Aerobic bacterial methane synthesis

Reports of biogenic methane (CH₄) synthesis associated with a range of organisms have steadily accumulated in the literature. This has not happened without controversy and in most cases the process is poorly understood at the gene and enzyme levels. In marine and freshwater environments, CH₄ supersaturation of oxic surface waters has been termed the “methane paradox” because biological CH₄ synthesis is viewed to be a strictly anaerobic process carried out by O₂-sensitive methanogens. Interest in this phenomenon has surged within the past decade because of the importance of understanding sources and sinks of this potent greenhouse gas. In our work on Yellowstone Lake in Yellowstone National Park, we demonstrate microbiological conversion of methylamine to CH₄ and isolate and characterize an Acidovorax sp. capable of this activity. Furthermore, we identify and clone a gene critical to this process (encodes pyridoxylamine phosphate-dependent aspartate aminotransferase) and demonstrate that this property can be transferred to Escherichia coli with this gene and will occur as a purified enzyme. This previously unrecognized process sheds light on environmental cycling of CH₄, suggesting that O₂-insensitive, ecologically relevant aerobic CH₄ synthesis is likely of widespread distribution in the environment and should be considered in CH₄ modeling efforts.

The “methane paradox” is well documented and has drawn significant attention from a broad array of biologists, biogeochemists, biochemists, and physical scientists (see review in ref. 1). This supersaturation phenomenon is inconsistent with the extreme oxygen sensitivity of methanogens, a phylogenetically constrained group of archaea, which have long been viewed to be the sole source of biogenic CH₄. Work in marine waters previously suggested this is due to methanogens active within anoxic microhabitats in suspended organic debris (2–5), digestive tracts, or fecal pellets of zooplankton (5–7) and/or of fish (8, 9). Recently, strong evidence for microbial dealkylation of methylphosphonate (referred to here as MPn) in marine (10, 11) and freshwater environments (12, 13) has been presented and thus provides at least a partial explanation for this phenomenon.Pelagic methane enriched zones (PMEZ, ref. 12) are well-defined CH₄ maxima occurring as a distinct region(s) of the water column in deep freshwater lakes (1, 12, 14). PMEZ offer a tractable environment in which to study the microbiology and biogeochemistry that underpins aerobic CH₄ synthesis. Our prior efforts on Yellowstone Lake using ¹³C-labeled methanogen substrates, 16S ribosomal RNA (rRNA) gene based microbial community characterization, and methyl-coenzyme M reductase (mcrA) gene-targeted PCRs demonstrated the absence of recognizable methanogens in PMEZ waters (12). Instead, MPn metabolism was strongly associated with distinct populations of Pseudomonas sp. as being important contributors to PMEZ formation (12).Efforts presented here summarize continuing studies that now identify aerobic metabolism of methylamine (MeA) as an important contributing metabolite to biogenic CH₄. Methylotrophic methanogens anaerobically convert MeA to CH₄ by disproportionation of MeA to carbon dioxide and CH₄ (15, 16). Methylotrophic bacteria can metabolize MeA as a carbon source (17, 18), nonmethylotrophs for nitrogen (17, 19) (20), a carbon and N source (21), or methylovores can use it as an energy source (22). MeA utilization is viewed to occur as an oxidation via either MeA dehydrogenase, MeA oxidase, or involving methy group transfer to tetrahydrofolate involving the formation of formation of γ‐glutamyl‐methylamide (GMA) and N‐methylglutamate (NMG) (20, 23). In the current study, we report the discovery of an alternate, simpler route of MeA metabolism that yields CH₄, involving a reaction catalyzed by a 5′pyridoxal-phosphate–dependent aspartate aminotransferase.     

正交试验设计优选九节龙皂苷I聚乳酸微球的制备工艺

目的 筛选溶剂蒸发法制备九节龙皂苷I聚乳酸微球（ADS-I-PLA-MS）最佳工艺。方法 采用HPLC-ELSD测定方法,以包封率和载药量为评价指标,W/O/W溶剂蒸发法制备微球;通过单因素和正交试验设计,考察内水相九节龙皂苷I（ADS-I）甲醇溶液的质量浓度、ADS-I甲醇溶液与聚乳酸（PLA）二氯甲烷溶液体积比、PLA二氯甲烷溶液质量浓度和聚乙烯醇（PVA）体积等因素对ADS-I-PLA-MS包封率及载药量的影响。结果 溶剂蒸发法制备ADS-I-PLA-MS的最佳工艺条件为ADS-I甲醇溶液质量浓度为8 mg/mL、ADS-I甲醇溶液与PLA二氯甲烷溶液体积比为1∶13、PLA二氯甲烷溶液质量浓度为90 mg/mL、PVA体积为500 mL。结论 优选出的ADS-I-PLA-MS制备工艺合理可行。     

The ability of betaine to reduce the irritating effects of detergents assessed visually, histologically and by bioengineering methods

Background/aims: A novel approach for reducing the undesired irritating properties of detergents on skin might be offered by betaine, which is a natural product derived from the sugar beet. The aim of the study was to explore the ability of betaine to reduce the irritating effects of two surfactants, sodium lauryl sulphate (SLS) and cocoamidopropylbetaine (CAPB). For evaluation of changes in skin reactions visual scoring, electrical impedance, transepidermal water loss and histology were used.
Methods: Twenty-one healthy subjects were patch tested for 24 h with SLS and CAPB alone and together with betaine, betaine alone, and the two controls distilled water and an unoccluded test site on both volar forearms. Responses were evaluated by measuring electrical impedance and transepidermal water loss before exposure and 24 h after the removal of the test substances, and also by visual inspection and histology. The electrical impedance device enables measurements at 31 frequencies and relevant information was extracted from the spectra using four indices.
Results: CAPB was found to be less irritating than SLS. The used detergents gave rise to distinctive impedance patterns also reflected by different types of histopathological skin responses. After the adding of betaine, the irritant reaction decreased for both detergents.
Conclusions: Betaine is a promising ingredient to reduce the side effects of detergents and electrical impedance is a suitable tool both to quantify the degree of irritation as well as to differentiate between various types of reactions.     

