Embryonic fibroblasts from Gpx4+/- mice: a novel model for studying the role of membrane peroxidation in biological processes

A previous study using mice null for Gpx4 indicates that PHGPx plays a critical role in antioxidant defense and is essential for the survival of the mouse. In the present study, we further analyzed the stress response of MEFs (murine embryonic fibroblasts) derived from mice heterozygous for the Gpx4 gene (Gpx4(+/-) mice). MEFs from Gpx4(+/-) mice have a 50% reduction in PHGPx expression without any changes in the activities of other major antioxidant defense enzymes. Compared to MEFs from Gpx4(+/+) mice, MEFs from Gpx4(+/-) mice were more sensitive to exposure to the oxidizing agent t-butyl hydroperoxide (t-BuOOH), and t-BuOOH exposure induced increased apoptosis in MEFs from Gpx4(+/-) mice. When cultured at low cell density, MEFs from Gpx4(+/-) mice also showed retarded growth under normal culture conditions (20% oxygen) that was reversed by culturing under low oxygen (2% oxygen). In addition, oxidative damage was increased in the MEFs from the Gpx4(+/-) mice, as indicated by increased levels of F(2)-isoprostanes and 8-oxo-2-deoxyguanosine in these cells. Our data demonstrate that MEFs from Gpx4(+/-) mice are more sensitive to oxidative stress because of reduced expression of PHGPx.     

Nitrite and nitrous oxide production by Methylosinus trichosporium

Conditions for the production of nitrite and nitrous oxide by an obligate methanotroph, Methylosinus trichosporium (OB 3b), were studied. The rate of nitrite production (V NO2-) was correlated with the concentration of ammonia up to 20 mM in the presence of sufficient amounts of oxygen and inversely correlated with the amounts of methane in the system. The rate of nitrous oxide (N2O) production (V N2O) was correlated positively with V NO2- and the amount of nitrite produced and inversely with the oxygen concentration in the system. Nitrite started to disappear when either oxygen or methane or both were depleted, but only a part of the loss could be accounted for by an increase in N2O. Maximum rates of nitrite and N2O production by Ms. trichosporium were 6.9 X 10(-16) and 2.2 X 10(-17) mol . cell-1 X day-1, respectively. These values are about 0.2 and 1.6% of the values for Nitrosomonas europaea. Therefore, production of nitrite and N2O by methanotrophs in aquatic environments may not be as significant as that of Nitrosomonas.     

Biodegradation of chloromethane by Pseudomonas aeruginosa strain NB1 under nitrate-reducing and aerobic conditions

Pseudomonas aeruginosa strain NB1 uses chloromethane (CM) as its sole source of carbon and energy under nitrate-reducing and aerobic conditions. The observed yield of NB1 was 0.20 (+/-0.06) (mean +/- standard deviation) and 0.28 (+/-0.01) mg of total suspended solids (TSS) mg of CM(-1) under anoxic and aerobic conditions, respectively. The stoichiometry of nitrate consumption was 0.75 (+/-0.10) electron equivalents (eeq) of NO(3)(-) per eeq of CM, which is consistent with the yield when it is expressed on an eeq basis. Nitrate was stoichiometrically converted to dinitrogen (0.51 +/- 0.05 mol of N(2) per mol of NO(3)(-)). The stoichiometry of oxygen use with CM (0.85 +/- 0.21 eeq of O(2) per eeq of CM) was also consistent with the aerobic yield. Stoichiometric release of chloride and minimal accumulation of soluble metabolic products (measured as chemical oxygen demand) following CM consumption, under anoxic and aerobic conditions, indicated complete biodegradation of CM. Acetylene did not inhibit CM use under aerobic conditions, implying that a monooxygenase was not involved in initiating aerobic CM metabolism. Under anoxic conditions, the maximum specific CM utilization rate (k) for NB1 was 5.01 (+/-0.06) micromol of CM mg of TSS(-1) day(-1), the maximum specific growth rate (micro(max)) was 0.0506 day(-1), and the Monod half-saturation coefficient (K(s)) was 0.067 (+/-0.004) microM. Under aerobic conditions, the values for k, micro(max), and K(s) were 10.7 (+/-0.11) micromol of CM mg of TSS(-1) day(-1), 0.145 day(-1), and 0.93 (+/-0.042) microM, respectively, indicating that NB1 used CM faster under aerobic conditions. Strain NB1 also grew on methanol, ethanol, and acetate under denitrifying and aerobic conditions, but not on methane, formate, or dichloromethane.     

