西南獐牙菜化学成分的研究

对龙胆科(Gentianaceae)獐牙菜属植物西南獐牙菜(Swertia cincta)进行化学成分研究,从中分离鉴定了15个化合物,包括5个裂环烯醚萜(苷),2个三萜,1个口山酮,4个芳香酸(醇),以及3个其它类成分.以上化合物分别为:獐牙菜苦苷(swertiamarin,1),龙胆苦苷(gentiopicroside,2),红白金花内酯(erythrocentaurin,3),(-)龙胆内酯((-)-gentiolactone,4),angelone(5),齐墩果酸(olcanolic acid,6),3-表-蒲公英赛醇(3-epi-tarax-erol,7),当药醇苷(swertianolin,8),间羟基苯甲醇(m-hydroxybenzyl alcohol,9),邻苯二甲酸二甲酯(dimethyl phthalate,10),邻苯二甲酸二异丁酯(diisobutyl phthalate,11),3,4-二羟基苯甲酸(12)和正三十一烷醇( n-hentria-contanol,13),β-谷甾醇(β-sitosterol,14)和胡萝卜苷(daucosterol,15).其中化合物4-5,7,9～13和15为首次从西南獐牙菜中分离得到.     

江淮丘陵地区油-稻轮作条件下土壤硫库变化研究

江淮丘陵地区下蜀系黄土母质发育的水稻土油2稻轮作试验表明, 油菜种植期间, 耕层土壤硫主要来源于耕层以下土层的补给, 其次是大气干湿沉降;该时期耕层土壤硫输出主要是油菜吸收, 其次是淋失.油菜种植期间耕层土壤硫输入量小于输出, 导致耕层土壤硫库下降8.76kg·hm^-2, 22%来自无机硫库的下降.水稻种植期间, 耕层土壤硫输入主要来自灌溉水, 其次是底土层的补给和大气干湿沉降;而硫输出主要是淋失, 其次是水稻吸收.耕层土壤硫输入量大于输出, 导致耕层土壤硫库增加18.69kg·hm^-2, 18%来自无机硫库的增加.全年油2稻轮作期间耕层土壤硫输入量大于输出, 导致耕层土壤硫库增加9.93kg·hm^-2, 13%来自无机硫库的增加.     

Effect of ethanol on activity of the plasma-membrane ATPase in, and accumulation of glycine by, Saccharomyces cerevisiae

The pH optimum of the ATPase activity in plasma membranes from Saccharomyces cerevisiae NCYC 431 from 8 h cultures was around 6.5 and that in membranes from organisms from 16 h cultures near 6.0. The Km[ATP] of the enzyme was virtually unaffected by the age of the culture from which organisms were harvested, although the Vmax of the enzyme in membranes from organisms from 8 h cultures was higher than that for organisms from 16 h cultures. Ethanol non-competitively inhibited ATPase activity in membranes, although the inhibition constant for the enzyme from organisms from 8 h cultures was lower than that from organisms from 16 h cultures. Glycine accumulation by the general amino acid permease was non-competitively inhibited by ethanol. Inhibition constants were virtually the same for glycine uptake by deenergized organisms from 8 h and 16 h cultures, but under energized conditions the value was greater for organisms from 16 h rather than 8 h cultures. The data indicate that inhibition of plasma-membrane ATPase activity by ethanol could account, at least in part, for inhibition of glycine accumulation by ethanol.     

银杏叶多酚的机械力辅助提取及其体外抗氧化活性研究

为优化银杏叶多酚提取工艺,通过单因素试验考察填充率、球磨转速、球磨时间、乙醇浓度、料液比、提取温度、提取时间七个因素对机械力辅助提取银杏叶多酚得率的影响,以银杏叶多酚得率为响应值,采用Box-Benhnken三因素三水平响应面设计优化工艺,同时比较了4种提取方法对银杏叶多酚提取得率和抗氧化活性的差异。结果表明,机械力辅助提取银杏叶多酚的最佳工艺条件为:填充率26%、球磨转速为400rpm、球磨时间为15min。在此条件下,银杏叶多酚的得率为7.33%。机械力辅助乙醇提取银杏叶多酚得率低于碱水提取法,但是抗氧化活性高于碱水法提取的银杏叶多酚;抗氧化活性与乙醇回流法提取的银杏叶多酚相当,但是提取得率高于乙醇回流法。此提取工艺高效可行,具有一定的参考价值。     

