A Simple,Large-Scale Overexpression Method of Deriving Carbon Monoxide Dehydrogenase II from Thermophilic Bacterium Carboxydothermus hydrogenoformans

We established an Na₂S-free, large-scale overexpression system of deriving CODH II from thermophilic bacterium Carboxydothermus hydrogenoformans in Escherichia coli using a large-scale fermentor. Recombinant-CODH II showed a CO oxidation activity of 9,600 U/mg. In addition, recombinant-CODH II exhibited considerable CO₂ reduction activity, of 16.9 U/mg.     

Characterization and purification of carbon monoxide dehydrogenase from Methanosarcina barkeri.

Carbon monoxide-dependent production of H2, CO2, and CH4 was detected in crude cell extracts of acetate-grown Methanosarcina barkeri. This metabolic transformation was associated with an active methyl viologen-linked CO dehydrogenase activity (5 to 10 U/mg of protein). Carbon monoxide dehydrogenase activity was inhibited 85% by 10 microM KCN and was rapidly inactivated by O2. The enzyme was nearly homogeneous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (ca. 5%). The native purified enzyme displayed a molecular weight of 232,000 and a two-subunit composition of 92,000 and 18,000 daltons. The enzyme was shown to contain nickel by isolation of radioactive CO dehydrogenase from cells grown in 63Ni. Analysis of enzyme kinetic properties revealed an apparent Km of 5 mM for CO and a Vmax of 1,300 U/mg of protein. The spectral properties of the enzyme were similar to those published for CO dehydrogenase from acetogenic anaerobes. The physiological functions of the enzyme are discussed.     

Superoxide and hydrogen peroxide suppression by metal ions and their EDTA complexes

Redox-active metal ions such as Fe(II)\(III) and Cu(I)\(II) have been proposed to activate reactive oxygen and nitrogen species (RONS) and thus, perpetuate oxidative damage. Here, we show that concentrations of metal ions and EDTA complexes with superoxide-destroying activities equivalent to 1 U SOD are Fe(III) 5.1 microM, Mn(II) 0.77 microM, Cu(II)-EDTA 3.55 microM, Fe(III)-EDTA 2.34 microM, and Mn(II)-EDTA 1.38 microM. The most active being the aquated Cu(II) species which exhibited superoxide-destroying activity equivalent to 2U of SOD at 0.29 microM. Hydrogen peroxide-destroying activities were as follows Fe(III)-EDTA ca. 70 U/mg and aquated Fe(III) 141 U/mg. In contrast, DTPA prevented superoxide-destroying activity and significantly depleted hydrogen peroxide-destroying activity. In conclusion, non-protein bound transition metal ions may have significant anti-oxidant effects in biological systems. Caution should be employed in bioassays when chelating metal ions. Our results demonstrate that DTPA is preferential to EDTA for inactivating redox-active metal ions in bioassays.     

一氧化碳吸入对脂多糖诱导大鼠急性肺损伤的保护作用

血红素氧合酶(heme oxygenase,HO)降解血红素的主要代谢产物一氧化碳(carbon monoxide,CO)具有抗氧化、抗炎症和抑制细胞凋亡作用,而脂多糖(lipopolysaccharide,LPS)诱导的肺组织过氧化、炎症性损伤及大量肺泡上皮和血管内皮细胞凋亡正是导致肺损伤(lung injury,LI)的关键.由此我们猜想,CO有可能通过上述机制对LI起保护作用.通过静脉注入LPS(5 mg/kg体重)诱导大鼠LI,观察吸入室内空气或2.5×10-4(V/V)CO 3 h后,肺氧化酶学、炎症细胞因子、细胞凋亡、HO-1表达及组织形态学变化.结果显示,静脉注入LPS诱导LI后,CO吸入组大鼠肺肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素6(interlukin-6,IL-6)、丙二醛(maleic dialdehyde,MDA)、髓过氧化物酶(myeloperoxidase,MPO)和细胞凋亡分别为(0.91±0.25)pg/mg蛋白、(0.64±0.05)pg/mg蛋白、(1.02±0.23)nmol/mg蛋白、(7.18±1.62)U/mg蛋白、(1.60±0.34)%,均显著低于LI组的(1.48±0.23)pg/mg蛋白、(1.16±0.26)pg/mg蛋白、(1.27+0.33)nmol/mg蛋白、(8.16+1.49)U/mg蛋白、(3.18±0.51)%(P＜0.05).CO吸入组HO-1、白细胞介素10(interlukin-10,IL-10)表达和超氧化物歧化酶(superoxide dismutase,SOD)活性分别为(5.43±0.92)、(0.26±0.07)pg/mg蛋白、(60.09±10.21)U/mg蛋白,它们均显著高于LI组的(3.08±0.82)、(0.15±0.03)pg/mg蛋白、(50.98±6.88)U/mg蛋白(P＜0.05).与LI组相比,CO吸入组肺损伤减轻.研究结果表明,低浓度CO吸入通过抗氧化、抗炎症、抑制细胞凋亡、上调HO-1表达而减轻LPS诱导的肺损伤.     

