Comparative Studies of the Pyrolytic and Kinetic Characteristics of Maize Straw and the Seaweed Ulva pertusa

Seaweed has attracted considerable attention as a potential biofuel feedstock. The pyrolytic and kinetic characteristics of maize straw and the seaweed Ulva pertusa were studied and compared using heating rates of 10, 30 and 50°C min⁻¹ under an inert atmosphere. The activation energy, and pre-exponential factors were calculated by the Flynn-Wall-Ozawa (FWO), Kissinger-Akahira-Sunose (KAS) and Popescu methods. The kinetic mechanism was deduced by the Popescu method. The results indicate that there are three stages to the pyrolysis; dehydration, primary devolatilization and residual decomposition. There were significant differences in average activation energy, thermal stability, final residuals and reaction rates between the two materials. The primary devolatilization stage of U. pertusa can be described by the Avramic-Erofeev equation (n = 3), whereas that of maize straw can be described by the Mampel Power Law (n = 2). The average activation energy of maize straw and U. pertusa were 153.0 and 148.7 KJ mol⁻¹, respectively. The pyrolysis process of U.pertusa would be easier than maize straw. And co-firing of the two biomass may be require less external heat input and improve process stability. There were minor kinetic compensation effects between the pre-exponential factors and the activation energy.     

Binding of β-lactam antibiotics to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R39

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl₂ buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10⁻⁶, 1.5×10⁻⁶ and 0.63×10⁻⁶s⁻¹ (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [¹⁴C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [¹⁴C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10⁻⁵s⁻¹ and 0.8×10⁻⁴s⁻¹ respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.     

Kinetics and thermodynamics of activation by pretreatment with guanosine 5′-[β-imido]triphosphate of smooth-muscle adenylate cyclase

Catalytic subunits (C) of uterine smooth-muscle adenylate cyclase were activated (C*) by incubating the enzyme with the GTP analogue guanosine 5′-[βγ-imido]triphosphate (p[NH]ppG), followed by treatment with GTP and washing at 2°C. Activation (C→C*) proceeded in a time- and temperature-dependent manner as disclosed by subsequent assay of the pretreated particles at 37°C. The properties of the activated subunits were a function of the pretreatment temperature and not those of the enzyme assay performed at 37°C. Over the range 6–24°C, activation by pretreatment with p[NH]ppG followed simple Michaelis–Menten kinetics, and increase in temperature increased the concentration of catalytic subunits in the C* state and decreased K_m for the guanosine nucleotide. Characterization of the temperature-dependent effects of pretreatment with p[NH]ppG suggested that activation of the catalytic subunit at the temperature in situ (37°C) was moderately endergonic (ΔH⁰ ~8kJ·mol⁻¹) and accompanied by an increase in entropy (ΔS⁰ ~146J·mol⁻¹·K⁻¹). The β-adrenergic catecholamine receptor, reflected by isoproterenol's effect on activation by pretreatment with p[NH]ppG, increased the concentration of catalytic subunits in the C* state but had an insignificant (P>0.05) effect on the K_m at every temperature. This result suggested that formation of the receptor–hormone complex produced an increase in the first-order rate constant without an appreciable effect on the actual catalytic-subunit activation step. The primary function of the β-adrenergic catecholamine receptor under these conditions appeared to be regulation of the concentration of activation sites available for binding of p[NH]ppG.     

Central Nervous System PET-CT Imaging Reveals Regional Impairments in Pediatric Patients with Wolfram Syndrome