Regulation of class II MHC gene expression by macrophages from Bcgr and Bcgs mice

The presence of class II mRNA was determined following stimulation of macrophages from Bcgr and Bcgs mice with rIFN-gamma. Despite the continuous expression of surface I-A glycoprotein by macrophages from Bcgr mice, class II mRNA was no longer present. The transient expression of I-A by macrophages from Bcgs mice, however, was accompanied by the disappearance of class II mRNA from the cells. Restimulation of macrophages from Bcgs mice, with rIFN-gamma resulted in the reappearance of class II mRNA and surface I-A expression. The reappearance of class II mRNA and the surface expression of I-A glycoprotein was inhibited by PGE2. These results indicate that differences in I-A expression by macrophages from Bcgr and Bcgs are not at the level of class II gene expression.     

An irreversible tissue inhibitor of collagenase in human amniotic fluid: Characterization and separation from fibronectin

Soluble fibronectin isolated from human plasma and amniotic fluid by gelatin-Sepharose affinity chromatography was tested for inhibitory activity against specific collagenase secreted by human and rabbit fibroblasts. The fibronectin preparation derived from plasma showed little inhibition, but the one derived from amniotic fluid contained potent inhibitory activity against collagenase. This activity was separated from fibronectin on a DE-52 cellulose column and did not cross-react with antibodies to fibronectin. The inhitor was a glycoprotein that was partially purified from amniotic fluid by concanavalin A-Sepharose affinity chromatography. Inhibition was irreversible and enzyme activity was not recovered after reaction with latent or activated collagenase by either trypsin or organomercurial treatment.     

Comparison of metabolism of free fatty acid by isolated perfused livers from male and female rats.

Livers from normal, fed male and female rats were perfused with different amounts of [1-14C]oleate under steady state conditions, and the rates of uptake and utilization of free fatty acid (FFA) were measured. The uptake of FFA by livers from either male or female rats was proportional to the concentration of FFA in the medium. The rate of uptake of FFA, per g of liver, by livers from female rats exceeded that of the males for the same amount of FFA infused. The incorporation by the liver of exogenous oleic acid into triglyceride, phospholipid, and oxidation products was proportional to the uptake of FFA. Livers from female rats incorporated more oleate into triglyceride (TG) and less into phospholipid (PL) and oxidation products than did livers from male animals. Livers from female rats secreted more TG than did livers from male animals when infused with equal quantities of oleate. The incorporation of endogenous fatty acid into TG of the perfusate was inhibite) by exogenous oleate. At low concentrations of perfusate FFA, however, endogenous fatty acids contributed substantially to the increased output of TG by livers from female animals. Production of 14CO2 and radioactive ketone bodies increased with increasing uptake of FFA. The partition of oleate between oxidative pathways (CO2 production and ketogenesis) was modified by the availability of the fatty acid substrate with livers from either sex. The percent incorporation of radioactivity into CO2 reached a maximum, whereas incorporation into ketone bodies continued to increase. The output of ketone bodies was dependent on the uptake of FFA, and output by livers from female animals was less than by livers from male rats. The increase in rate of ketogenesis was dependent on the influx of exogenous FFA, while ketogenesis from endogenous sources remained relatively stable. The output of glucose by the liver increased with the uptake of FFA, but no difference due to sex was observed. The output of urea by livers from male rats was unaffected by oleate, while the output of urea by livers from females decreased as the uptake of FFA increased. A major conclusion to be derived from this work is that oleate is not metabolized identically by livers from the two sexes, but rather, per gram of liver, livers from female rats take up and esterify more fatty acid to TG and oxidize less than do livers from male animals; livers from female animals synthesize and secrete more triglyceride than do livers from male animals when provided with equal quantities of free fatty acid.     