滤液法和沉淀法测定地骨皮醇提物中的甜菜碱含量

目的建立地骨皮醇提物中甜菜碱的含量测定方法。方法基于甜菜碱与雷氏盐的反应 ,采用分光光度法测定地骨皮醇提物中甜菜碱含量。结果沉淀法与滤液法的平均回收率分别为 98.4 0 %与 98.2 1% ,RSD分别为0 .97%与 1.0 4 % (n =6 )。结论滤液法与沉淀法用于甜菜碱的含量测定 ,所得结果基本一致 ,滤液法操作相对简便     

HPLC-ELSD法测定枸杞子中甜菜碱

目的:研究以HPLC-ELSD法测定枸杞子中甜菜碱的含量。方法:采用HPLC-ELSD法。Luna5u SCX100A色谱柱(4.6mm×250mm,5μm),流动相甲醇-0.10mol·L-1醋酸铵(15∶85),体积流量1.0mL·min-1,柱温30℃,检测器参数为雾化器温度60℃,蒸发器温度90℃,蒸发器气流1.0L·min-1。结果:甜菜碱线性范围为0.75～3.75μg(r=0.9997),平均回收率为99.86%,RSD为2.05%(n=6)。结论:该方法快速,准确,适用于测定枸杞子中甜菜碱的含量。     

离子交换－比色法测定杞菊地黄丸中甜菜碱含量

杞菊地黄丸水提取物依次通过强阳离子交换树脂柱、强阴离子交换树脂柱分离得到甜菜碱（Ｂａｔａｉｎｅ），然后与雷氏盐形成沉淀，沉淀用７００ｇ／Ｌ丙酮溶解，用比色法在λｍａｘ５２５ｎｍ处测定其含量，两个厂家生产的杞菊地黄丸中甜菜碱的含量分别为０．０６６％，０．０７４％，平均回收率为９７．００％，相对标准差为０．８３％，相关系数为０．９９９８．     

Effects of substrate and potassium on the betaine-synthesizing enzyme glycine sarcosine dimethylglycine N-methyltransferase from a halophilic methanoarchaeon Methanohalophilus portucalensis

Methanohalophilus portucalensis FDF1 can synthesize the compatible solute betaine de novo through the methylation of glycine, sarcosine and dimethylglycine with the methyl group from S-adenosylmethionine. After separation by DEAE-Sephacel ion chromatography using a KCl step gradient, glycine, sarcosine and dimethylglycine methytransfer (GSDMT) activities were detected in a single peak. The estimated molecular weight of GSDMT was 240 kDa and 2-D gel analysis indicated it was separated into four subunits (52 kDa) with different pI. The PBE94 chromatofocusing column also separated GSDMT into four protein peaks A, B, C, D. Both peak B and D proteins possessed GSDMT activity, while the peak A protein only exhibited SDMT activity. The multiple methyltransferase activities of the large complex appear to be unique compared to other methyltransferases used in betaine synthesis. Further methyltransferase assays in response to different concentrations of KCl indicated that the peak D protein exhibited low GSDMT activity only when K(+) < or = 0.4 M. The peak B protein exhibited a higher GSDMT activity at 0.4 M K(+), while the peak A protein exhibited SDMT activity only at higher K(+) (0.8 M). These results suggest that the internal K(+) concentration regulates GSDMT activities and affects the net betaine accumulation in the cells.     

Effect of Folic Acid,Betaine, Vitamin B6, and Vitamin B12 on Homocysteine and Dimethylglycine Levels in Middle-Aged Men Drinking White Wine

Moderate regular consumption of alcoholic beverages is believed to protect against atherosclerosis but can also increase homocysteine or dimethylglycine, which are putative risk factors for atherosclerosis. We aimed (1) to investigate the effect of alcohol consumption on vitamins and several metabolites involved in one-carbon metabolism; and (2) to find the most effective way of decreasing homocysteine during moderate alcohol consumption. Methods: Male volunteers (n = 117) were randomly divided into five groups: the wine-only group (control, 375 mL of white wine daily for one month) and four groups combining wine consumption with one of the supplemented substances (folic acid, betaine, and vitamins B₁₂ or B₆). Significant lowering of homocysteine concentration after the drinking period was found in subjects with concurrent folate and betaine supplementation. Vitamin B₁₂ and vitamin B₆ supplementation did not lead to a statistically significant change in homocysteine. According to a multiple linear regression model, the homocysteine change in the wine-only group was mainly determined by the interaction between the higher baseline homocysteine concentration and the change in dimethylglycine levels. Folate and betaine can attenuate possible adverse effects of moderate alcohol consumption. Dimethylglycine should be interpreted together with data on alcohol consumption and homocysteine concentration.