The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N2-fixing bacterium Clostridium pasteurianum

The H₂ uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H₂ evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N₂-fixing conditions have the highest H₂ uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction (M_r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H₂ oxidation and H₂ evolution at rates of 3000 and 5.9 μmol H₂ consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.     

Purification and some properties of two proteases from Pseudomonas aeruginosa No. 700/75

Two extracellular proteases have been isolated from the culture supernatant of a virulent strain of Pseudomonas aeruginosa. The enzymes were purified in a three-step procedure involving ammonium sulfate fractionation, acetone precipitation and column chromatography on DE-52 cellulose. The specific activity of protease I was 22.2 U/mg of protein and protease II 6.6 U/mg of protein. Immunological properties and electrophoretic mobilities of the two forms were different. The two forms differ in substrate specificity (only from I exhibited elastinolytic activity) and pH optimum (pH 7.5 and pH 10 for form I and II, respectively).     

New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) III: Endogenous Inhibition of Angiotensin Converting Enzyme (ACE) Provides Protection against Cardiovascular Diseases

ACE inhibitor drugs decrease mortality by up to one-fifth in cardiovascular patients. Surprisingly, there are reports dating back to 1979 suggesting the existence of endogenous ACE inhibitors. Here we investigated the clinical significance of this potential endogenous ACE inhibition.ACE concentration and activity was measured in patient''s serum samples (n = 151). ACE concentration was found to be in a wide range (47–288 ng/mL). ACE activity decreased with the increasing concentration of the serum albumin (HSA): ACE activity was 56±1 U/L in the presence of 2.4±0.3 mg/mL HSA, compared to 39±1 U/L in the presence of 12±1 mg/mL HSA (values are mean±SEM). Effects of the differences in ACE concentration were suppressed in human sera: patients with ACE DD genotype exhibited a 64% higher serum ACE concentration (range, 74–288 ng/mL, median, 155.2 ng/mL, n = 52) compared to patients with II genotype (range, 47–194 ng/mL, median, 94.5 ng/mL, n = 28) while the difference in ACE activities was only 32% (range, 27.3–59.8 U/L, median, 43.11 U/L, and range 15.6–55.4 U/L, median, 32.74 U/L, respectively) in the presence of 12±1 mg/mL HSA. No correlations were found between serum ACE concentration (or genotype) and cardiovascular diseases, in accordance with the proposed suppressed physiological ACE activities by HSA (concentration in the sera of these patients: 48.5±0.5 mg/mL) or other endogenous inhibitors.Main implications are that (1) physiological ACE activity can be stabilized at a low level by endogenous ACE inhibitors, such as HSA; (2) angiotensin II elimination may have a significant role in angiotensin II related pathologies.     