Wolfram syndrome (WFS) is inherited as an autosomal recessive disease with main clinical features of diabetes mellitus, optic atrophy, diabetes insipidus and deafness. However, various neurological defects may also be detected. The aim of this study was to evaluate aspects of brain structure and function using PET-CT (positron emission tomography and computed tomography) and MRI (magnetic resonance imaging) in pediatric patients with WFS. Regional changes in brain glucose metabolism were measured using standardized uptake values (SUVs) based on images of (¹⁸F) fluorodeoxyglucose (FDG) uptake in 7 WFS patients aged 10.1–16.0 years (mean 12.9±2.4) and in 20 healthy children aged 3–17.9 years (mean 12.8±4.1). In all patients the diagnosis of WFS was confirmed by DNA sequencing of the WFS1 gene. Hierarchical clustering showed remarkable similarities of glucose uptake patterns among WFS patients and their differences from the control group. SUV data were subsequently standardized for age groups <13 years old and>13 years old to account for developmental differences. Reduced SUVs in WFS patients as compared to the control group for the bilateral brain regions such as occipital lobe (−1.24±1.20 vs. −0.13±1.05; p = 0.028) and cerebellum (−1.11±0.69 vs. −0.204±1.00; p = 0.036) were observed and the same tendency for cingulate (−1.13±1.05 vs. −0.15±1.12; p = 0.056), temporal lobe (−1.10±0.98 vs. −0.15±1.10; p = 0.057), parietal lobe (−1.06±1.20 vs. −0.08±1.08; p = 0.058), central region (−1.01±1.04 vs. −0.09±1.06; p = 0.060), basal ganglia (−1.05±0.74 vs. −0.20±1.07; p = 0.066) and mesial temporal lobe (−1.06±0.82 vs. −0.26±1.08; p = 0.087) was also noticed. After adjusting for multiple hypothesis testing, the differences in glucose uptake were non-significant. For the first time, regional differences in brain glucose metabolism among patients with WFS were shown using PET-CT imaging.     

Genetic Determinants of Long-Term Changes in Blood Lipid Concentrations: 10-Year Follow-Up of the GLACIER Study

Recent genome-wide meta-analyses identified 157 loci associated with cross-sectional lipid traits. Here we tested whether these loci associate (singly and in trait-specific genetic risk scores [GRS]) with longitudinal changes in total cholesterol (TC) and triglyceride (TG) levels in a population-based prospective cohort from Northern Sweden (the GLACIER Study). We sought replication in a southern Swedish cohort (the MDC Study; N = 2,943). GLACIER Study participants (N = 6,064) were genotyped with the MetaboChip array. Up to 3,495 participants had 10-yr follow-up data available in the GLACIER Study. The TC- and TG-specific GRSs were strongly associated with change in lipid levels (β = 0.02 mmol/l per effect allele per decade follow-up, P = 2.0×10⁻¹¹ for TC; β = 0.02 mmol/l per effect allele per decade follow-up, P = 5.0×10⁻⁵ for TG). In individual SNP analysis, one TC locus, apolipoprotein E (APOE) rs4420638 (β = 0.12 mmol/l per effect allele per decade follow-up, P = 2.0×10⁻⁵), and two TG loci, tribbles pseudokinase 1 (TRIB1) rs2954029 (β = 0.09 mmol/l per effect allele per decade follow-up, P = 5.1×10⁻⁴) and apolipoprotein A-I (APOA1) rs6589564 (β = 0.31 mmol/l per effect allele per decade follow-up, P = 1.4×10⁻⁸), remained significantly associated with longitudinal changes for the respective traits after correction for multiple testing. An additional 12 loci were nominally associated with TC or TG changes. In replication analyses, the APOE rs4420638, TRIB1 rs2954029, and APOA1 rs6589564 associations were confirmed (P≤0.001). In summary, trait-specific GRSs are robustly associated with 10-yr changes in lipid levels and three individual SNPs were strongly associated with 10-yr changes in lipid levels.     

Transporting Antitumor Drug Tamoxifen and Its Metabolites, 4-Hydroxytamoxifen and Endoxifen by Chitosan Nanoparticles

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K _tam-ch-15  = 8.7 (±0.5)×10³ M⁻¹, K _tam-ch-100  = 5.9 (±0.4)×10⁵ M⁻¹, K _tam-ch-200  = 2.4 (±0.4)×10⁵ M⁻¹ and K _{hydroxytam-ch-15}  = 2.6(±0.3)×10⁴ M⁻¹, K _{hydroxytam – ch-100}  = 5.2 (±0.7)×10⁶ M⁻¹ and K _{hydroxytam-ch-200}  = 5.1 (±0.5)×10⁵ M⁻¹, K _endox-ch-15  = 4.1 (±0.4)×10³ M⁻¹, K _endox-ch-100  = 1.2 (±0.3)×10⁶ M⁻¹ and K _endox-ch-200  = 4.7 (±0.5)×10⁵ M⁻¹ with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH₂ residues with drug OH and NH₂ groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.     