Photooxidation of pterin in aqueous solutions: biological and biomedical implications

Studies of the photochemical reactivity of pterin (= 2-aminopteridin-4(3H)-one; PT) in acidic (pH 5.0-6.0) and alkaline (pH 10.2-10.8) aqueous solutions have been performed. The photochemical reactions were followed by UV/VIS spectrophotometry, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), and an enzymatic method for H2O2 determination. PT is not light-sensitive in the absence of molecular oxygen, but it undergoes photooxidation in the presence of O2, yielding several nonpteridinic products. The quantum yields for PT disappearance were found to be 8.2 (+/-0.6) x 10(-4) and 1.2 (+/-0.2) x 10(-3) in acidic and alkaline media, respectively. H2O2 was detected and quantified in irradiated solutions of PT; and its importance from a biomedical point of view is discussed. The rate constant of the chemical reaction between singlet oxygen ((1)O2) and PT was determined to be 2.5 (+/-0.2) x 10(5) l mol(-1) s(-1) in alkaline medium, and the role of (1)O2 in the photooxidation of pterin was evaluated.     

Whole animal and gill tissue oxygen uptake in the Eastern oyster, Crassostrea virginica: Effects of hypoxia, hypercapnia, air exposure, and infection with the protozoan parasite Perkinsus marinus(1)

The Eastern oyster, Crassostrea virginica, lives in shallow coastal waters and experiences many different environmental extremes including hypoxia, hypercapnia and air exposure and many oysters are infected with the protozoan parasite Perkinsus marinus. The effects of these conditions on oyster metabolism, as measured by oxygen uptake, were investigated. Mild hypercapnia had no effect on the ability of oysters to regulate oxygen uptake in hypoxic water, as measured by the B2 coefficient of oxygen regulation. The average B2 was -0.060x10(-3) (+/-0.01x10(-3) S.E.M.; n=20; low and high CO(2) treatments combined) in oysters uninfected with P. marinus and -0.056x10(-3) (+/-0.01x10(-3) S.E.M.; n=16; low and high CO(2) treatments combined) in infected oysters. There was no significant effect of light to moderate infections of P. marinus on oxygen regulation. Nor did the presence of P. marinus have an effect on the rate of oxygen uptake of whole animals in well-aerated water. In well-aerated conditions, oxygen uptake was significantly reduced by moderate hypercapnia in oysters when data from uninfected and infected oysters were combined. Mean oxygen uptake of infected oysters under hypercapnia (pCO(2)=6-8 Torr; pH 7) was 9.10 μmol O(2) g ww(-1) h(-1) +/-0.62 S.E.M. (n=9), significantly different from oxygen uptake under normocapnia (pCO(2) 相似文献   

Inhibition of dopamine beta-hydroxylase by bidentate chelating agents

1-2H-Phthalazine hydrazone (hydralazine; HYD), 2-1H-pyridinone hydrazone (2-hydrazinopyridine; HP), 2-quinoline-carboxylic acid (QCA), 1-isoquinolinecarboxylic acid (IQCA), 2,2'-bi-1H-imidazole (2,2'-biimidazole; BI), and 1H-imidazole-4-acetic acid (imidazole-4-acetic acid; IAA) directly and reversibly inhibit homogeneous soluble bovine dopamine beta-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1). HYD, QCA and IAA show competitive allosteric inhibition of dopamine beta-hydroxylase with respect to ascorbate (Kis = 5.7(+/- 0.9) microM, 0.14(+/- 0.03) mM, 0.80(+/- 0.20) mM; nH = 1.4(+/- 0.1), 1.8(+/- 0.4), 2.8(+/- 0.6), respectively). HYD and IAA show slope and intercept mixed-type allosteric inhibition of dopamine beta-hydroxylase with respect to tyramine. QCA shows allosteric uncompetitive inhibition of dopamine beta-hydroxylase with respect to tyramine. HP, BI and IQCA all show linear competitive inhibition (Kis = 1.9(+/- 0.3) microM, 21(+/- 6) microM, and 0.9(+/- 0.3) microM, respectively) with respect to ascorbate. HP and BI show linear mixed-type while IQCA shows linear uncompetitive inhibition of dopamine beta-hydroxylase with respect to tyramine. In the presence of HP, HYD or IAA intersecting double-reciprocal plots of the initial velocity as a function of tyramine concentration at differing fixed levels of ascorbate are observed. These findings are consistent with a uni-uni-ping-pong-ter-bi kinetic mechanism for dopamine beta-hydroxylase that involves a ternary enzyme-ascorbate-tyramine-oxygen complex. The results for HYD, QCA and IAA are the first examples of allosteric inhibitor interactions with dopamine beta-hydroxylase.     