CO2 and O2 dependence of PS II activity in C4 plants having genetically produced deficiencies in the C3 or C4 cycle

The CO2 dependence of rates of CO2 fixation (A) and photochemistry of PS II at 5, 15 and 30% O2 were analyzed in the C4 plant Amaranthus edulis having a C4 cycle deficiency [phosphoenolpyruvate carboxylase (PEPC) mutants], and in the C4 plant Flaveria bidentis having a C3 cycle deficiency [Rubisco small subunit antisense (SSU)]. In the wild type (WT) A. edulis and its heterozygous mutant having less than 50% WT PEPC activity there was a similar dependence of A and PS II photochemistry on varying CO2, although the CO2 saturated rates were 25% lower in heterozygous plants. The homozygous plants having less than 2% PEPC of the WT had significant levels of photorespiration at ambient levels of CO2 and required about 30 times ambient levels for maximum rates of A. Despite variation in the capacity of the C4 cycle, more than 91% of PS II activity was linearly associated with A under varying CO2 at 5, 15 and 30% O2. However, the WT plant had a higher PS II activity per CO2 fixed under saturating CO2 than the homozygous mutant, which is suggested to be due to elimination of the C4 cycle and its associated requirement for ATP from a Mehler reaction. In the SSU F. bidentis plants, a decreased rate of A (35%) and PS II activity (33%) accompanied a decrease in Rubisco capacity. There was some increase in alternative electron sinks at high CO2 when the C3 cycle was constrained, which may be due to increased flux through the C4 cycle via an ATP generating Mehler reaction. Nevertheless, even with constraints on the function of the C4 or C3 cycle by genetic modifications, analyses of CO2 response curves under varying levels of O2 indicate that CO2 assimilation is the main determinant of PS II activity in C4 plants.     

Characterization of recombinant barley oxalate oxidase expressed by Pichia pastoris

Oxalate oxidase catalyzes the oxidation of oxalate to carbon dioxide and hydrogen peroxide, making it useful for clinical analysis of oxalate in biological fluids. An artificial gene for barley oxalate oxidase has been used to produce functional recombinant enzyme in a Pichia pastoris heterologous expression system, yielding 250 mg of purified oxalate oxidase from 5 L of fermentation medium. The recombinant oxalate oxidase was expressed as a soluble, hexameric 140 kDa glycoprotein containing 0.2 g-atom Mn/monomer with a specific activity of 10 U/mg, similar to the properties reported for enzyme isolated from barley. No superoxide dismutase activity was detected in the recombinant oxalate oxidase. EPR spectra indicate that the majority of the manganese in the protein is present as Mn(II), and are consistent with the six-coordinate metal center reported in the recent X-ray crystal structure for barley oxalate oxidase. The EPR spectra change when bulky anions such as iodide bind, indicating conversion to a five-coordinate complex. Addition of oxalate perturbs the EPR spectrum of the Mn(II) sites, providing the first characterization of the substrate complex. The optical absorption spectrum of the concentrated protein contains features associated with a minor six-coordinate Mn(III) species, which disappears on addition of oxalate. EPR spin-trapping experiments indicate that carboxylate free radicals (CO2*-) are transiently produced by the enzyme in the presence of oxalate, most likely during reduction of the Mn(III) sites. These features are incorporated into a turnover mechanism for oxalate oxidase.     

Removal and mineralization of polycyclic aromatic hydrocarbons by litter-decomposing basidiomycetous fungi

Nine strains of litter-decomposing fungi, representing eight species of agaric basidiomycetes, were tested for their ability to remove a mixture of three polycyclic aromatic hydrocarbons (PAHs) (total 60 mg l(-1)) comprising anthracene, pyrene and benzo(a)pyrene (BaP) in liquid culture. All strains were able to convert this mixture to some extent, but considerable differences in degradative activity were observed depending on the species, the Mn(II) concentration, and the particular PAH. Stropharia rugosoannulata was the most efficient degrader, removing or transforming BaP almost completely and about 95% of anthracene and 85% of pyrene, in cultures supplemented with 200 micro M Mn(II), within 6 weeks. In contrast less than 40, 18, and 50% BaP, anthracene and pyrene, respectively, were degraded in the absence of supplemental Mn(II). In the case of Stropharia coronilla, the presence of Mn(II) led to a 20-fold increase of anthracene conversion. The effect of manganese could be attributed to the stimulation of manganese peroxidase (MnP). The maximum activity of MnP increased in S. rugosoannulata cultures from 10 U l(-1) in the absence of Mn(II) to 320 U l(-1) in Mn(II)-supplemented cultures. The latter degraded about 6% of a (14)C-labeled BaP into (14)CO(2) whereas only 0.7% was mineralized in the absence of Mn(II). In solid-state straw cultures, S. rugosoannulata, S. coronilla and Agrocybe praecox mineralized between 4 and 6% of (14)C-labeled BaP within 12 weeks.     