Isoniazid Inhibits the Heme-Based Reactivity of Mycobacterium tuberculosis Truncated Hemoglobin N

Isoniazid represents a first-line anti-tuberculosis medication in prevention and treatment. This prodrug is activated by a mycobacterial catalase-peroxidase enzyme called KatG in Mycobacterium tuberculosis), thereby inhibiting the synthesis of mycolic acid, required for the mycobacterial cell wall. Moreover, isoniazid activation by KatG produces some radical species (e.g., nitrogen monoxide), that display anti-mycobacterial activity. Remarkably, the ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of (pseudo-)enzymatic detoxification systems, including truncated hemoglobins (trHbs). Here, we report that isoniazid binds reversibly to ferric and ferrous M. tuberculosis trHb type N (or group I; Mt-trHbN(III) and Mt-trHbN(II), respectively) with a simple bimolecular process, which perturbs the heme-based spectroscopic properties. Values of thermodynamic and kinetic parameters for isoniazid binding to Mt-trHbN(III) and Mt-trHbN(II) are K = (1.1±0.1)×10⁻⁴ M, k _on = (5.3±0.6)×10³ M⁻¹ s⁻¹ and k _off = (4.6±0.5)×10⁻¹ s⁻¹; and D = (1.2±0.2)×10⁻³ M, d _on = (1.3±0.4)×10³ M⁻¹ s⁻¹, and d _off = 1.5±0.4 s⁻¹, respectively, at pH 7.0 and 20.0°C. Accordingly, isoniazid inhibits competitively azide binding to Mt-trHbN(III) and Mt-trHbN(III)-catalyzed peroxynitrite isomerization. Moreover, isoniazid inhibits Mt-trHbN(II) oxygenation and carbonylation. Although the structure of the Mt-trHbN-isoniazid complex is not available, here we show by docking simulation that isoniazid binding to the heme-Fe atom indeed may take place. These data suggest a direct role of isoniazid to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism.     

Effects of Abiotic Factors on Acetylene Reduction by Cyanobacteria Epiphytic on Moss at a Subantarctic Island

Acetylene reduction (AR) rates by cyanobacteria epiphytic on a moss at Marion Island (46°54′ S, 37°45′ E) increased from −5°C to a maximum at 25 to 27°C. Q₁₀ values between 0 and 25°C were between 2.3 and 2.9, depending on photosynthetic photon flux density. AR rates declined sharply at temperatures above the optimum and were lower at 35°C than at 0°C. Photosynthetic photon flux density at low levels markedly influenced AR, and half of the maximum rate occurred at 84 μmol m⁻² s⁻¹, saturation occurring at ca. 1,000 μmol m⁻² s⁻¹. Higher photosynthetic photon flux density levels decreased AR rates. AR increased up to the highest sample moisture content investigated (3,405%), and the pH optimum was between 5.9 and 6.2. The addition of P, Co, and Mo, individually or together, depressed AR.     

Growth Kinetics and Yield Coefficients of the Extreme Thermophile Thermothrix thiopara in Continuous Culture

