Characterization and biosynthesis of soluble and membrane-bound carbonic anhydrase in brain

Carbonic anhydrase from both the cytoplasmic and membrane fractions of the forebrains of rats was characterized with respect to enzymatic activity, immunoreactivity, and in vitro biosynthesis. A procedure for the rapid purification of both membrane-bound and soluble brain carbonic anhydrase is presented that permits retention of full enzymatic activity. Both forms of the enzyme were found to show specific activities of approximately 5500 Units/mg protein when CO2 hydrating activity was determined. In addition, they exhibited similar esterase activity when assayed with p-nitrophenyl acetate. The membrane-bound form, although requiring detergent for extraction from membranes, was freely soluble in aqueous buffers after purification. The molecular weights of both soluble and membrane-bound carbonic anhydrase are 30,000 daltons, and mixing experiments failed to show any significant differences with respect to size. The two forms also exhibit isoelectric points of 7.2. However, the two proteins were found to differ in two respects. Complement fixation indicated that antibodies to soluble carbonic anhydrase had a higher affinity for the soluble form than for the membrane-bound form. The failure to observe any precursor-product relationship between these two proteins with pulse chase studies and the establishment that carbonic anhydrase-like proteins are synthesized on both free polysomes and the rough endoplasmic reticulum indicated that these proteins are synthesized by two separate mechanisms. In vitro synthesis on both free and bound polysomes was determined by two independent methods using different antibodies and different analytical procedures. The basis for these findings and their physiologic importance are discussed.     

Lipid content and related fatty acid composition in fallow and cultivated soil in Southern Italy

Summary Lipid content and fatty acid composition were studied on two soil samples—one fallow and one cultivated with durum wheat. Differences between the cultivated and the fallow subunits in respect of total lipid content and the saponifiable fraction were found to be statistically significant. The same observations were made on some fatty acids showing either accumulation or decrease after three years. The interaction years x cultivation for total lipid content and the saponifiable fraction was significant showing that accumulation and reduction of lipids in the cultivated subunit are in equilibrium. The interaction dates of sampling x cultivation was significant for C150, C160, C180, C240 as their contents increase in the cultivated subunit in July as a result of the decomposition of plant tissues.Results discussed in this paper have been partially presented at the 7th International Symposium Humus et Planta Brno (Czechoslovakia) on August 20–25th 1979. This research has been partially supported by the National Research Council of Italy and carried out at the Experimental Institute of Agronomy — Bari — Italy (Director: F. Lanza).     

两种农田土壤不同组分呼吸及其温度敏感性

为探讨农田土壤不同组分呼吸及其对温度变化的响应,选取山东平邑旱耕土和湖南桃江水稻土为供试土壤,设置4个温度水平(5、15、25、35 ℃),对两种土壤的轻组、重组及全土进行63 d的培养试验.结果表明: 两种土壤全土的呼吸均高于轻组和重组.旱耕土重组的呼吸高于轻组,水稻土重组和轻组的呼吸在5～25 ℃温度水平下无显著差异,但35 ℃下重组高于轻组.在不同温度水平下,旱耕土轻组、重组和全土累积呼吸量分别占其初始碳的0.3%～2.8%、0.4%～3.7%和0.6%～7.0%,水稻土分别占其初始碳的0.4%～3.0%、0.3%～3.8%和0.7%～5.3%.两种土壤全土及轻、重组呼吸的温度敏感性(Q₁₀)均随温度升高和培养时间延长而降低;水稻土重组的Q₁₀高于轻组,旱耕土重组和轻组Q₁₀的差异无明显规律.在5～25 ℃温度水平下,旱耕土全土Q₁₀显著高于水稻土,但在25～35 ℃下低于水稻土.说明平邑旱耕土有机碳矿化强度高于桃江水稻土,且对温度变化的响应总体比水稻土更敏感.     