Color removal ability of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in relation to lignin peroxidase and manganese peroxidase produced in molasses wastewater

Decolorization of molasses wastewater (MWW) from an ethanolic fermentation plant by Phanerochaete chrysosporium was studied. By diluting MWW properly (10%v/v) and incubating it with an appropriate concentration of the spores (2.5 × 10⁶/ml), extensive decolorization occurred (75%) on day 5 of the incubation. The colour removal ability was found to be correlated to the activity of ligninolytic enzyme system: lignin peroxidase (LiP) activity was 185 U/l while manganese peroxidase (MnP) activity equaled 25 U/l. Effects of some selected operating variables were studied: manganese(II), veratryl alcohol (VA), glucose as a carbon source and urea and ammonium nitrate, each as a source of nitrogen. Results showed that the colour reduction and LiP activity were highest (76% and 186 U/l, respectively) either when no Mn(II) was added or added at the lowest level tested (0.16 mg/l to provide 0.3 mg/l). Activity of MnP was highest (25 U/l) when Mn(II) added to the diluted MWW at the highest level (100 ppm) while activity of LiP was lowest (7.1 U/l) at this level of added Mn(II). The colour reduction in the presence of the added VA was shown to be little less than in its absence (70 vs. 75%). When urea as an organic source of nitrogen for the fungus, was added to the MWW, the decolorizing activity of P. chrysosporium decreased significantly (15 vs. 75%) and no activities were detected for LiP and MnP. Use of ammonium nitrate as an inorganic source of nitrogen did not show such a decelerating effects, although no improvements in the metabolic behavior of the fungus (i.e., LiP and MnP activities) deaccelerating was observed. Effects of addition of glucose was also discussed.     

尾加压素Ⅱ对正常及缺血-再灌注离体大鼠心脏的影响

在正常Langendorff灌流与缺血-再灌注(停灌20 min-复灌20 min)离体大鼠心脏模型,观察尾加压素Ⅱ(urotensin Ⅱ,UⅡ)对冠脉流量、心功能和心肌代谢的影响以及心肌UⅡ受体的功能,以探讨UⅡ的心脏效应。对正常心脏给予0.1、1和10 nmol/L UⅡ各5 min,然后换洗5 min,对停灌缺血-再灌注心脏在再灌注期给予1或10nmol/L UⅡ。监测心率、左室内压和左室内压升降的最大变化率等心功能指标,计算冠脉流量,测定冠脉流出液中总蛋白和肌红蛋白含量以及乳酸脱氢酶(LDH)活性。灌流结束后,测定心肌丙二醛(MDA)含量和质膜UⅡ结合位点(放射性配基结合法)。结果如下:(1)正常心脏灌流UⅡ后,冠脉流量和心功能呈浓度依赖下降,换洗后没有完全恢复。心肌蛋白、肌红蛋白和LDH漏出随UⅡ浓度的增加而增加,换洗后迅速减少。UⅡ组心肌MDA含量与对照组差异无显著性。(2)缺血-再灌注后,冠脉流量显著减少,心功能显著抑制,再灌注期心肌蛋白、肌红蛋白和LDH明显漏出;给予UⅡ后,上述变化增强,且高浓度组更强,与对照组差异有显著性(P<<0.01),再灌注后心肌MDA含量亦显著高于对照(P<0.01)。(3)缺血-再灌注心肌质膜UⅡ受体的B_(max)显著高于正常对照心肌(14.65±1.78vs20.53±1.98 fmol/mg pr,P<0.01),Kd值变化无统计学意义。上述结果表明,在正常     

Effects of acetazolamide and 4-aminoprydine on the responses of deflationary slowly adapting pulmonary stretch receptors to CO2 inhalation in the rat