Thermothrix thiopara did not appear to be stressed at high temperature (72°C). Both the actual and theoretical yields were higher than those of analogous mesophilic sulfur bacteria, and the specific growth rate (μ_max) was more rapid than that of most autotrophs. The specific growth rate (0.58 h⁻¹), specific maintenance rate (0.11 h⁻¹), actual molar growth yield at μ_max (Y_max = 16 g mol⁻¹), and theoretical molar growth yield (Y_G = 24 g mol⁻¹) were all higher for T. thiopara (72°C) than for mesophilic (25 to 30°C) Thiobacillus spp. The growth efficiencies for T. thiopara at 70 and 75°C (0.84 and 0.78) were significantly higher than at 65°C (0.47). Corresponding specific maintenance rates were highest at 65°C (0.41 h⁻¹) and lowest at 70 and 75°C (0.11 and 0.15 h⁻¹, respectively). Growth efficiencies of metabolically similar mesophiles were generally higher than for T. thiopara. However, the actual yields at μ_max were higher for T. thiopara because its theoretical yield was higher. Thus, at 70°C, T. thiopara was capable of deriving more metabolically useful energy from thiosulfate than were mesophilic sulfur bacteria at 25 and 30°C. The low growth efficiency of T. thiopara reflected higher maintenance expenditures. T. thiopara had higher maintenance rates than Thiobacillus ferroxidans or Thiobacillus denitrificans, but also attained higher molar growth yields. It is concluded that sulfur metabolism may be more efficient overall at extremely high temperatures due to increased theoretical yields despite increased maintenance requirements.     

Metabolic Depression in Cunner (Tautogolabrus adspersus) Is Influenced by Ontogeny,and Enhances Thermal Tolerance

To examine the effect of ontogeny on metabolic depression in the cunner (Tautogolabrus adspersus), and to understand how ontogeny and the ability to metabolically depress influence this species'' upper thermal tolerance: 1) the metabolic rate of 9°C-acclimated cunner of three size classes [0.2–0.5 g, young of the year (YOY); 3–6 g, small; and 80–120 g, large (adult)] was measured during a 2°C per day decrease in temperature; and 2) the metabolic response of the same three size classes of cunner to an acute thermal challenge [2°C h⁻¹ from 10°C until Critical Thermal Maximum, CT_Max] was examined, and compared to that of the Atlantic cod (Gadus morhua). The onset-temperature for metabolic depression in cunner increased with body size, i.e. from 5°C in YOY cunner to 7°C in adults. In contrast, the extent of metabolic depression was ∼80% (Q₁₀ = ∼15) for YOY fish, ∼65% (Q₁₀ = ∼8) for small fish and ∼55% (Q₁₀ = ∼5) for adults, and this resulted in the metabolic scaling exponent (b) gradually increasing from 0.84 to 0.92 between 9°C to 1°C. All size classes of cunner had significantly (approximately 60%) lower routine metabolic rates at 10°C than Atlantic cod. However, there was no species'' difference in the temperature-induced maximum metabolic rate, and this resulted in factorial metabolic scope values that were more than two-fold greater for cunner, and CT_Max values that were 6–9°C higher (∼21 vs. 28°C). These results: 1) show that ontogeny influences the temperature of initiation and the extent of metabolic depression in cunner, but not O₂ consumption when in a hypometabolic state; and 2) suggest that the evolution of cold-induced metabolic depression in this northern wrasse species has not resulted in a trade-off with upper thermal tolerance, but instead, an enhancement of this species'' metabolic plasticity.     

The PSEN1, p.E318G Variant Increases the Risk of Alzheimer's Disease in APOE-ε4 Carriers

The primary constituents of plaques (Aβ42/Aβ40) and neurofibrillary tangles (tau and phosphorylated forms of tau [ptau]) are the current leading diagnostic and prognostic cerebrospinal fluid (CSF) biomarkers for AD. In this study, we performed deep sequencing of APP, PSEN1, PSEN2, GRN, APOE and MAPT genes in individuals with extreme CSF Aβ42, tau, or ptau levels. One known pathogenic mutation (PSEN1 p.A426P), four high-risk variants for AD (APOE p.L46P, MAPT p.A152T, PSEN2 p.R62H and p.R71W) and nine novel variants were identified. Surprisingly, a coding variant in PSEN1, p.E318G (rs17125721-G) exhibited a significant association with high CSF tau (p = 9.2×10⁻⁴) and ptau (p = 1.8×10⁻³) levels. The association of the p.E318G variant with Aβ deposition was observed in APOE-ε4 allele carriers. Furthermore, we found that in a large case-control series (n = 5,161) individuals who are APOE-ε4 carriers and carry the p.E318G variant are at a risk of developing AD (OR = 10.7, 95% CI = 4.7–24.6) that is similar to APOE-ε4 homozygous (OR = 9.9, 95% CI = 7.2.9–13.6), and double the risk for APOE-ε4 carriers that do not carry p.E318G (OR = 3.9, 95% CI = 3.4–4.4). The p.E318G variant is present in 5.3% (n = 30) of the families from a large clinical series of LOAD families (n = 565) and exhibited a higher frequency in familial LOAD (MAF = 2.5%) than in sporadic LOAD (MAF = 1.6%) (p = 0.02). Additionally, we found that in the presence of at least one APOE-ε4 allele, p.E318G is associated with more Aβ plaques and faster cognitive decline. We demonstrate that the effect of PSEN1, p.E318G on AD susceptibility is largely dependent on an interaction with APOE-ε4 and mediated by an increased burden of Aβ deposition.     