A three-step method for isolating a few to several hundred milligrams of protein

An operationally simple general protein isolation method was devised from three previously available separation tools, and was tested by application to two demanding fractionation problems and for yield. One test system was the isolation by gel electrofocusing of two model proteins with pI values of 4.6 and 4.8, bovine serum albumin and ovalbumin, with a load of 220 mg each. The other test was the isolation of 10 mg of human growth hormone isohormone B from a mixture of closely migrating other isohormones. The three-step procedure comprises of: (1) separation into zones of homogeneous protein by gel electrofocusing; (2) excision of the zones of homogeneous protein from the gel followed by concentration of the protein to a small volume of solution by means of Steady-State Stacking; (3) purification from polyacrylamide-like contaminants and non-volatile buffers by gel filtration followed by lyophilization. The average overall recovery was 70--80%.     

Secretion and uptake of nascent hepatic very low density lipoprotein by perfused livers from fed and fasted rats

Livers from fed or 24-hr fasted male rats were perfused in a recycling system. VLDL labeled with [1-14C]oleate (95% in triglyceride), produced in separate perfusions of livers from fed rats, was added to the medium as a pulse. Uptake of VLDL 14C-labeled triglyceride by livers from fasted rats was less than that from fed rats regardless of addition of oleate. During the interval in which radioactive triglyceride was taken up, the mass of triglyceride in the medium increased, indicative of the synthesis and net secretion of triglycerides. The rates of secretion of VLDL and uptake of VLDL were both more rapid in livers from fed rats in comparison to those from fasted animals. It was calculated that about 50% of the triglyceride synthesized and secreted by the liver was taken back by livers from fed rats. The VLDL from livers of fasted rats did not contain any apoE detectable by SDS gel electrophoresis or by radioimmunoassay when no fatty acid or 166 mumol of oleic acid was infused. In contrast, apoE comprised 6% of the VLDL apoprotein derived from perfusion of livers from fed animals in the absence of added fatty acid, and 20% when the fed livers were infused with 166 mumol of oleic acid. However, the net output (accumulation) of apoE by fasted liver was only two-thirds that from fed livers. When lipoprotein-free rat plasma containing apoE (4 mg/dl) was used in place of bovine serum albumin, the VLDL secreted by livers from either fed or fasted rats contained apoE and was taken up to a similar extent by such livers. These data suggested that the apoE of the d greater than 1.21 g/ml fraction was transferred to newly secreted VLDL which then stimulated uptake of the VLDL by livers from fasted rats. With further stimulation of secretion of VLDL triglyceride by infusion of 332 mumol of oleic acid/hr, the percent of apoE in the VLDL secreted by livers from fasted rats increased to 20%, which was similar to that of the VLDL produced by livers from fed rats when either 166 or 332 mumol/hr was infused. These data suggest a relationship between rates of hepatic secretion of VLDL (TG) and apoE, and the association of apoE with the secreted VLDL. During fasting, reduced secretion of both VLDL and apoE resulted in a VLDL particle that was considerably diminished in content of apoE and, therefore, that would be taken up by the liver at a reduced rate, in comparison to that observed in the fed animal.     

Metabolism of daidzein by intestinal bacteria from rhesus monkeys (Macaca mulatta)

PURPOSE: To identify the metabolites produced from an isoflavonoid, daidzein, by colonic bacteria of rhesus monkeys. METHODS: The metabolism of daidzein by the fecal bacteria of nine monkeys was investigated. Daidzein was incubated anaerobically with fecal bacteria, and the metabolites were analyzed by use of liquid chromatography and mass spectrometry. RESULTS: The fecal bacteria of all of the monkeys metabolized daidzein to various extents. Dihydrodaidzein was found in cultures of fecal bacteria from two monkeys; dihydrodaidzein and equol were found in cultures from four monkeys; dihydrodaidzein, equol, and an unknown metabolite (MW = 244) were found in cultures from one monkey; and dihydrodaidzein and the unknown metabolite were found in cultures from two monkeys. CONCLUSIONS: Similar to that in humans, variation was evident in the metabolism of isoflavonoids by fecal bacteria from rhesus monkeys. Some metabolites produced by fecal bacteria from monkeys were the same as those produced by fecal bacteria from humans.     