Quantitative determination of glycopyrrolate in human plasma by liquid chromatography-electrospray ionization mass spectrometry: the use of a volatile ion-pairing agent during both liquid-liquid extraction and liquid chromatography

The work presented here deals with the development of a quantitative tool for the determination of the quaternary ammonium anticholinergic glycopyrrolate in human plasma samples. Mepenzolate was used as an internal standard. The plasma samples were subjected to a suitable sample clean-up consisting of a simple and relatively fast, two step liquid-liquid ion-pair extraction procedure. The chromatography, using the same volatile ion-pair reagent heptafluorobutyric acid (HFBA), takes only 10 min. Relative standard deviation of retention times was never above 2.26% (n=36). The method was fully validated based on the US FDA Bioanalytical Method Validation Guidance for Industry. As such, a quantitative ESI-LC-MS(/MS) (TOF mass spectrometry) method was optimized for the absolute quantification of glycopyrrolate in human plasma in a concentration range from 0.101 to 101 ng/mL using a quadratic calibration function (R(2)=0.9995), y=-2.21 x 10(-4) (+/-3.93 x 10(-5))xx(2)+5.85 x 10(-2) (+/-5.27 x 10(-3))xx+4.08 x 10(-3) (+/-4.82 x 10(-4)). For the three QC concentrations (QC(1) 0.252, QC(2) 2.52, and QC(3) 25.2ng/mL) and the LLOQ (0.101 ng/mL), total precision was under 20% (18.0% (n=6) at the LLOQ) and maximum accuracy was 112% (88.9% for the LLOQ, n=6). Absolute matrix effect (maximum 133%+/-9.59, n=3), absolute recovery (better than 41.8%+/-2.22, n=3), relative (inter-subject) matrix effect (maximum 10.9%+/-1.45, n=4) and process efficiency (better than 45.2%+/-5.74, n=3) too were assessed at the 3 QC concentrations.     

Performance characteristics of a two-stage dark fermentative system producing hydrogen and methane continuously

The performance of a mesophilic two-stage system generating hydrogen and methane continuously from sucrose (10-30 g/L) was investigated. A hydrogen-generating CSTR followed by an upflow anaerobic filter were both inoculated with anaerobically digested sewage sludge, and ORP, pH, gas output, %H(2), %CH(4) and %CO(2) monitored. pH was controlled with NaOH, KOH or Ca(OH)(2). Using NaOH as alkali with 10 g/L sucrose, yields of 1.62 +/- 0.2 mol H(2)/mol hexose added and 323 mL CH(4)/gCOD added to the hydrogen and methane reactors respectively were achieved. The overall chemical oxygen demand (COD) reduction was 92.6% with 0.90 +/- 0.1 g/L sodium and 316 +/- 40 mg/L residual acetate in the methane reactor. Operation at 20 g/L sucrose and NaOH as alkali led to impaired volatile fatty acid (VFA) degradation in the methane reactor with 2.23 +/- 0.2 g/L sodium, 1,885 mg/L residual acetate, a hydrogen yield of 1.47 +/- 0.1 mol/mol hexose added, a methane yield of 294 mL/gCOD added and an overall COD reduction of 83%. Using Ca(OH)(2) as alkali with 20 g/L sucrose gave a hydrogen yield of 1.29 +/- 0.3 mol/mol hexose added, a methane yield of 337 mL/gCOD added and improved the overall COD reduction to 91% with residual acetate concentrations of 522 +/- 87 mg/L. Operation at 30 g/L sucrose with Ca(OH)(2) gave poorer overall COD reduction (68%), a hydrogen yield of 1.47 +/- 0.2 mol/mol hexose added, a methane yield of 138 mL/gCOD added and residual acetate 7,343 +/- 715 mg/L. It was shown that sodium toxicity and overloading are important issues for successful anaerobic digestion of effluent from biohydrogen reactors in high rate systems.     