Detection of Melamine in Feed Using Liquid-Liquid Extraction Treatment Combined with Surface-Enhanced Raman Scattering Spectroscopy

A rapid, selective, and sensitive method to determine the melamine content in animal feeds was developed using surface-enhanced Raman scattering spectroscopy on aggregated 55 nm Au nanoparticles with liquid–liquid extraction sample preparation. Butyl alcohol was used as the initial extraction solvent, and liquid–liquid extraction was performed twice using HCl (pH 3–4) and 6∶1 (v/v) n-butyl alcohol/ethyl acetate. The intensity of the matrix-based peak at 731 cm⁻¹ was set at 100 as a basis for the feeds, and the peak at 707 cm⁻¹ was the characteristic peak of melamine used in the calculations. Sufficient linearity was obtained in the range 2–10 µg·g⁻¹ (R ² = 0.991). Limits of detection and quantification in the feeds were 0.5 and 2 µg·g⁻¹, respectively. The recovery rates were 82.5–90.2% with coefficients of variation below 4.02%. This new protocol could be easily developed for the routine monitoring of on-site feed quality and market surveillance.     

Genetic Variants Associated with Serum Thyroid Stimulating Hormone (TSH) Levels in European Americans and African Americans from the eMERGE Network

Thyroid stimulating hormone (TSH) hormone levels are normally tightly regulated within an individual; thus, relatively small variations may indicate thyroid disease. Genome-wide association studies (GWAS) have identified variants in PDE8B and FOXE1 that are associated with TSH levels. However, prior studies lacked racial/ethnic diversity, limiting the generalization of these findings to individuals of non-European ethnicities. The Electronic Medical Records and Genomics (eMERGE) Network is a collaboration across institutions with biobanks linked to electronic medical records (EMRs). The eMERGE Network uses EMR-derived phenotypes to perform GWAS in diverse populations for a variety of phenotypes. In this report, we identified serum TSH levels from 4,501 European American and 351 African American euthyroid individuals in the eMERGE Network with existing GWAS data. Tests of association were performed using linear regression and adjusted for age, sex, body mass index (BMI), and principal components, assuming an additive genetic model. Our results replicate the known association of PDE8B with serum TSH levels in European Americans (rs2046045 p = 1.85×10⁻¹⁷, β = 0.09). FOXE1 variants, associated with hypothyroidism, were not genome-wide significant (rs10759944: p = 1.08×10⁻⁶, β = −0.05). No SNPs reached genome-wide significance in African Americans. However, multiple known associations with TSH levels in European ancestry were nominally significant in African Americans, including PDE8B (rs2046045 p = 0.03, β = −0.09), VEGFA (rs11755845 p = 0.01, β = −0.13), and NFIA (rs334699 p = 1.50×10⁻³, β = −0.17). We found little evidence that SNPs previously associated with other thyroid-related disorders were associated with serum TSH levels in this study. These results support the previously reported association between PDE8B and serum TSH levels in European Americans and emphasize the need for additional genetic studies in more diverse populations.     