Average attributable fractions: a coherent theory for apportioning excess risk to individual risk factors and subpopulations

The attributable fraction in a population and the attributable fraction in exposed are different epidemiologic measures for quantifying the contribution of a risk factor to the risk of disease. While the attributable fraction in a population depends on both the relative risk of disease and the risk of being exposed in the population, the attributable fraction in exposed depends only on the relative risk. Similar relationships apply to the combined attributable fraction in a population and in exposed, respectively, for quantifying the total contribution of a group of risk factors. Eide and Gefeller (1995) showed how the sequential and average attributable fractions could be applied to quantify the contributions of the individual risk factors to a combined attributable fraction in a population. The present paper shows how this methodology can be extended to the combined attributable fraction in exposed. The resulting average attributable fractions in exposed are compared to other proposed methods. The relationship between the average attributable fractions in a population and in exposed is outlined, thus establishing a coherent theory for apportioning attributable fractions in individuals, groups of individuals and populations, to single risk factors or groups of risk factors like modifiable versus nonmodifiable factors.     

射血分数保留的老年心力衰竭患者的临床特征

目的:分析和比较射血分数保留的心力衰竭(HFp EF)、射血分数中间值(HFmr EF)及射血分数降低的老年心力衰竭(HFr EF)患者临床特征的差异。方法:选取2017年9月至2018年8月哈尔滨市第一医院收治的老年慢性心力衰竭患者共287例,根据心动超声所测左室舒张末期内径(LVEF)值将其分为3组:HFpEF组175例、HFmr EF组50例和HFr EF组62例。比较各组患者一般情况、心动超声检查结果、血清学指标的差异。结果:(1)与HFr EF组患者比较,HFpEF组患者年龄、性别、吸烟史、体重指数(BMI)、原发冠心病、高血压、2型糖尿病患者比例、房颤发生率及心功能分级构成比均具有统计学差异(P0.05);(2)与HFr EF组相比较,HFpEF组患者的E/A比值,左房内径、肺动脉内径、LVEDD较小,而室间隔厚度较厚(P0.05);(3)与HFr EF组患者相比,HFpEF组血清总胆固醇、甘油三酯较高;血肌酐、血尿素氮、血尿酸、超敏C反应蛋白、N-末端脑钠肽前体水平较低,具有统计学差异(P0.05)。结论:老年HFpEF心力衰竭患者以女性居多,体重指数较大,以向心性肥胖为主,血压水平较高,心功能II级者比例高,有明显的舒张功能不全,易发生房性心律失常,房颤发生率高,主要病因为高血压。     

TRPC6 participates in the regulation of cytosolic basal calcium concentration in murine resting platelets

Cytosolic-free Ca^2 + plays a crucial role in blood platelet function and is essential for thrombosis and hemostasis. Therefore, cytosolic-free Ca^2 + concentration is tightly regulated in this cell. TRPC6 is expressed in platelets, and an important role for this Ca^2 + channel in Ca^2 + homeostasis has been reported in other cell types. The aim of this work is to study the function of TRPC6 in platelet Ca^2 + homeostasis. The absence of TRPC6 resulted in an 18.73% decreased basal [Ca^2 +]_c in resting platelets as compared to control cells. Further analysis confirmed a similar Ca^2 + accumulation in wild-type and TRPC6-deficient mice; however, passive Ca^2 + leak rates from agonist-sensitive intracellular stores were significantly decreased in TRPC6-deficient platelets. Biotinylation studies indicated the presence of an intracellular TRPC6 population, and subcellular fractionation indicated their presence on endoplasmic reticulum membranes. Moreover, the presence of intracellular calcium release in platelets stimulated with 1-oleoyl-2-acetyl-sn-glycerol further suggested a functional TRPC6 population located on the intracellular membranes surrounding calcium stores. However, coimmunoprecipitation assay confirmed the absence of STIM1–TRPC6 interactions in resting conditions. This findings together with the absence of extracellular Mn^2 + entry in resting wild-type platelets indicate that the plasma membrane TRPC6 fraction does not play a significant role in the maintenance of basal [Ca^2 +]_c in mouse platelets. Our results suggest an active participation of the intracellular TRPC6 fraction as a regulator of basal [Ca^2 +]_c, controlling the passive Ca^2 + leak rate from agonist-sensitive intracellular Ca^2 + stores in resting platelets.     