Methanogenesis, fires and the regulation of atmospheric oxygen

The Gaia hypothesis states that the composition, oxidation-reduction potential and the temperature of the Earth's lower atmosphere are modulated by and for the biota living on the surface (Lovelock, 1972; Margulis and Lovelock, 1974). A corollary is that atmospheric oxygen is presently regulated at about 21% for the dominant life forms today: vascular plants and metazoa. We suggest that the enormous annual production of methane (of the order of 10¹⁴ mol) is directly related to the short term modulation of oxygen concentration. Atmospheric oxygen results from the burial of reduced carbon; methanogenesis and subsequent atmospheric oxidation of methane prevents that burial. We also present experimental work on the probability of ignition of vegetation as a function of increasing oxygen concentration (Watson, 1978). Both the experiments and consideration of the fossil record lead us to conclude that oxygen has been regulated by methane (and perhaps by N₂O and others) at about 10–25% for very long periods relative to the atmospheric residence times of these reactive gases.     

Evaluation of support materials for the immobilization of sulfate-reducing bacteria and methanogenic archaea

This paper reports on the adhesion of sulfate-reducing bacteria (SRB) and methanogenic archaea on polyurethane foam (PU), vegetal carbon (VC), low-density polyethylene (PE) and alumina-based ceramics (CE). Anaerobic differential reactors fed with a sulfate-rich synthetic wastewater were used to evaluate the formation of a biofilm. The PU presented the highest specific biomass concentration throughout the experiment, achieving 872 mg TVS/g support, while 84 mg TVS/g support was the maximum value obtained for the other materials. FISH results showed that bacterial cells rather than archaeal cells were predominant on the biofilms. These cells, detected with EUB338 probe, accounted for 76.2% (+/-1.6%), 79.7% (+/-1.3%), 84.4% (+/-1.4%) and 60.2% (+/-1.0%) in PU, VC, PE and CE, respectively, of the 4'6-diamidino-2-phenylindole (DAPI)-stained cells. From these percentages, 44.8% (+/-2.1%), 55.4% (+/-1.2%), 32.7% (+/-1.4%) and 18.1% (+/-1.1%), respectively, represented the SRB group. Archaeal cells, detected with ARC915 probe, accounted for 33.1% (+/-1.6%), 25.4% (+/-1.3%), 22.6% (+/-1.1%) and 41.9% (+/-1.0%) in PU, VC, PE and CE, respectively, of the DAPI-stained cells. Sulfate reduction efficiencies of 39% and 45% and mean chemical oxygen demand (COD) removal efficiencies of 86% and 90% were achieved for PU and VC, respectively. The other two supports, PE and CE, provided mean COD removal efficiencies of 84% and 86%, respectively. However, no sulfate reduction was observed with these supports.     

Effect of inspired oxygen on periodic breathing in methy-CpG-binding protein 2 (Mecp2) deficient mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (Mecp2) that encodes a DNA binding protein involved in gene silencing. Periodic breathing (Cheyne-Stokes respiration) is commonly seen in RTT. Freely moving mice were studied with continuous recording of pleural pressure by telemetry. Episodes of periodic breathing in heterozygous Mecp2 deficient (Mecp2(+/-)) female mice (9.4 +/- 2.2 h(-1)) exceeded those in wild-type (Mecp2(+/+)) animals (2.5 +/- 0.4 h(-1)) (P = 0.010). Exposing Mecp2(+/-) animals to 40% oxygen increased the amount of periodic breathing from 118 +/- 25 s/30 min in air to 242 +/- 57 s/30 min (P = 0.001), and 12% oxygen tended to decrease it (67 +/- 29 s/30 min, P = 0.14). Relative hyperoxia and hypoxia did not affect the incidence of periodic breathing in Mecp2(+/+) animals. The ventilation/apnea ratio (V/A) was less at all levels of oxygen in heterozygous Mecp2(+/-) females compare with wild type (P = 0.003 to P < 0.001), indicating that their loop gain is larger. V/A in Mecp2(+/-) fell from 2.42 +/- 0.18 in normoxia to 1.82 +/- 0.17 in hyperoxia (P = 0.05) indicating an increase in loop gain with increased oxygen. Hyperoxia did not affect V/A in Mecp2(+/+) mice (3.73 +/- 0.28 vs. 3.5 +/- 0.28). These results show that periodic breathing in this mouse model of RTT is not dependent on enhanced peripheral chemoreceptor oxygen sensitivity. Rather, the breathing instability is of central origin.     

Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (K(A)) of 10(+/-7)x10(6), 5.7(+/-3)x10(6), 2.0(+/-2)x10(6) and 2.0(+/-3)x10(4) M(-1) for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(+/-2)x10(6), 3.2(+/-2)x10(4), 1.76(+/-1)x10(5) and 1.5(+/-2)x10(3) M(-1) respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 degrees C). The stability of metal ion binding to the sensory site follows the Irving-Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents.     

Associations of methanotrophs with the roots and rhizomes of aquatic vegetation.

Results of an in vitro assay revealed that root-associated methane consumption was a common attribute of diverse emergent wetland macrophytes from a variety of habitats. Maximum potential uptake rates (Vmaxp) varied between about 1 and 10 micromol g (dry weight)-1 h-1, with no obvious correlation between rate and gross morphological characteristics of the plants. The Vmaxp corresponded to about 2 x 10(8) to 2 x 10(9) methanotrophs g (dry weight)-1, assuming that the root-associated methanotrophs have cell-specific activities comparable to those of known isolates. Vmaxp varied seasonally for an aquatic grass, Calamogrostis canadensis, and for the cattail, Typha latifolia, with highest rates in the late summer. Vmaxp was well correlated with ambient temperature for C. canadensis but weakly correlated for T. latifolia. The seasonal changes in Vmaxp, as well as inferences from apparent half-saturation constants for methane uptake (Kapp; generally 3 to 6 microM), indicated that oxygen availability might be more important than methane as a rate determinant. In addition, roots incubated under anoxic conditions showed little or no postanoxia aerobic methane consumption, indicating that root-associated methanotrophic populations might not tolerate variable oxygen availability. Hybridization of oligodeoxynucleotide probes specific for group I or group II methylotrophs also varied seasonally. The group II-specific probe consistently hybridized to a greater extent than the group I probe, and the relative amount of group II probe hybridization to C. canadensis root extracts was positively correlated with Vmaxp.     

Influence of rumen protozoa on methane emission in ruminants: a meta-analysis approach

A meta-analysis was conducted to evaluate the effects of protozoa concentration on methane emission from ruminants. A database was built from 59 publications reporting data from 76 in vivo experiments. The experiments included in the database recorded methane production and rumen protozoa concentration measured on the same groups of animals. Quantitative data such as diet chemical composition, rumen fermentation and microbial parameters, and qualitative information such as methane mitigation strategies were also collected. In the database, 31% of the experiments reported a concomitant reduction of both protozoa concentration and methane emission (g/kg dry matter intake). Nearly all of these experiments tested lipids as methane mitigation strategies. By contrast, 21% of the experiments reported a variation in methane emission without changes in protozoa numbers, indicating that methanogenesis is also regulated by other mechanisms not involving protozoa. Experiments that used chemical compounds as an antimethanogenic treatment belonged to this group. The relationship between methane emission and protozoa concentration was studied with a variance−covariance model, with experiment as a fixed effect. The experiments included in the analysis had a within-experiment variation of protozoa concentration higher than 5.3 log₁₀ cells/ml corresponding to the average s.e.m. of the database for this variable. To detect potential interfering factors for the relationship, the influence of several qualitative and quantitative secondary factors was tested. This meta-analysis showed a significant linear relationship between methane emission and protozoa concentration: methane (g/kg dry matter intake)=−30.7+8.14×protozoa (log₁₀ cells/ml) with 28 experiments (91 treatments), residual mean square error=1.94 and adjusted R²=0.90. The proportion of butyrate in the rumen positively influenced the least square means of this relationship.     