Effects of Low-Temperature Acclimation and Oxygen Stress on Tocopheron Production in Euglena gracilis Z

The effects of low-temperature acclimation and oxygen stress on tocopheron production were examined in the unicellular phytoflagellate Euglena gracilis Z. Cells were cultured photoheterotrophically at 27.5 ± 1°C with 5% carbon dioxide-95% air and 740 microeinsteins m⁻² s⁻¹ (photosynthetically active radiation) and served as controls. Low-temperature acclimation (12.5 ± 1°C) and high-oxygen stress (5% carbon dioxide-95% oxygen) were individually examined in the mass culturing of the algae. Chromatographic analyses demonstrated a six-to sevenfold enhancement of α-tocopherol production in temperature-stressed cells, along with a concomitant decline in the levels of α-tocotrienol and the absence of other tocopherol homologs. Oxygen-stressed cultures demonstrated the presence of high levels of α-tocopherylquinone; α-tocopheron and its homologs and precursors were absent or declined markedly. These findings are discussed in terms of the feasibility of microbial production of natural tocopherols. In addition, these results lend themselves to speculation regarding the biological role(s) of tocopherols as antioxidants and free radical scavengers in reducing photo-induced oxidative damage or lipid peroxidation toxicities or both in photosynthetically active E. gracilis Z.     

Nitrite-Reductase and Peroxynitrite Isomerization Activities of Methanosarcina acetivorans Protoglobin

Within the globin superfamily, protoglobins (Pgb) belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb), since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies) to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb*) are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II)) is biphasic and values of the second-order rate constant for the reduction of NO₂ ^– to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II) (Ma-Pgb*-Fe(II)-NO) are k _app1 = 9.6±0.2 M^–1 s^–1 and k _app2 = 1.2±0.1 M^–1 s^–1 (at pH 7.4 and 20°C). The k _app1 and k _app2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III)) is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III) and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are h _app = 3.8×10⁴ M^–1 s^–1 and h ₀ = 2.8×10^–1 s^–1 (at pH 7.4 and 20°C). The pH-dependence of h _on and h ₀ values reflects the acid-base equilibrium of peroxynitrite (pK _a = 6.7 and 6.9, respectively; at 20°C), indicating that HOONO is the species that reacts preferentially with the heme-Fe(III) atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species.     

Genome-Wide Association Study Identifies Variants in PMS1 Associated with Serum Ferritin in a Chinese Population

Only a small proportion of genetic variation in serum ferritin has been explained by variant genetic studies, and genome-wide association study (GWAS) for serum ferritin has not been investigated widely in Chinese population. We aimed at exploring the novel genetic susceptibility to serum ferritin, and performed this two stage GWAS in a healthy Chinese population of 3,495 men aged 20–69 y, including 1,999 unrelated subjects in the first stage and 1,496 independent individuals in the second stage. Serum ferritin was measured with electrochemiluminescence immunoassay, and DNA samples were collected for genotyping. A total of 1,940,243 SNPs were tested by using multivariate linear regression analysis. After adjusting for population stratification, age and BMI, the rs5742933 located in the 5′UTR region of PMS1 gene on chromosome 2 was the most significantly associated with ferritin concentrations (P-combined = 2.329×10⁻¹⁰) (β = −0.11, 95% CI: −0.14, −0.07). Moreover, this marker was about 200kb away from the candidate gene SLC40A1 which is responsible for iron export. PMS1 gene was the novel genetic susceptibility to serum ferritin in Chinese males and its relation to SLC40A1 needs further study.     

NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina

Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min⁻¹ mg⁻¹ and 10.0 ± 0.5 nmol min⁻¹ mg⁻¹, respectively. The highest k_cat/K_m value of ω3 and ω6 desaturase was 0.41 µM⁻¹ min⁻¹ and 0.09 µM⁻¹ min⁻¹ when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.     

