猴头菌菌丝富硒特性及富硒深层发酵的研究

为获得高产的富硒猴头菌菌丝体,采用平板法,通过Na_2SeO_3浓度梯度实验,从H2、H99、NK 3种猴头菌中筛选出耐硒品种;以猴头菌菌丝干重为指标,采用单因素试验结合正交试验优化培养基的碳源、氮源、无机盐和维生素,并进一步通过均匀设计试验确定各营养元素的配比;以菌丝干重和有机硒含量为指标,确定深层发酵培养基中Na_2SeO_3的浓度。结果表明:3种猴头菌中H2最耐硒,在含6μg/mL的Na_2SeO_3平板培养基中培养的菌丝适合作为后续研究的菌种;优化的液体培养基成分为5.8%可溶性淀粉、5.8%黄豆粉、0.04%CaCl_2、0.024%VB_1+VB_2+VB_6,此时菌丝干重理论值可达2.37 g/100 mL;当液体培养基含12μg/mL的Na_2SeO_3时,菌丝生长不受抑制,有机硒含量最高,达136.8 mg/kg菌丝。该研究为富硒猴头菌产品的开发和利用奠定了基础。     

亚硒酸钠对烟草冠瘿组织生长的影响及其与内源激素的关系

低浓度(0.1～1μmol/L)亚硒酸钠(Na_2-ScO_s)对烟草冠瘿组织的生长有促进作用;但浓度较高时(5～20μmol/L)不仅对生长有抑制作用,同时能促使其内源吲哚乙酸(IAA)和玉米素(ZA)含量下降,脱落酸(ABA)含量增加,IAA氧化酶活性增强。适宜浓度的外源2,4-D,IAA,6-BA,ZA都可部分缓解较高浓度Na_2SeO_3对生长的抑制作用,其中2,4-D,6-BA的缓解能力分别大于IAA和ZA。Na_2SeO_3还引起烟草冠瘿组织过氧化物同工酶谱发生显著变化,这种变化可被适宜浓度的外源6-BA逆转,并向正常组织方向转化,缓解较高浓度Na_2SeO_3对生长的抑制作用与逆转其过氧化物同工酶谱变化的6-BA浓度范围完全一致。     

亚硒酸钠对AH_(22)小鼠腹水型肝癌细胞中组织特异酶CPSⅠ的诱导及对细胞增殖酶ACT的抑制

通过连续四天腹腔给硒(1μgNa_2SeO_3/克体重)后,腹水型肝癌细胞中与细胞分化相关的CPS_(ase)Ⅰ活性显著上升,同时与细胞增殖相关的ACT_(ase)活性明显下降。而在正常鼠肝中,按相同方式给硒的结果是ACT_(ase)活性明显增高,CPS_(ase)Ⅰ活性则略有下降。该结果表明硒可能涉及对细胞分化与增殖的调控。     

硒对红细胞膜稳定作用的浓度依赖性

 Na_2SeO_3对人红细胞膜骨架具有稳定作用,但这种作用依赖于Na_2SeO_3的浓度。在低离子强度下,4℃透析人红细胞膜,实验组加入不同浓度的Na_2SeO_3,对照组不加Na_2SeO_3。结果表明,0.1—0.8ppm Na_2SeO_3的存在比对照组具有较高的Na~+,K~+-ATP酶活性、膜脂流动性。用N-[3-芘]-马来酰胺作探针,反映两者构象也有差异。如果在透析液中加入较高浓度的Na_2SeO_3(>1.0ppm)则会产生与低浓度相反的结果。人红细胞膜~31P-NMR的测试也表明,加入0.4ppm与4.0ppm Na_2SeO_3会产生不同的结果。与对照组相比较,低浓度使化学位移各向异性值(△σ)下降,而高浓度则使△σ增加。     