Fewer metabolites of dietary choline reach the blood of rats after treatment with lithium

Choline is an important precursor for the biosynthesis of acetylcholine, phosphatidylcholine and sphingomyelin. It is also a major source of labile methyl groups. Lithium is an important component of the treatment of bipolar affective illness, and it inhibits choline transport across membranes. We studied the effect of lithium treatment upon the appearance in blood, liver and intestine of metabolites formed from dietary choline. Rats were treated for 9 days with 2 mEq/kg lithium carbonate or water. Animals were fasted overnight, and on the 10th day were fed with a solution containing radiolabeled choline chloride. The lithium-treated groups also received 2.0 mEq/kg lithium as part of this solution. After an oral dose of 1 ml of a 1 mM choline solution, the lithium-treated animals had significantly lower levels of choline-derived radiolabel in blood than did controls at 30, 60, 120, and 180 minutes (47% (+/- 5%; SEM), 51% (+/- 7%), 59% (+/- 4%) and 74% (+/- 9%), respectively). We observed similar decreases of the accumulation in blood, at 180 minutes after the dose, of choline-derived radiolabel when choline was administered at lower or higher concentrations. After an oral treatment containing 0.1, 1 or 10 mM choline, lithium treated animals accumulated 69% (+/- 6%; SEM), 66% (+/- 11%) and 72% (+/- 7%) as much radiolabel in serum as did controls. Most of the radiolabel found in blood at 180 minutes was in metabolites of choline which are formed within liver (betaine and phosphatidylcholine). The diminished accumulation of radiolabel in serum after lithium treatment was not due to increased accumulation of label by erythrocytes, liver or gut wall. We suggest that lithium influences the release by liver of betaine and phosphatidylcholine.     

Protein unfolding in drug-RNase complexes

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of P-O5' bonds in RNA on the 3' side of pyrimidine to form cyclic 2', 5'-phosphates. It has several high affinity binding sites that make it possible target for many organic and inorganic molecules. Ligand binding to RNase A can alter protein secondary structure and its catalytic activity. In this review, the effects of several drugs such as AZT (anti-AIDS), cis-Pt (antitumor), aspirin (anti-inflammatory), and vitamin C (antioxidant) on the stability and conformation of RNase A in vitro are compared. The results of UV-visible, FTIR, and CD spectroscopic analysis of RNase complexes with aspirin, AZT, cis-Pt, and vitamin C at physiological conditions are discussed here. Spectroscopic results showed one major binding for each drug-RNase adduct with KAZT=5.29 (+/-1.6)x10(4) M(-1), Kaspirin=3.57 (+/-1.4)x10(4) M(-1), Kcis-Pt=5.66 (+/-1.9)x10(3) M(-1), and Kascorbate=3.50 (+/-1.5)x10(3) M(-1). Major protein unfolding occurred with reduction of alpha-helix from 29% (free protein) to 20% and increase of beta-sheet from 39% (free protein) to 45% in the aspirin-, ascorbate-, and cis-Pt-RNase complexes, while minor increase of alpha-helix was observed for AZT-RNase adduct.     

Volumetric scale-up of a three stage fermentation system for food waste treatment

In this study, a volumetric scale-up of this system was designed and built on a field pilot-scale (total digester volume 10 m(3)), with the results from the field pilot-scale experiments compared with those from the bench-scale (total digester volume 0.4 m(3)) process prior to scale-up. The reduction rate of total chemical oxygen demand (tCOD) and the maximum methane content produced in the biogas from the bench-scale system were 90.6% and 72%; whereas those from the field pilot-scale system were 90.1% and 68%, respectively. The estimated methane yields were 282 and 254 l CH(4)/kg tCOD(degraded) in bench and field pilot-scale fermentation systems, respectively. These results indicate that the three stage fermentation system developed in this study can be applied as a commercial process for the disposal of food waste in view of process stability.     

Electrostatically mediated interactions between cationic lipid-DNA particles and an anionic surface.

In an effort to model the interaction of lipid-based DNA delivery systems with anionic surfaces, such as a cell membrane, we have utilized microelectrophoresis to characterize how electrokinetic measurements can provide information on surface charge and binding characteristics. We have established that cationic lipids, specifically N-N-dioleoyl-N,N-dimethylammonium chloride (DODAC), incorporated into liposomes prepared with 1, 2-dioleoyl-i-glycero-3-phosphoethanolamine (DOPE) or 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at 50 mol%, change the inherent electrophoretic mobility of anionic latex polystyrene beads. Self-assembling lipid-DNA particles (LDPs), prepared at various cationic lipid to negative DNA phosphate charge ratios, effected no changes in bead mobility when the LDP charge ratio (+/-) was equal to or less than 1. Increasing the LDP concentration in a solution of 0.1% (w/v) anionic beads resulted in a charge reversal effect when a net charge of LDP to total bead charge ratio (+/-) of 1:1 was observed. LDP formulations, utilizing either DOPE or DOPC, showed similar titration profiles with a charge reversal observed at a 1:1 net LDP to bead charge ratio (+/-). It was confirmed through centrifugation studies that the DNA in the LDP was associated with the anionic latex beads through electrostatic interactions. LDP binding, rather than the binding of dissociated cationic lipids, resulted in the observed electrophoretic mobility changes of the anionic latex beads.     

