHSM molecules may locally accumulate to form a DHSM-rich domain due to a DHSM-induced hydrogen bond. On the other hand, excess accumulation of DHSM should be prevented because the difference in the curvature between DHSM and DOPC assemblies causes elastic constraint at the domain boundary between the DHSM-rich and DOPC-rich domains. Competition between the energetic advantages provided by formation of the hydrogen bond and the energetic disadvantage conferred by elastic constraints likely results in L_α/L_α phase separation within a limited compositional range.     

Kinetic study of the plastoquinone pool availability correlated with H2O2 release in seawater and antioxidant responses in the red alga Kappaphycus alvarezii exposed to single or combined high light, chilling and chemical stresses

Under biotic/abiotic stresses, the red alga Kappaphycus alvarezii reportedly releases massive amounts of H₂O₂ into the surrounding seawater. As an essential redox signal, the role of chloroplast-originated H₂O₂ in the orchestration of overall antioxidant responses in algal species has thus been questioned. This work purported to study the kinetic decay profiles of the redox-sensitive plastoquinone pool correlated to H₂O₂ release in seawater, parameters of oxidative lesions and antioxidant enzyme activities in the red alga Kappaphycus alvarezii under the single or combined effects of high light, low temperature, and sub-lethal doses of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which are inhibitors of the thylakoid electron transport system. Within 24 h, high light and chilling stresses distinctly affected the availability of the PQ pool for photosynthesis, following Gaussian and exponential kinetic profiles, respectively, whereas combined stimuli were mostly reflected in exponential decays. No significant correlation was found in a comparison of the PQ pool levels after 24 h with either catalase (CAT) or ascorbate peroxidase (APX) activities, although the H₂O₂ concentration in seawater (R = 0.673), total superoxide dismutase activity (R = 0.689), and particularly indexes of protein (R = 0.869) and lipid oxidation (R = 0.864), were moderately correlated. These data suggest that the release of H₂O₂ from plastids into seawater possibly impaired efficient and immediate responses of pivotal H₂O₂-scavenging activities of CAT and APX in the red alga K. alvarezii, culminating in short-term exacerbated levels of protein and lipid oxidation. These facts provided a molecular basis for the recognized limited resistance of the red alga K. alvarezii under unfavorable conditions, especially under chilling stress.     

Brain edema in diseases of different etiology

Cerebral edema is a potentially life-threatening complication shared by diseases of different etiology, such as diabetic ketoacidosis, acute liver failure, high altitude exposure, dialysis disequilibrium syndrome, and salicylate intoxication. Pulmonary edema is also habitually present in these disorders, indicating that the microcirculatory disturbance causing edema is not confined to the brain. Both cerebral and pulmonary subclinical edema may be detected before it becomes clinically evident. Available evidence suggests that tissue hypoxia or intracellular acidosis is a commonality occurring in all of these disorders. Tissue ischemia induces physiological compensatory mechanisms to ensure cell oxygenation and carbon dioxide removal from tissues, including hyperventilation, elevation of red blood cell 2,3-bisphosphoglycerate content, and capillary vasodilatation. Clinical, laboratory, and necropsy findings in these diseases confirm the occurrence of low plasma carbon dioxide partial pressure, increased erythrocyte 2,3-bisphosphoglycerate concentration, and capillary vasodilatation with increased vascular permeability in all of them. Baseline tissue hypoxia or intracellular acidosis induced by the disease may further deteriorate when tissue oxygen requirement is no longer matched to oxygen delivery resulting in massive capillary vasodilatation with increased vascular permeability and plasma fluid leakage into the interstitial compartment leading to edema affecting the brain, lung, and other organs. Causative factors involved in the progression from physiological adaptation to devastating clinical edema are not well known and may include uncontrolled disease, malfunctioning adaptive responses, or unknown factors. The role of carbon monoxide and local nitric oxide production influencing tissue oxygenation is unclear.