亚硒酸钠抗红细胞膜蛋白交联作用的机理探讨

邻苯二酚氧化处理人红细胞膜会导致膜蛋白交联,产生高分子聚合物(HMP)。用N—乙基马来酰胺(NEM)封闭膜蛋白硫基,则不产生HMP。预先用Na SeO_3(0.05mol/L)处理红细胞膜,也同样不产生HMP。用N—(3-芘)马来酰胺(N-〔3-P〕NEM)标记红细胞膜来测试不同浓度Na_2SeO_3对荧光强度的影响。结果表明,随着Na SeO_3浓度增高荧光强度相应降低。Na_2SeO_3对红细胞膜的预处理时间和荧光强度的变化有关。经Na SeO_3处理的红细胞膜ESR谱提示了Na_2SeO_3与材相互作用有关。用荧光法测定膜结合硒含量表明,Na_2SeO_3处理红细胞膜可导致膜结合硒含量增高。推测,Na SeO_3很可能与膜蛋白疏基作用形成结合硒,从而起到抗膜蛋白交联作用。     

灵芝菌丝体富硒条件优化及其硒多糖抗氧化活性研究

利用正交试验对灵芝菌丝体液体发酵富硒条件进行优化,研究培养基中不同碳源、氮源和亚硒酸钠浓度对菌丝体生物量、硒含量、富硒率以及胞外酶活性的影响,此外还对硒多糖的抗氧化活性进行了初步研究。结果表明:当培养基中碳源为20%马铃薯、氮源为2%麸皮、Na_2SeO_3浓度为5 mg/L时,培养基中菌丝生物量最高达到3.86 g/L,硒含量最高为0.576 mg/g,富硒率也达到最高为44.47%。Na_2SeO_3对菌丝生长的影响大于碳源和氮源;同样的Na_2SeO_3对胞外酶活性的影响最大,最优组的POD和PPO酶活性都较其他组高,且随着Na_2SeO_3浓度的升高,其酶活性逐渐降低。富硒灵芝菌丝体粗多糖都具有较强的体外抗氧化活性,通过相关性分析发现,其粗多糖的抗氧化活性与硒含量、富硒率具有显著的正相关。显示硒多糖在食品和药品等领域具有良好的开发利用前景。     

高生物量富硒酵母的选育及培养条件初步优化

通过筛选、单倍体分离、诱变和原生质体融合,从融合子中选育了一株高生物量富硒酵母菌株（编号为ZFF-28）,其细胞硒总含量分别是原始亲株ZY-67和ZY-198的2.8倍和2.0倍。通过单因素实验和正交试验设计,确定了优化培养条件：6%糖浓度的蔗糖糖蜜,添加0.5% (NH4)2SO4、0.1% H3PO4、60μg/mL Se,pH60～6.5,装液量50mL/250mL三角瓶,接种量10%,培养时间25h。在优化培养条件下,菌株ZFF_28的生物量可达8.2g/L,细胞中硒的含量达2050μg/g,硒总含量达到了16810μg/L,是培养条件优化前的1.3倍。细胞硒含量的91%为有机硒。     

枳实提取物及其药效组分对ox-LDL损伤的人脐静脉内皮细胞ICAM-1表达和NO释放的影响

目的:研究枳实提取物及其药效组分橙皮苷和新橙皮苷对氧化低密度脂蛋白(oxidized low density lipoprotein,Ox-LDL)损伤的人脐静脉内皮细胞(human umbilical vein endothelial cells line,HUVEC)细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)表达和一氧化氮(nitric oxide,NO)释放的影响。方法:体外培养HUVEC,50μg/mL ox-LDL制造HUVEC损伤模型。以MTS染色法检测细胞毒性确定用药浓度。细胞ELISA法测定细胞表面ICAM-1的含量,试剂盒测定细胞培养上清液中NO含量。结果:①枳实提取物小于等于2 mg/mL时,橙皮苷浓度小于等于0.03125 mg/mL时,新橙皮苷浓度小于等于0.25 mg/mL时,HUVEC存活率分别大于80%。②2.0 mg/mL和1.0 mg/mL两个浓度的枳实提取物、15.625μg/mL的橙皮苷和0.2500 mg/mL新橙皮苷对ox-LDL诱导的HUVEC的ICAM-1表达有显著抑制作用。③2.0 mg/mL枳实提取物显著提高ox-LDL诱导的HUVEC和正常HUVEC培养液中的NO含量;7.813μg/mL、15.625μg/mL和31.250μg/mL 3个浓度的橙皮苷能显著提高ox-LDL诱导的HUVEC培养液中的NO含量,31.250μg/mL的橙皮苷能促进正常HUVEC的NO释放;0.2500 mg/mL和0.1250 mg/mL 2个浓度的新橙皮苷能显著提高ox-LDL诱导的HUVEC培养液中的NO含量。结论:枳实提取物及其药效组分橙皮苷、新橙皮苷能抑制ox-LDL诱导的HUVEC的ICAM-1表达,促进ox-LDL诱导的HUVEC的NO释放。     