Pathway of gluconeogenesis from D- and L-glycerate in rat hepatocytes

Synthesis of glucose from 10 mM D-glycerate by hepatocytes isolated from rats fasted 24 h was inhibited by 3-aminopicolinate (3AP), 3-mercaptopicolinate (3MP), and aminoxyacetate (AOA). The inhibition by AOA can be exerted over that caused by either of the picolinates. The effects of 3AP and 3MP were not additive, and cannot be ascribed totally to inhibition of reducing equivalent transfer by their inhibitory effect on phosphoenolpyruvate carboxykinase. The time course and dependence on substrate concentration of gluconeogenesis from L-glycerate was similar to those from D-glycerate, even though the cellular content of D-glycerate was markedly less when L-glycerate was the substrate. Gluconeogenesis from L-glycerate was not significantly affected by 3AP or 3MP, but was inhibited by AOA. The degree of inhibition by AOA of glucose synthesis from L-glycerate was greater than that from D-glycerate. Studies employing [14C]paraformaldehyde revealed that hydroxypyruvate may be converted to dihydroxyacetone in the hepatocyte. A hitherto unknown inhibition site of 3AP and 3MP is inferred and a dual pathway of D- and L-glycerate gluconeogenesis consistent with these results is proposed.     

Chemiosmotic lysis and insulin secretion: studies of isolated granules, intact and permeabilised rat islets of Langerhans

The possible involvement of chemiosmotic lysis of secretory granules in the exocytosis of insulin from pancreatic beta cells was investigated by comparing insulin release from isolated secretory granules, from intact islets of Langerhans, and from electrically permeabilised islets. Lysis of isolated granules was stimulated by ATP in the presence of Mg2+. ATP-induced granule lysis was pH and temperature dependent and was inhibited by collapsing the pH gradient across the granule membrane by removal of permeant anions, or by increasing the extragranular osmolarity. However, insulin secretion from intact islets in response to glucose, a phosphodiesterase inhibitor or a Ca2+ ionophore was only partially inhibited by anion replacement, while Ca2+ -induced insulin release from electrically permeabilised islets was not affected by altering the extragranular or intragranular pH. These results suggest that studies of the stability of isolated granules in vitro do not necessarily relate to insulin release from whole cells, and do not support a major role for chemiosmotic lysis of secretory granules in the exocytotic release of insulin.     

Isolation and partial characterization of crispacin A, a cell-associated bacteriocin produced by Lactobacillus crispatus JCM 2009

Crispacin A, a cell-associated bacteriocin produced by Lactobacillus crispatus JCM 2009, was purified from culture broth by ammonium sulfate precipitation, followed by ion exchange and reversed-phase chromatography. Crispacin A was also purified from the cells of L. crispatus JCM 2009 by acid extraction and reversed-phase chromatography. Purified crispacin A was determined to be 5393 Da by mass spectrometry and found not to show sequence homology with other bacteriocins from lactic acid bacteria.     

Membrane-bound and soluble extracellular alpha-amylase from Bacillus subtilis.

Extracellular alpha-amylase was purified to homogeneity from a Marburg strain of Bacillus subtilis. The enzyme is a single polypeptide chain of molecular weight approximately 67,000. Its NH2-terminal amino acid sequence is Leu-Thr-Ala-Pro-Ser-Ile-Lys. A membrane-derived alpha-amylase was solubilizing from membrane vesicles by treatment with Triton X-100 and was highly purified by chromatography on an anti-alpha-amylase-protein A-Sepharose column. Membrane-derived alpha-amylase was indistinguishable from the soluble extracellular enzyme by sodium dodecyl sulfate-gel electrophoresis and radioimmunoassay. The membrane-derived enzyme contains phospholipid. Approximately 30 to 80% of the phospholipid was extracted from the purified enzyme by chloroform:methanol. The extracted phospholipid was predominately phosphatidylethanolamine. Treatment with phospholipase D released phosphatidic acid. Membrane-bound alpha-amylase was latent in membrane vesicles. Release of membrane-bound alpha-amylase from vesicles by an endogenous enzyme was maximal at pH 8.5, was inhibited by metal chelators and diisopropyl fluorophosphate and was stimulated by Ca2+ and Mg2+. The amount of membrane-bound alpha-amylase was related to the level of secretion.     