The inhibitory effect of CO(2) on deflationary slowly adapting pulmonary stretch receptors (deflationary SARs) was investigated before and after administration of acetazolamide, a carbonic anhydrase (CA) inhibitor, or 4-aminopyridine (4-AP), a K(+) channel blocker, in anesthetized, artificially ventilated rats after unilateral vagotomy. CO(2) inhalation (maximum tracheal CO(2) concentration ranging from 9 to 12%) for approximately 60 s decreased the impulse activity of deflationary SARs but had no significant effect on tracheal pressure (P(T)) as an index of bronchomotor tone. Acetazolamide treatment (20 mg/kg) diminished the inhibitory response of deflationary SARs to CO(2) inhalation. 4-AP (0.7 and 2.0 mg/kg) dose-dependently attenuated the decrease in deflationary SAR activity induced by CO(2) inhalation. When comparing the maximum attenuation due to 4-AP (2.0 mg/kg) and acetazolamide (20 mg/kg) in CO(2)-induced deflationary SAR inhibition, blockade of K(+) channels had a more pronounced effect. These results suggest that inhibition of deflationary SARs by CO(2) inhalation may be largely mediated by the stimulating action of 4-AP-sensitive K(+) currents in the nerve terminals of the receptors.     

12 alpha-hydroxysteroid dehydrogenase from Clostridium group P, strain C 48-50. Production, purification and characterization

NADP(H)-dependent 12 alpha-hydroxysteroid dehydrogenase (HSDH) from Clostridium group P, strain C 48-50, is still expressed at unusual high level (approximately 1% of total protein) under cultivation conditions where the usual expensive brain/heart infusion complex medium is replaced by inexpensive technical grade yeast autolysate. An inexpensive anaerobic bioprocess for the production of HSDH was developed provisionally up to 900-1 scale (9000 U/l, 7 g HSDH, specific activity 1.0 U/mg crude protein, 55 U/g wet cells). By a simple two-step affinity chromatography procedure, easily adaptable to a large-scale operation, using columns of small dimensions of Sephacryl-S-400-Procion-orange-P-2R (5 cm x 28 cm) and Sephacryl-S-400-Procion-red-HE-7B (2.6 cm x 14 cm) approximately 140 mg (1.8 x 10(4) U), HSDH was purified to apparent homogeneity and concentrated directly from a crude cell extract (overall yield 53%, specific activity 128 U/mg). As confirmed by fast native and SDS/PAGE, isoelectric focussing and electron microscopy, HSDH has a molecular mass of approximately 105 kDa and consists of four flattened tetrahedrically arranged identical subunits (26 kDa). The enzyme exhibits a rather low isoelectric point of 4.6, a pH optimum of 8.5-9.5 and a temperature optimum of approximately 55 C for the oxidation of cholic acid. Inhibition by SH reagents and pyridoxal 5'-phosphate has been observed. Chelating agents have no inhibitory effect. The presence of NADP increases considerably the thermostability (t 1/2 4-10 d, 25 C; 2-5 d, 37 C). Steady-state kinetic analysis for both reaction directions indicated that the reaction proceeds through an ordered bi bi mechanism with NADP(H) binding first to the free enzyme. Km, Vmax [forward (Vf) and reverse reactions (Vr)] and the dissociation constants Kd for the binary complexes with NADP and NADPH were as follows. NADP, Km = 35 microns, Kd = 35 microns; cholic acid, Km = 72 microns, deoxycholic acid, Km = 45 microns, Vf = 160 U mg; NAPDH, Kd = 16 microns; 12-oxochenodeoxylic acid, Km = 12 microns, 66 U/mg (conditions, 0.1 M potassium phosphate, pH 8.0, 25 degrees C). N6-functionalized NADP derivatives, e.g. N6-(2-aminoethyl)NADP (Km = 4.5 mM) are poorly accepted as coenzyme by HSDH.     

Glucose-induced secretion of Trichoderma reesei xylanases.

