Particulate methane monooxygenase from Methylosinus trichosporium is a copper-containing enzyme

Particulate methane monooxygenase (pMMO) has been exfoliated and isolated from membranes of the Methylosinus trichosporium IMV 3011. It appears that the stability of pMMO in the exfoliation process is increased with increasing copper concentration in the growth medium, but extensive intracytoplasmic membrane formed under higher copper concentration may inhibit the exfoliation of active pMMO from membrane. The highest total activity of purified pMMO is obtained with an initial concentration of 6 microM Cu in the growth medium. The purified MMO contains only copper and does not utilize NADH as electron donor. Treatment of purified pMMO with EDTA resulted in little change in copper level, suggesting that the copper in the pMMO is tightly bound with pMMO.     

A decade of boreal rich fen greenhouse gas fluxes in response to natural and experimental water table variability

Rich fens are common boreal ecosystems with distinct hydrology, biogeochemistry and ecology that influence their carbon (C) balance. We present growing season soil chamber methane emission (F_CH4), ecosystem respiration (ER), net ecosystem exchange (NEE) and gross primary production (GPP) fluxes from a 9‐years water table manipulation experiment in an Alaskan rich fen. The study included major flood and drought years, where wetting and drying treatments further modified the severity of droughts. Results support previous findings from peatlands that drought causes reduced magnitude of growing season F_CH4, GPP and NEE, thus reducing or reversing their C sink function. Experimentally exacerbated droughts further reduced the capacity for the fen to act as a C sink by causing shifts in vegetation and thus reducing magnitude of maximum growing season GPP in subsequent flood years by ~15% compared to control plots. Conversely, water table position had only a weak influence on ER, but dominant contribution to ER switched from autotrophic respiration in wet years to heterotrophic in dry years. Droughts did not cause inter‐annual lag effects on ER in this rich fen, as has been observed in several nutrient‐poor peatlands. While ER was dependent on soil temperatures at 2 cm depth, F_CH4 was linked to soil temperatures at 25 cm. Inter‐annual variability of deep soil temperatures was in turn dependent on wetness rather than air temperature, and higher F_CH4 in flooded years was thus equally due to increased methane production at depth and decreased methane oxidation near the surface. Short‐term fluctuations in wetness caused significant lag effects on F_CH4, but droughts caused no inter‐annual lag effects on F_CH4. Our results show that frequency and severity of droughts and floods can have characteristic effects on the exchange of greenhouse gases, and emphasize the need to project future hydrological regimes in rich fens.     

Influence of temperature on the δ13C values and distribution of methanotroph‐related hopanoids in Sphagnum‐dominated peat bogs

Methane emissions from peat bogs are mitigated by methanotrophs, which live in symbiosis with peat moss (e.g. Sphagnum). Here, we investigate the influence of temperature and resultant changes in methane fluxes on Sphagnum and methanotroph‐related biomarkers, evaluating their potential as proxies in ancient bogs. A pulse‐chase experiment using ¹³C‐labelled methane in the field clearly showed label uptake in diploptene, a biomarker for methanotrophs, demonstrating in situ methanotrophic activity in Sphagnum under natural conditions. Peat cores containing live Sphagnum were incubated at 5, 10, 15, 20 and 25°C for two months, causing differences in net methane fluxes. The natural δ¹³C values of diploptene extracted from Sphagnum showed a strong correlation with temperature and methane production. The δ¹³C values ranged from ?34‰ at 5°C to ?41‰ at 25°C. These results are best explained by enhanced expression of the methanotrophic enzymatic isotope effect at higher methane concentrations. Hence, δ¹³C values of diploptene, or its diagenetic products, potentially provide a useful tool to assess methanotrophic activity in past environments. Increased methane fluxes towards Sphagnum did not affect δ¹³C values of bulk Sphagnum and its specific marker, the C₂₃ n‐alkane. The concentration of methanotroph‐specific bacteriohopanepolyols (BHPs), aminobacteriohopanetetrol (aminotetrol, characteristic for type II and to a lesser extent type I methanotrophs) and aminobacteriohopanepentol (aminopentol, a marker for type I methanotrophs) showed a non‐linear response to increased methane fluxes, with relatively high abundances at 25°C compared to those at 20°C or below. Aminotetrol was more abundant than aminopentol, in contrast to similar abundances of aminotetrol and aminopentol in fresh Sphagnum. This probably indicates that type II methanotrophs became prevalent under the experimental conditions relative to type I methanotrophs. Even though BHP concentrations may not directly reflect bacterial activity, they may provide insight into the presence of different types of methanotrophs.     