Radial Gradients in Wood Specific Gravity,Water and Gas Content in Trees of a Mexican Tropical Rain Forest

Cell walls, water, and gas that have mechanical and physiological functions in wood, and wood specific gravity (WSG) is related to demographic traits. To understand variation in wood structure and function, we analyzed radial changes in WSG, in the gas and the water fractions from trees growing in four different habitats in a southern Mexican rain forest. Mean WSG was 0.55 ± 0.16, slightly lower than reported for other tropical forests. In 27 species, WSG decreased and in two species, it increased from pith to bark with a strong (r² = 0.65) negative correlation between WSG in the center of the tree and the radial WSG gradient. Habitat had some effect on mean WSG and trees growing on karst had significantly higher WSG than the same species growing on alluvial soil. The cell wall, water, and gas fractions accounted for 35 percent (range: 16–50), 42 percent (28–65), and 23 percent (2–56), respectively, of wood volume, with a negative correlation between the gas and the cell wall and between the gas and the water fractions, but not between the cell wall and water fractions. Radially increasing WSG is advantageous for pioneer trees with fast initial growth. We found that the water displacement method may result in biased WSG estimates. To increase the accuracy of WSG data, we suggest to measure sample volume geometrically using a constant diameter (that of the borer tip), to include radial variation in WSG, and to consider for possible site effects on species‐specific WSG.     

抚育间伐对长白落叶松人工林土壤碳、氮及其组分的影响

抚育间伐作为重要的森林经营措施之一,能够改变林分结构和稳定性,进而影响森林生态系统的生物地球化学循环.然而,抚育间伐对森林土壤碳、氮循环的影响程度如何尚不明确,尤其缺少长期试验结果报道.本研究以黑龙江省孟家岗林场经过4种不同强度和频度的抚育间伐处理后的60年生长白落叶松人工林为研究对象(4次低强度的间伐,LT4;3次中等强度的间伐,MT3;2次高强度间伐,HT2;不进行间伐的对照,CK),从酸水解法划分土壤碳、氮库(活性碳、氮库Ⅰ,活性碳、氮库Ⅱ和惰性碳、氮库)的角度研究了抚育间伐对长白落叶松人工林土壤总有机碳、全氮的影响机制.结果表明: 抚育间伐显著增加了土壤有机碳和全氮含量,增幅分别高达48.7%～50.3%和28.9%～42.7%.抚育间伐均增加了3种碳、氮组分的含量,而增加的程度因碳、氮组分和抚育间伐措施的不同而异.与活性碳库Ⅰ和活性碳库Ⅱ的增加程度相比,惰性碳库的增加程度最大,LT4、MT3和HT2处理下惰性碳库分别增加71%、69%和75%.此外,抚育间伐也显著增加了惰性碳占土壤总有机碳的比例.LT4显著提高了土壤微生物生物量碳、氮含量和微生物熵,而MT3和HT2对微生物生物量碳、氮和微生物熵却无显著影响.抚育间伐可能通过产生较多的粗木质残体于土体中,增加土壤木栓质和木质素等顽固组分的输入,进而导致土壤惰性碳含量增加,降低有机质的分解,最终导致土壤有机碳增加.     

Simultaneous quantitative determination method for ceramide species from crude cellular extracts by high-performance liquid chromatography-thermospray mass spectrometry