To produce two xylanases with Trichoderma reesei grown on glucose, recombinant strains which carry either the xyn1 or the xyn2 (xylanase I and II [XYN I and XYN II]-encoding) structural genes under the expression signals of the homologous pki1 (pyruvate kinase-encoding) gene were constructed. The two types of transformants secreted XYN I or II, respectively, during growth on glucose, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining. The corresponding specific xylanase activities of the best transformants on glucose were 76 and 145 U/mg of protein for XYN I and XYN II, respectively, as opposed to that obtained by the parent strain (26 U/mg of protein). When related to the amount of biomass formed, however, they produced only about 4 to 5 U/g, in contrast to much higher activities (10 to 12 U/g) during growth on xylan. The ultrastructural location of XYN II in the transformant strain producing the highest constitutive XYN II formation (ATX2-12) was investigated by immunoelectron microscopy and compared with that in the wild-type strain growing on xylan. Cell extracts from both types of transformants grown on glucose exhibited a higher intracellular xylanase activity than did the parent strain grown on xylan. By using electron microscopy and immunogold labelling, XYN II was detected in the endoplasmic reticulum, Golgi-like vesicles, secretory vesicles, vacuoles, and cell walls. The immunolabel in the vacuoles was detected preferentially in subapical cells. When a recombinant strain which expressed xyn2 from the pki1 promoter was compared with the parent strain during growth on xylan, the former exhibited a less proliferated endoplasmic reticulum and a smaller number of secretory vesicles; however, a higher density of labelling was observed. The relationship of these findings to the efficacy of protein secretion during growth on glucose is discussed.     

Carbon Monoxide (CO) Released from Tricarbonyldichlororuthenium (II) Dimer (CORM-2) in Gastroprotection against Experimental Ethanol-Induced Gastric Damage

The physiological gaseous molecule, carbon monoxide (CO) becomes a subject of extensive investigation due to its vasoactive activity throughout the body but its role in gastroprotection has been little investigated. We determined the mechanism of CO released from its donor tricarbonyldichlororuthenium (II) dimer (CORM-2) in protection of gastric mucosa against 75% ethanol-induced injury. Rats were pretreated with CORM-2 30 min prior to 75% ethanol with or without 1) non-selective (indomethacin) or selective cyclooxygenase (COX)-1 (SC-560) and COX-2 (celecoxib) inhibitors, 2) nitric oxide (NO) synthase inhibitor L-NNA, 3) ODQ, a soluble guanylyl cyclase (sGC) inhibitor, hemin, a heme oxygenase (HO)-1 inductor or zinc protoporphyrin IX (ZnPPIX), an inhibitor of HO-1 activity. The CO content in gastric mucosa and carboxyhemoglobin (COHb) level in blood was analyzed by gas chromatography. The gastric mucosal mRNA expression for HO-1, COX-1, COX-2, iNOS, IL-4, IL-1β was analyzed by real-time PCR while HO-1, HO-2 and Nrf2 protein expression was determined by Western Blot. Pretreatment with CORM-2 (0.5–10 mg/kg) dose-dependently attenuated ethanol-induced lesions and raised gastric blood flow (GBF) but large dose of 100 mg/kg was ineffective. CORM-2 (5 mg/kg and 50 mg/kg i.g.) significantly increased gastric mucosal CO content and whole blood COHb level. CORM-2-induced protection was reversed by indomethacin, SC-560 and significantly attenuated by celecoxib, ODQ and L-NNA. Hemin significantly reduced ethanol damage and raised GBF while ZnPPIX which exacerbated ethanol-induced injury inhibited CORM-2- and hemin-induced gastroprotection and the accompanying rise in GBF. CORM-2 significantly increased gastric mucosal HO-1 mRNA expression and decreased mRNA expression for iNOS, IL-1β, COX-1 and COX-2 but failed to affect HO-1 and Nrf2 protein expression decreased by ethanol. We conclude that CORM-2 released CO exerts gastroprotection against ethanol-induced gastric lesions involving an increase in gastric microcirculation mediated by sGC/cGMP, prostaglandins derived from COX-1, NO-NOS system and its anti-inflammatory properties.     