NO-cGMP Signaling and Regenerative Medicine Involving Stem Cells

Nitric oxide (NO) is a short lived diatomic free radical species synthesized by nitric oxide synthases (NOS). The physiological roles of NO depend on its local concentrations as well as availability and the nature of downstream target molecules. At low nanomolar concentrations, activation of soluble guanylyl cyclase (sGC) is the major event initiated by NO. The resulting elevation in the intracellular cyclic GMP (cGMP) levels serves as signals for regulating diverse cellular and physiological processes. The participation of NO and cGMP in diverse physiological processes is made possible through cell type specific spatio-temporal regulation of NO and cGMP synthesis and signal diversity downstream of cGMP achieved through specific target selection. Thus cyclic GMP directly regulates the activities of its downstream effectors such as Protein Kinase G (PKG), Cyclic Nucleotide Gated channels (CNG) and Cyclic nucleotide phosphodiesterases, which in turn regulate the activities of a number of proteins that are involved in regulating diverse cellular and physiological processes. Localization and activity of the NO-cGMP signaling pathway components are regulated by G-protein coupled receptors, receptor and non receptor tyrosine kinases, phosphatases and other signaling molecules. NO also serves as a powerful paracrine factor. At micromolar concentrations, NO reacts with superoxide anion to form reactive peroxinitrite, thereby leading to the oxidation of important cellular proteins. Extensive research efforts over the past two decades have shown that NO is an important modulator of axon outgrowth and guidance, synaptic plasticity, neural precursor proliferation as well as neuronal survival. Excessive NO production as that evoked by inflammatory signals has been identified as one of the major causative reasons for the pathogenesis of a number of neurodegenerative diseases such as ALS, Alzheimers and Parkinson diseases. Regenerative therapies involving transplantation of embryonic stem cells (ES cells) and ES cell derived lineage committed neural precursor cells have recently shown promising results in animal models of Parkinson disease (PD). Recent studies from our laboratory have shown that a functional NO-cGMP signaling system is operative early during the differentiation of embryonic stem cells. The cell type specific, spatio-temporally regulated NO-cGMP signaling pathways are well suited for inductive signals to use them for important cell fate decision making and lineage commitment processes. We believe that manipulating the NO-cGMP signaling system will be an important tool for large scale generation of lineage committed precursor cells to be used for regenerative therapies. Special issue dedicated to John P. Blass.     

Vanillin production using <Emphasis Type="Italic">Escherichia coli</Emphasis> cells over-expressing isoeugenol monooxygenase of <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20°C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.     

辽河口滨海湿地CH4排放特征及其影响因素

采用静态箱-气相色谱法,于2016年6—11月连续观测辽河口芦苇湿地、翅碱蓬湿地和裸滩湿地的CH_4排放速率,同时测定温度、氧化还原电位(Eh)、pH值和电导率(EC)等相关环境因子的动态变化。结果表明,3种类型湿地的CH_4排放具有明显的季节变化特征,均呈先上升后下降趋势。芦苇湿地、翅碱蓬湿地(涨潮前)和裸滩湿地(涨潮前)CH_4排放通量变化范围分别为0.447—10.40、0.045—0.509 mg m~(-2) h~(-1)和0.016—0.593 mg m~(-2) h~(-1),观测期内排放通量均值相应为(3.699±3.679)、(0.165±0.156) mg m~(-2) h~(-1)和(0.198±0.191) mg m~(-2) h~(-1),不同类型湿地之间差异显著(P0.01),芦苇湿地裸滩湿地(涨潮前)翅碱蓬湿地(涨潮前)。涨潮过程中,翅碱蓬湿地和裸滩湿地的排放速率分别变化在0.009—0.353 mg m~(-2) h~(-1)和0.018—0.335 mg m~(-2) h~(-1),观测期间其排放速率均值分别为(0.119±0.132) mg m~(-2) h~(-1)和(0.131±0.103) mg m~(-2) h~(-1),明显低于涨潮前(P0.01)。不同湿地类型间CH_4排放通量与电导率(EC)呈显著负相关(P0.01)。研究结果表明,潮汐和电导率均为影响辽河口不同类型湿地中CH_4排放的关键因子。     