β-Carotene Attenuates Angiotensin II-Induced Aortic Aneurysm by Alleviating Macrophage Recruitment in Apoe?/? Mice

Abdominal aortic aneurysm (AAA) is a common chronic degenerative disease characterized by progressive aortic dilation and rupture. The mechanisms underlying the role of α-tocopherol and β-carotene on AAA have not been comprehensively assessed. We investigated if α-tocopherol and β-carotene supplementation could attenuate AAA, and studied the underlying mechanisms utilized by the antioxidants to alleviate AAA. Four-months-old Apoe^−/− mice were used in the induction of aneurysm by infusion of angiotensin II (Ang II), and were orally administered with α-tocopherol and β-carotene enriched diet for 60 days. Significant increase of LDL, cholesterol, triglycerides and circulating inflammatory cells was observed in the Ang II-treated animals, and gene expression studies showed that ICAM-1, VCAM-1, MCP-1, M-CSF, MMP-2, MMP-9 and MMP-12 were upregulated in the aorta of aneurysm-induced mice. Extensive plaques, aneurysm and diffusion of inflammatory cells into the tunica intima were also noticed. The size of aorta was significantly (P = 0.0002) increased (2.24±0.20 mm) in the aneurysm-induced animals as compared to control mice (1.17±0.06 mm). Interestingly, β-carotene dramatically controlled the diffusion of macrophages into the aortic tunica intima, and circulation. It also dissolved the formation of atheromatous plaque. Further, β-carotene significantly decreased the aortic diameter (1.33±0.12 mm) in the aneurysm-induced mice (β-carotene, P = 0.0002). It also downregulated ICAM-1, VCAM-1, MCP-1, M-CSF, MMP-2, MMP-9, MMP-12, PPAR-α and PPAR-γ following treatment. Hence, dietary supplementation of β-carotene may have a protective function against Ang II-induced AAA by ameliorating macrophage recruitment in Apoe^−/− mice.     

Electrodiffusion,Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl⁻ conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl⁻ conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K⁺ and Cl⁻ were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl⁻ effluxes, induced by valinomycin or gramicidin at varied [K⁺]_o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K⁺]_o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P_K/P_Cl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P_Cl. Fitting the constant field current-voltage equation to the measured Cl⁻ currents yields P _Cl = 0.13 h⁻¹ with DIDS, compared to 0.49 h⁻¹ without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl⁻ current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.     

Genetic Modulation of Lipid Profiles following Lifestyle Modification or Metformin Treatment: The Diabetes Prevention Program

Weight-loss interventions generally improve lipid profiles and reduce cardiovascular disease risk, but effects are variable and may depend on genetic factors. We performed a genetic association analysis of data from 2,993 participants in the Diabetes Prevention Program to test the hypotheses that a genetic risk score (GRS) based on deleterious alleles at 32 lipid-associated single-nucleotide polymorphisms modifies the effects of lifestyle and/or metformin interventions on lipid levels and nuclear magnetic resonance (NMR) lipoprotein subfraction size and number. Twenty-three loci previously associated with fasting LDL-C, HDL-C, or triglycerides replicated (P = 0.04–1×10⁻¹⁷). Except for total HDL particles (r = −0.03, P = 0.26), all components of the lipid profile correlated with the GRS (partial |r| = 0.07–0.17, P = 5×10⁻⁵–1×10⁻¹⁹). The GRS was associated with higher baseline-adjusted 1-year LDL cholesterol levels (β = +0.87, SEE±0.22 mg/dl/allele, P = 8×10⁻⁵, P _interaction = 0.02) in the lifestyle intervention group, but not in the placebo (β = +0.20, SEE±0.22 mg/dl/allele, P = 0.35) or metformin (β = −0.03, SEE±0.22 mg/dl/allele, P = 0.90; P _interaction = 0.64) groups. Similarly, a higher GRS predicted a greater number of baseline-adjusted small LDL particles at 1 year in the lifestyle intervention arm (β = +0.30, SEE±0.012 ln nmol/L/allele, P = 0.01, P _interaction = 0.01) but not in the placebo (β = −0.002, SEE±0.008 ln nmol/L/allele, P = 0.74) or metformin (β = +0.013, SEE±0.008 nmol/L/allele, P = 0.12; P _interaction = 0.24) groups. Our findings suggest that a high genetic burden confers an adverse lipid profile and predicts attenuated response in LDL-C levels and small LDL particle number to dietary and physical activity interventions aimed at weight loss.