Hormonal stimulation of cyclic AMP accumulation and glycogen phosphorylase activity in calcium-depleted hepatocytes from euthyroid and hypothyroid rats.

Activation of glycogen phosphorylase by hormones was examined in hepatocytes isolated from euthyroid and hypothyroid female rats and incubated by Ca2+-free buffer containing 1 mM-EGTA. Basal glycogen phosphorylase activity was decreased in Ca2+-free buffer. However, the activation of hepatocyte glycogen phosphorylase, in the absence of extracellular Ca2+, in response to adrenaline, glucagon or phenylephrine was slightly lower, whereas that by vasopressin was abolished. The activation of glycogen phosphorylase by phenylephrine, adrenaline or isoproterenol (isoprenaline) in hepatocytes from euthyroid rats incubated in the absence of Ca2+ was not accompanied by any detectable increase in total cyclic AMP. The log-dose/response curves for activation of phosphorylase by phenylephrine or low concentrations of adrenaline were the same in hepatocytes from hypothyroid as compared wit euthyroid rats, whereas the response to isoproterenol was greater in hepatocytes from hypothyroid rats. However, the increases in total cyclic AMP accumulation caused by adrenaline or isoproterenol were greater in hepatocytes from hypothyroid rats than in hepatocytes from euthyroid rats. The increases in cyclic AMP accumulation caused by adrenaline or isoproterenol in Ca2+-depleted hepatocytes from hypothyroid rats were blocked by propranolol, a beta-adrenergic antagonist. In contrast, propranolol was only partially effective asan inhibitor of the activation of glycogen phosphorylase by phenylephrine or adrenaline in hepatocytes from hypothyroid rats and ineffective on phosphorylase activation in cells from euthyroid rats. These data indicate that the alpha-adrenergic activation of glycogen phosphorylase is not affected by the absence of extracellular Ca2+, and the extent to which total cyclic AMP was increased by adrenergic amines did not correlate with glycogen phosphorylase activation.     

Examination by chromatography and immunodiffusion of an adenovirus 3 isolated from humans with infectious hepatitis

Prototype adenovirus 3 and adenovirus SC8, which was found in feces from a patient with infectious hepatitis and which was classified as adenovirus 3 by standard procedures, were compared by chromatography and immunodiffusion techniques. When the radioactive adenovirus moiety in SC8 had been separated from other radioactive components of tissue culture by gel filtration, a smaller infectious agent was detected, whereas with prototype adenovirus 3 one infectious agent was found. The large agent from SC8 was classified as adenovirus type 3 by serum neutralization tests, but results from homologous and heterologous immunodiffusion tests and heat sensitivity tests indicated that this agent was different from the classical prototype adenovirus 3. Similar precipitin patterns obtained in homologous and heterologous reactions by immunodiffusion suggested a similarity between the smaller particle and an unidentified agent isolated without adenoviruses from blood clots from overt cases of hepatitis. With the present evidence, it was not possible to relate the smaller agent to adeno-associated viruses; however, its similarity to an agent isolated from blood of overt cases implies a possible relationship with hepatitis. The continued recovery of the variant strain of adenovirus type 3 from patients with hepatitis, although at relatively low rates of isolation, suggested a possible undetermined relation to the disease.     