Successful in vitro culture of early cleavage stage embryos recovered from superovulated red deer (Cervus elaphus)

Three separate embryo culture systems were evaluated for their ability to support development of early cleavage stage red deer (Cervus elaphus ) embryos: ligated sheep oviducts (Treatment A); cervine oviduct epithelial monolayer in TCM 199 + 10% deer serum (Treatment B); synthetic oviduct fluid + 20% human serum at 7% O(2) atmosphere (Treatment Q. In addition, 2 superovulation protocols were compared for their efficacy in producing early cleavage stage embryos. Twenty red deer (2 to 7 yr old) were synchronized in April with intravaginal CIDR devices for 12 d. All animals received a total of 0.4 units of ovine FSH administered in 8 equal doses, 12 h apart, beginning 72 h before removal of CIDR devices. The deer additionally received 200 IU PMSG, either with the first FSH injection (Group 1, n = 10) or with the last FSH injection (Group 2, n = 10). Hinds were placed with fertile stags following withdrawal of CIDR devices. Ova were collected by surgical recovery 63 h post CIDR removal. At the time of collection, animals in Group 2 had a significantly greater mean (+/- SEM) ovulation rate (11.2 +/- 2.4 vs 5.3 +/- 2.4), with more animals responding to treatment (>1 ovulation), than the animals in Group 1 (10/10 vs 4/10). Late in the breeding season (June), 10 additional red deer (Group 3, Experiment 2) were superovulated using the same protocol as for the deer in Group 2, with ova collection advanced by 24 h. Mean (+/- SEM) ovulation rate was 6.4 +/- 1.2 with 9 10 animals responding. Ova recovery did not differ among the groups (range 73 to 87%). Superovulation treatment did not affect cultured embryo development to the morula/blastocyst stage. Furthermore, there was no difference among the 3 culture systems in their support of development either to the morula (range 50 to 58%) or to the blastocyst (range 22 to 26%) stage. After laparoscopic transfer of 4 morula/blastocyst embryos to recipient red deer (2 from Treatment B and 2 from Treatment C) 2 live calves were born from embryos cultured in Treatment B.     

Quantitative structure toxicity relationships for catechols in isolated rat hepatocytes

One- and two-parameter quantitative structure toxicity relationship (QSTR) equations were obtained to describe the cytotoxicity of isolated rat hepatocytes induced by 23 catechols in which LD(50) represents the catechol concentration required to induce 50% cytotoxicity in 2 h. A QSTR equation logLD(50) (microM = - 0.464(+/-0.065) log P + 3.724(+/-0.114) (n = 20, r(2) = 0.740, s(y,x) = 0.372, P < 1 x 10(-6), outliers: 4-methoxycatechol, 3-methoxycatechol, L-dopa) was derived where logP represents octanol/water partitioning. Outliers were determined by adopting a statistical method to standardize the identification of outliers. When pK(a1), the first ionization constant, was considered as a contributing parameter a two-parameter QSTR equation was derived: logLD(50) (microM = - 0.343(+/-0.058) log P - 0.116(+/-0.041) pK(a1)+4.389 (+/-0.315) (n = 22, r(2) = 0.738, s(y,x) = 0.375, P < 0.01, outlier: 4-methoxycatechol). Replacing logP with logD(7.4), the partition coefficient at pH 7.4, improved the first correlation by limiting the outlier to 4-methoxycatechol: logLD(50) (microM)=-0.252(+/-0.039) logD(7.4)+3.168(+/-0.090) (n = 22, r(2) = 0.671, s(y,x) = 0.420, P < 1 x 10(-5). In this study, 4-methoxycatechol (readily autooxidizable) was found to be an outlier for all QSTR equations derived. These findings point to lipophilicity and pK(a1) as two important characteristics of catechols that can be used to predict their cytotoxicity towards isolated rat hepatocytes. The catechols with the higher lipophilicity/distribution coefficient, the lower degree of ionization and the higher pK(a(catechol)) were more toxic towards hepatocytes than the other catechols.