I have developed a simple method which enabled simultaneous analysis of ceramides in the subcellular fractions from cultured cells by HPLC-thermospray mass spectrometry. The HPLC-thermospray mass spectra from ceramide standards were characterized by the high intensity of the MNa(+) and MH(+)-H(2)O ions. As the other minor ions, MK(+), MH(+) and m/z 282 ions were detected. Although the preponderance of MNa(+) ions compared with the MH(+)-H(2)O ions was detected in non-hydroxy fatty acid-ceramides, the preponderance of MH(+)-H(2)O ions based on the elimination of the hydroxyl group introduced at the alpha-position of acyl-portion compared with the MNa(+) ions was detected in alpha-hydroxy fatty acid-ceramides. In calibrations for authentic ceramides using N-octanoylsphingosine as an internal standard, an approximately linear relationship existed between the ratios of peak-areas of each ceramide to that of the internal standard and the known amounts of each ceramide. The factor (f) of each ceramide was calculated as follows; N-oleoyl-D-sphingosine (f=0.45), N-palmitoyl-D-sphingosine (f=0.40), N-stearoyl-D-sphingosine (f=0.39), N-nervonoyl-D-sphingosine (f=0.39) and N-lignoceroyl-D-sphingosine (f=0.35). In subcellular fractions from A549 and HepG2 cells, although ceramide species content per mg protein was high in the nuclear envelope fractions, the 7000 g pellet fractions and the 100000 g pellet fractions, a large portion of the ceramide species was concentrated in the nuclear envelope fraction. In addition, this method was applied to a mild alkaline hydrolyzate of total ceramides from pig stratum corneum, and MNa(+)/MH(+)-H(2)O ions corresponding to several omega-hydroxyacyl-ceramides were detected.     

The occurrence of multiple forms of cytochrome P-450 in hepatic microsomes from untreated rats and mice

The hepatic microsomes of rat and mice were subfractionated by the procedure of Dallner. When a 1.3 M sucrose lower layer was used for the two-step discontinuous gradient, no differences in spectral characteristics were noted between subfractions, though the smooth fractions (SER) had higher oxidative activity towards the substrates tested. When lower layers of 1.05, 1.1 or 1.15 M sucrose were used, the SER isolated contained cytochrome P-450 with significantly different spectral characteristics from that of the rough fraction (RER). The SER cytochrome P-450 had a wavelength maximum in the carbon-monoxide reduced difference spectrum that was significantly lower (ca. 1.0 nm) than that in the RER. In addition, the type I:CO-reduced spectral ratio of these fractions is significantly elevated. These data indicate that liver microsomes from untreated rats and mice contain more than one cytochrome P-450 and that these cytochromes may be located in different parts of the endoplasmic reticulum.     

Adriamycin and daunomycin induce interstrand DNA crosslinks in HeLa S3 cells

By using a new mild procedure for detecting DNA crosslinks it has been shown that adriamycin and daunomycin are able to form interstrand DNA crosslinks in HeLa cells. This effect seems to be preceded by transformation of the parent antibiotics in the cell to active forms. In addition, interstrand DNA crosslinks formed by adriamycin and daunomycin were found to be temperature- and alkali-labile.     

Intracellular distribution of selenium and the growth of mammary cells in culture

Retention of Se in CMT-13 cells increased with an increase in the concentration of selenite in the incubation medium, the duration of exposure, and the density of the culture. The enhanced toxicity of selenite coincided with a proportional increase in Se in both the cytoplasm and nucleus. About 90% of the accumulated Se was isolated with cytoplasmic macromolecules. Increased nuclear Se retention correlated with increased cytoplasmic Se retention. Greater quantities of cytosolic Se-containing proteins (74, 55, 41, 34, and 28 kDa) and a nuclear Se-containing protein (56 kDa) were detected as the quantity of Se within CMT-13 cells increased. These findings suggest that cellular retention and distribution of Se are determinanants of the degree of cellular growth inhibition caused by this trace element.     

Improving and evaluating the soil cover indicator for agricultural land in Canada