Characteristics of ATPase from sugarcane protoplast and vacuole membranes

Characteristics of membrane-associated ATPase from commercial Hawaiian varieties of sugarcane ( Saccharum spp. hybrids) were investigated in preparations from sugarcane cell suspension culture and from stalk tissues of the intact plant. In order to examine comparable preparations, protoplasts and vacuoles, in turn, were obtained from both sources. ATPase from preparations of crude protoplast membranes and tonoplast had a pH optimum of 6 to 6.5. The relative effectiveness of divalent cations in stimulating ATPase was Mg²⁺ > Mn²⁺≥ Co²⁺ > Ca²⁺≥ Zn²⁺. Enzyme activity was not stimulated by K⁺, nor by other monovalent cations. Protoplasts and vacuoles from both sources showed significant acid phosphatase activity. Acid phosphatase activity was inhibited by molybdate, but ATPase activity was unaffected. Membrane preparations from protoplasts contained inorganic pyrophosphatase, but enzyme activity was low or not present in tonoplast preparations. Cell suspension and stalk tissue preparations hydrolyzed a large number of nucleoside di- and triphosphates. The hydrolysis is most likely due to a series of enzymes rather than a single enzyme. ATPase from protoplast and tonoplast preparations was inhibited 30–50% by diethylstilbestrol and sodium ortho-vanadate and was unaffected by ionophores. This study illustrates the complexity of phosphohydrolase activities in membrane preparations from sugarcane. The study, however, also illustrates substantial similarity in the behavior of these enzymes, whether they are derived from the plant itself or from cell cultures originating from comparable tissues of the plant.     

Fe2+-Induced Lysis and Lipid Peroxidation of Chromaffin Granules

Chromaffin granules, the catecholaminergic storage granules from adrenal chromaffin cells, lysed in 10(-9)-10(-7) M Fe2+. Lysis was accompanied by the production of malondialdehyde which results from lipid peroxidation. Both chromaffin granule lysis and malondialdehyde production were inhibited by the free radical trapping agent butylated hydroxytoluene but not by catalase and/or superoxide dismutase. The results suggest that lysis resulted from a direct transfer of electrons from Fe2+ to a component of the chromaffin granule membrane without the participation of either superoxide or hydrogen peroxide and may have resulted from lipid peroxidation. In some experiments, ascorbate alone induced chromaffin granule lysis which was inhibited by EDTA, EGTA, or deferoxamine. The lysis was probably caused by trace amounts of reducible polyvalent cation. Lysis sometimes occurred when Ca2+ was added with EGTA (10 microM free Ca2+ concentration) and was consistently observed together with malondialdehyde production in the presence of Ca2+, EGTA, and 10 microM Fe2+ (total concentration). The apparent Ca2+ dependency for chromaffin granule lysis and malondialdehyde production was probably caused by a trace reducible polyvalent ion displaced by Ca2+ from EGTA and not by a Ca2+-dependent reaction involving the chromaffin granule.     

Comparison of the carbon-sequestering abilities of pineapple leaf residue chars produced by controlled combustion and by field burning

This study was undertaken to compare the chemical properties and yields of pineapple leaf residue (PLR) char produced by field burning (CF) with that produced by a partial combustion of air-dried PLR at 340 °C for 3 h in a furnace (CL). Higher total C, lignin content, and yield from CL as well as the presence of aromatic compounds in the Fourier Transform Infrared spectra of the char produced from CL suggest that the CL process was better in sequestering C than was the CF process. Although the C/N ratio of char produced from CL was low indicating a high N content of the char, the C in the char produced from CL was dominated by lignin suggesting that the decomposition of char produced from CL would be slow. To sequester C by char application, the PLR should be combusted in a controlled process rather than by burning in the field.     

Purification and some properties of chalcone synthase from a carrot suspension culture induced for anthocyanin synthesis and preparation of its specific antiserum

Chalcone synthase was purified to homogeneity by polyacrylamide gel electrophoresis from cell suspension cultures of carrot in which anthocyanin synthesis was induced by transferring the cells from a medium containing 2,4-dichlorophenoxy-acetic acid (2,4-D) to one lacking it. A molecular weight of 80,000-85,000 for the enzyme was determined by gel filtration and disc-gel polyacrylamide electrophoresis, and one of about 40,600 for the subunit by SDS slab-gel electrophoresis. The primary reaction product was chalcone and the pH optimum of the reaction was 8.0. The Km values for 4-coumaroyl-CoA and malonyl-CoA were 5.7 microM and 18 microM, respectively. These properties of carrot chalcone synthase were discussed in comparison to those of that from cell cultures of parsley reported previously. Antiserum against chalcone synthase from carrot was obtained from mice bred under specific pathogen free conditions. Crossreactivity was examined by Western-blotting, and the high specificity of the antiserum against chalcone synthase was demonstrated.