Physiological responses of different olive genotypes to drought conditions

Gas exchange rates, chlorophyll fluorescence, pressure–volume relationships, photosynthetic pigments, total soluble sugars, starch, soluble proteins and proline concentrations were investigated in five Olea europaea L. cultivars with different geographical origins (Arbequina, Blanqueta, Cobran?osa, Manzanilla and Negrinha) grown under Mediterranean field conditions. We found considerable genotypic differences among the cultivars. Comparing the diurnal gas exchange rates, we observed that Cobran?osa, Manzanilla and Negrinha had high photosynthetic rate than Arbequina and Blanqueta. The first group reveals to be better acclimated to drought conditions, and appears to employ a prodigal water-use strategy, whereas Blanqueta and Arbequina, with high water-use efficiency, appear to employ a conservative water-use strategy. The degree of midday depression in photosynthesis was genotype dependent, with a maximum in Arbequina and a minimum in Negrinha. The reductions in the photosynthetic rate were dependent from both stomatal and non-stomatal limitations. Elastic adjustment plays an important role as drought tolerance mechanism. The group of cultivars that employ a prodigal water-use strategy revealed high tissue elasticity, whereas Arbequina and Blanqueta revealed high tissue rigidity. We also identified the existence of drought tolerance mechanisms associated with soluble proteins accumulation in the foliage. The high levels of soluble proteins in Arbequina may represent an increased activity of oxidative stress defence enzymes and may also represent a reserve for post stress recovery. In all cultivars, especially in Manzanilla, free proline was accumulated in the foliage. The discussed aspects of drought stress metabolism may have an adaptative meaning, supporting the hypothesis that olive cultivars native to dry regions, such as Cobran?osa, Manzanilla and Negrinha, have more capability to acclimate to drought conditions than cultivars originated in regions with a more temperate climate, like Arbequina and Blanqueta.     

燃料乙醇制造的“零能耗零污染”趋势

酒精蒸馏废液有充足的非淀粉生物质可供沼气转化,沼液营养丰富可作酵母发酵工艺用水。通过酒精高浓度发酵、沼气高效转化、沼气热电联产、差压蒸馏、环形过程工艺等产能、节能、无废技术的研发和集成,将最终完成木薯原料燃料酒精制造向“零能耗、零污染”生产技术的转型。     

Quantitative trait locus analysis of circulating adhesion molecules in hyperlipidemic apolipoprotein E-deficient mice

Circulating soluble adhesion molecules have been suggested as useful markers to predict several clinical conditions such as atherosclerosis, type 2 diabetes, obesity, and hypertension. To determine genetic factors influencing plasma levels of soluble vascular cell adhesion molecule-1 (VCAM-1) and P-selectin, quantitative trait locus (QTL) analysis was performed on an intercross between C57BL/6J (B6) and C3H/HeJ (C3H) mouse strains deficient in apolipoprotein E-deficient (apoE^−/−). Female F₂ mice were fed a western diet for 12 weeks. One significant QTL, named sVcam1 (71 cM, LOD 3.9), on chromosome 9 and three suggestive QTLs on chromosomes 5, 13 and 15 were identified to affect soluble VCAM-1 levels. Soluble P-selectin levels were controlled by one significant QTL, named sSelp1 (8.5 cM, LOD 3.4), on chromosome 16 and two suggestive QTLs on chromosomes 10 and 13. Both adhesion molecules showed significant or an apparent trend of correlations with body weight, total cholesterol, and LDL/VLDL cholesterol levels in the F₂ population. These results indicate that plasma VCAM-1 and P-selectin levels are complex traits regulated by multiple genes, and this regulation is conferred, at least partially, by acting on body weight and lipid metabolism in hyperlipidemic apoE^−/− mice. Zuobiao Yuan and Zhiguang Su contributed equally.     

中华绒螯蟹蜕壳过程中肌肉的组织学、超微结构及主要蛋白质含量的变化

为探讨中华绒螯蟹(Eriocheir sinensis)蜕壳前后肌肉组织的形态特征变化, 采用石蜡切片、电镜及生物化学方法, 研究了中华绒螯蟹蜕皮过程中步行足和腹部肌肉的组织学、超微结构及主要蛋白质含量的变化。结果显示: 相对于蜕皮间期, 步行足在蜕皮前后组织学形态特征无明显变化; 超微结构在蜕皮前无明显变化, 蜕皮后可见肌原纤维纵裂及肌小节横裂现象, 表明蜕皮后外骨骼硬化的过程伴随着肌肉的生长。相对于蜕皮间期, 腹部肌肉在蜕皮前后组织学特征变化明显: 蜕皮前肌束间隙增大, 蜕皮后肌束内肌纤维间隙增大。电子显微镜观察显示, 蜕皮前肌原纤维在内部降解, 出现空洞, 肌原纤维边缘降解, 导致肌原纤维间隙增大; 蜕皮后肌原纤维重新组装、重建, 恢复到间期正常形态。生物化学研究发现, 蜕皮前后步行足和腹部肌肉中肌原纤维蛋白和可溶性蛋白含量的变化同其结构特征的变化相一致。以上研究结果表明, 中华绒螯蟹肌肉组织的结构特征同蜕皮周期密切相关。