A soil cover days (SCD) model has been developed by Agriculture and Agri-Food Canada for use as an agri-environmental indicator to monitor the relationship between agricultural production activities and agri-environmental quality. The SCD indicator integrates information on crops, soils, climate, and field activities to estimate the total equivalent number of days that agricultural soils are covered by crop canopy, crop residue and snow in a given year. Daily cover fractions of plant and residue for a given crop in an ecoregion are simulated using typical crop calendar and field management practices, and the equivalent number of days that soil is covered by snow in winter is derived from long term climate normals. The equivalent SCD for a spatial unit is then derived as the area-weighted sum of different crops and different management practices within the unit. This paper presents the SCD framework, details an assessment of the accuracy of the model and outlines future improvements. Annual snow days derived from 30-year climate normals as used in the model was strongly correlated (excluding mountain areas) with that derived from satellite data (R² = 0.45, n = 48), even though the remote sensing product showed significant temporal and spatial variability. Crop residue fraction estimated by the model was strongly correlated with field data collected over major crop areas and crop types (R² = 0.74, n = 55), and modelled plant cover fraction was well correlated with that derived from remote sensing data (R² = 0.57, n = 57). Large discrepancies were observed for some samples due to deviation of the actual crop calendar from that estimated using climate normals. National map showing the change in the indicator from 1981 to 2011 reveals changes in crop and residue management practices.     

Learning-based biomarker-assisted rules for optimized clinical benefit under a risk constraint

Novel biomarkers, in combination with currently available clinical information, have been sought to improve clinical decision making in many branches of medicine, including screening, surveillance, and prognosis. Statistical methods are needed to integrate such diverse information to develop targeted interventions that balance benefit and harm. In the specific setting of disease detection, we propose novel approaches to construct a multiple-marker-based decision rule by directly optimizing a benefit function, while controlling harm at a maximally tolerable level. These new approaches include plug-in and direct-optimization-based algorithms, and they allow for the construction of both nonparametric and parametric rules. A study of asymptotic properties of the proposed estimators is provided. Simulation results demonstrate good clinical utilities for the resulting decision rules under various scenarios. The methods are applied to a biomarker study in prostate cancer surveillance.     

耕作方式对紫色水稻土轻组有机碳的影响

以位于西南大学实验农场的长期免耕试验田为研究对象,通过0—60cm分层采集土壤样品的方法,探讨了不同耕作方式———冬水田平作(DP)、水旱轮作(SH)、垄作免耕(LM)及垄作翻耕(LF)对土壤轻组有机碳的影响。结果表明,重组有机碳是土壤总有机碳的主体,约占土壤总有机碳的69.56%—95.66%,在土壤剖面上随土壤深度的增加其分配比例逐渐升高;其次是自由轻组有机碳,约占土壤总有机碳的5.03%—26.43%,从土壤表层向下,其分配比例迅速下降;闭合轻组有机碳最低,仅占土壤总有机碳的1.37%—4.93%,其分配比例随土壤深度的变化不明显。在0—60 cm土壤深度内,不同耕作方式下自由轻组有机碳的平均含量为LM(4.36 g/kg)>DP(2.11 g/kg)>LF(1.74 g/kg)>SH(1.46 g/kg),相应的有机碳分配比例分别为17.1%、14.0%、12.2%和11.3%;闭合轻组有机碳的平均含量为LM(0.82 g/kg)>DP(0.51 g/kg)>LF(0.36 g/kg)>SH(0.34g/kg),相应的有机碳分配比例分别为3.36%、3.45%、2.71%和3.00%。因此,在西南地区紫色水稻土上实行垄作免耕能提高轻组有机碳的含量及其分配比例,从而改善土壤有机碳质量。另外,与土壤总有机碳、重组有机碳及闭合轻组有机碳相比,自由轻组有机碳对耕作方式的变化最敏感,是指示土壤有机碳变化的良好指标。     

Proteomic profiling of epicardial fat in heart failure with preserved versus reduced and mildly reduced ejection fraction