施氮和大气CO2浓度升高对小麦旗叶光合电子传递和分配的影响

采用开顶式气室盆栽培养小麦,设计2个大气CO2浓度(正常:400 μmol.mol-1;高:760 μmol·mol-1)、2个氮素水平(0和200 mg·kg-1土)的组合处理,通过测定小麦抽穗期旗叶氮素和叶绿素浓度、光合速率(Pn)-胞间CO2浓度(C1)响应曲线及荧光动力学参数,来测算小麦叶片光合电子传递速率等,研究了高大气CO2浓度下施氮对小麦旗叶光合能量分配的影响.结果表明:与正常大气CO2浓度相比,高大气CO2浓度下小麦叶片氮浓度和叶绿素浓度降低,高氮处理的小麦叶片叶绿素a/b升高.施氮后小麦叶片PSⅡ最大光化学效率(Fv/Fm)、PSⅡ反应中心最大量子产额(Fv'/Fm')、PSⅡ反应中心的开放比例(qr)和PSⅡ反应中心实际光化学效率(φPSⅡ)在大气CO2浓度升高后无明显变化,虽然叶片非光化学猝灭系数(NPQ)显著降低,但PSⅡ总电子传递速率(JF)无明显增加;不施氮处理的Fv'/Fm'、φPSⅡ和NPQ在高大气CO2浓度下显著降低,尽管Fv/Fm和qp无明显变化,JF仍显著下降.施氮后小麦叶片JF增加,参与光化学反应的非环式电子流传递速率(Jc)明显升高.大气CO2浓度升高使参与光呼吸的非环式电子流传递速率(J0)、Rubisco氧化速率(V0)、光合电子的光呼吸/光化学传递速率比(J0/Jc)和Rubisco氧化/羧化比(V0/Vc)降低,但使Jc和Rubisco羧化速率(Vc)增加.因此,高大气CO2浓度下小麦叶片氮浓度和叶绿素浓度降低,而增施氮素使通过PSⅡ反应中心的电子流速率显著增加,促进了光合电子流向光化学方向的传递,使更多的电子进入Rubisco羧化过程,Pn显著升高.     

Control of Carbon and Electron Flow in Clostridium acetobutylicum Fermentations: Utilization of Carbon Monoxide to Inhibit Hydrogen Production and to Enhance Butanol Yields

Extracts prepared from non-solvent-producing cells of Clostridium acetobutylicum contained methyl viologen-linked hydrogenase activity (20 U/mg of protein at 37°C) but did not display carbon monoxide dehydrogenase activity. CO addition readily inhibited the hydrogenase activity of cell extracts or of viable metabolizing cells. Increasing the partial pressure of CO (2 to 10%) in unshaken anaerobic culture tube headspaces significantly inhibited (90% inhibition at 10% CO) both growth and hydrogen production by C. acetobutylicum. Growth was not sensitive to low partial pressures of CO (i.e., up to 15%) in pH-controlled fermentors (pH 4.5) that were continuously gassed and mixed. CO addition dramatically altered the glucose fermentation balance of C. acetobutylicum by diverting carbon and electrons away from H₂, CO₂, acetate, and butyrate production and towards production of ethanol and butanol. The butanol concentration was increased from 65 to 106 mM and the butanol productivity (i.e., the ratio of butanol produced/total acids and solvents produced) was increased by 31% when glucose fermentations maintained at pH 4.5 were continuously gassed with 85% N₂-15% CO versus N₂ alone. The results are discussed in terms of metabolic regulation of C. acetobutylicum saccharide fermentations to achieve maximal butanol or solvent yield.     

Enzymatic properties of terephthalate 1,2-dioxygenase of Comamonas sp. strain E6

The tphA1 II and tphA2 II A3 II genes of Comamonas sp. E6 perhaps code for the terephthalate (TPA) 1,2-dioxygenase (TPADO). To characterize E6 TPADO, these genes were expressed in a His-tagged form in Escherichia coli, and the recombinant proteins were purified. TPADO activity was reconstituted from TphA1 II and TphA2 II A3 II, indicating that TPADO consists of a reductase (TphA1 II) and a terminal oxygenase component (TphA2 II and TphA3 II). TphA1(II) contains FAD, and the presence of a plant-type [2Fe-2S] cluster was suggested. These results indicate that TPADO is a class IB aromatic ring-hydroxylating dioxygenase. NADH and NADPH were effective as electron donors for TphA1 II, but NADPH appeared to be the physiological electron donor, based on the kinetic parameters. TPADO showed activity only toward TPA, and Fe2+ was required for it. The Km values for TPA and the Vmax were determined to be 72+/-6 microM and 9.87+/-0.06 U/mg respectively.