In order to explore the proteomic signatures of epicardial adipose tissue (EAT) related to the mechanism of heart failure with reduced and mildly reduced ejection fraction (HFrEF/HFmrEF) and heart failure (HF) with preserved ejection fraction (HFpEF), a comprehensive proteomic analysis of EAT was made in HFrEF/HFmrEF (n = 5) and HFpEF (n = 5) patients with liquid chromatography–tandem mass spectrometry experiments. The selected differential proteins were verified between HFrEF/HFmrEF (n = 20) and HFpEF (n = 40) by ELISA (enzyme-linked immunosorbent assay). A total of 599 EAT proteins were significantly different in expression between HFrEF/HFmrEF and HFpEF. Among the 599 proteins, 58 proteins increased in HFrEF/HFmrEF compared to HFpEF, whereas 541 proteins decreased in HFrEF/HFmrEF. Of these proteins, TGM2 in EAT was down-regulated in HFrEF/HFmrEF patients and was confirmed to decrease in circulating plasma of the HFrEF/HFmrEF group (p = 0.019). Multivariate logistic regression analysis confirmed plasma TGM2 could be an independent predictor of HFrEF/HFmrEF (p = 0.033). Receiver operating curve analysis indicated that the combination of TGM2 and Gensini score improved the diagnostic value of HFrEF/HFmrEF (p = 0.002). In summary, for the first time, we described the proteome in EAT in both HFpEF and HFrEF/HFmrEF and identified a comprehensive dimension of potential targets for the mechanism behind the EF spectrum. Exploring the role of EAT may offer potential targets for preventive intervention of HF.     

Microbial immobilization of cadmium released from CdO in the soil

The effect of microorganisms on the fate of Cd introduced into the soil as cadmium oxide (CdO) was investigated. Cadmium oxide (875 µg Cd per gram of soil) was added to -irradiated (sterile) and non-sterile soils. The soils were incubated for 90 days at 18 °C under aerobic conditions with moisture kept at 60% of water-holding capacity. Half of the samples in each treatment were supplemented with starch (0.5%, w/w) in order to stimulate microbial growth in the non-sterile soil. After various time intervals (7- or 10-day), soil samples from each treatment were extracted with deionized distilled water (ratio 1:40) or 0.25 M CaCl₂ (ratio 1:5). The results indicated that during the incubation period the amount of Cd extracted from the non-sterile soil with either solvent was markedly lower than that extracted from the -irradiated sterile control. The addition of starch to the non-sterile soil reduced the concentration of Cd in the 0.25 M CaCl₂ extracts without affecting the Cd-content in the water extracts. Short-term experiments in which Cd was added to the soil as a solution of Cd(NO₃)₂ indicated that irradiation did not affect the sorption of Cd to the soil. The addition of bacterial mass (1 mg of dry weight g^–1 soil) decreased the amount of Cd extracted with water as well as that extracted with 0.25 M CaCl₂. Under sterile conditions the solubility of CdO in soil extracts was higher than in the other extractants. The addition of glucose (0.5%, w/w) or a glucose/starch mixture (0.5%, w/w of each) to the sterile soil increased the amount of extractable Cd after a short incubation (18 h at 18 °C). The obtained results suggest that primarily physicochemical reactions are involved in dissolving CdO in the soil but that microbial activity may be responsible for the immobilization of the released metal.     

Purification and Characterization of a Ca2+- and Calmodulin-Dependent Protein Kinase from Rat Brain

Abstract: A Ca²⁺- and calmodulin-dependent protein kinase was purified from rat brain cytosol fraction to apparent homogeneity at approximately 800-fold and with a 5% yield. The purified enzyme had a molecular weight of 640,000 as determined by gel filtration analysis on Sephacryl S-300 and a sedimentation coefficient of 15.3 S by sucrose density gradient centrifugation, and resulted in a single protein band of MW 49,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the native enzyme has a large molecular weight and consists of 11 to 14 identical subunits. The purified enzyme exhibited K _m values of 109 and 30 μM for ATP and chicken gizzard myosin light chain, respectively, and K _a values of 12 n M and 1.9 μM for brain calmodulin and Ca²⁺, respectively. In addition to myosin light chain, myelin basic protein, casein, arginine-rich histone, microtubule protein, and synaptosomal proteins were phosphorylated by the enzyme in a Ca²⁺- and calmodulin-dependent manner. The purified enzyme was phosphorylated without the addition of the catalytic subunit of cyclic AMP-dependent protein kinase. Our findings indicate that there is a multifunctional Ca²⁺- and calmodulin-dependent protein kinase in the brain and that this enzyme may regulate the reactions of various endogenous proteins.