硒与巯基作用稳定红细胞膜骨架

Na_2SeO_3可以减少人红细胞膜血影收缩蛋白(Spectrin)从膜骨架上解离下来.当含有巯基的二硫苏糖醇(DTT)存在下或红细胞膜用碘代乙酰胺(IAA)封闭巯基后.Na_2SeO_3的效应就不再呈现.用与巯基结合的荧光探针:N-[3-芘]-马来酰胺(N-[3-P]-M)来测试不同浓度Na_2SeO_3存在下红细胞膜的荧光强度.结果表明,随着Na_2SeO_3浓度增加,荧光强度就相应降低,这都说明硒是通过与红细胞膜巯基相作用来发挥效应的.~(31)P-NMR测试表明,人红细胞膜在加硒条件下透析,化学位移各向异性(Δσ)值低于不加硒的样品,这提示硒与红细胞膜蛋白巯基作用后红细胞膜脂-蛋白质的相互作用发生了变化.根据实验结果对硒作用后可能引起膜蛋白构象的变化也进行了讨论.     

L型多聚赖氨酸和D型多聚赖氨酸对神经细胞分化的影响

目的:比较L型多聚赖氨酸(L-PL)和D型多聚赖氨酸(D-PL)浸泡的玻片培养小鼠大脑皮层神经元细胞对其分化的影响。方法:取昆明白小鼠的大脑皮层细胞,分别用L-PL和D-PL进行培养,统计和比较细胞的分化比率、突起数量以及突起生长长度。结果:L-PL和D-PL培养的神经细胞第1、2天的分化率比较差异具有统计学意义(P0.05),而突起数和突起长度比较差异无统计学意义(P0.05)。当L-PL、D-PL的浓度分别为20μL/m L、5μL/mL时,其对1、2天细胞的分化产生显著影响,并且D型多聚赖氨酸培养的细胞分化率高于L型。500、100、20μg/m L的L-PL中,100μg/ml L-PL培养细胞的分化率最高,而125、25、5μg/m L的D型多聚赖氨酸培养细胞分化率比较差异虽然无统计学意义,但25μg/ml D-PL培养神经细胞的分化率总体趋势高于其他浓度。D-PL所形成的粒径大小与L-PL不同,且D-PL的总体趋势稍大于L-PL。结论:L型多聚赖氨酸和D型多聚赖氨酸会在神经细胞生长早期对分化率产生影响,但不影响突起数和突起长度。     

N-nitrosodiethylamine genotoxicity evaluation: a cytochrome P450 induction study in rat hepatocytes

In rats, N-nitrosodiethylamine (NDEA) induces tumors mainly in the liver. This could be because various enzymes are responsible for the metabolic activation of NDEA, besides the hepatic NDEA metabolizing enzyme, CYP2E1. We examined NDEA genotoxicity and cytotoxicity in primary cultures of female rat hepatocytes; we also looked at how it affected CYP mRNA expression. Single incubation with 0.9% NaCl resulted in a mean of 0.2% apoptotic cells, which doubled with 105 μg NDEA/mL. The frequency of necrosis with NDEA treatment was also doubled. Besides the cytotoxic effects, there was also a 4-fold decrease in mitotic index and a 3-fold decrease in the percentage of cells with micronuclei. A significant increase in micronucleus cells when hepatocytes were incubated with 2.1 μg NDEA/mL suggests that DNA repair was inactive. The chromosomal aberration evaluation revealed a discrete dose-response curve. Treatment with NDEA induced increases in CYP mRNA: CYP2B2 (1.8 times) and CYP2E1 (1.6 times) with non-cytotoxic NDEA concentrations (0.21-21 μg/mL). CYP2B1 mRNA levels decreased at 0.21 μg NDEA/mL (2.5-fold), while CYP4A3 mRNA decreased 1.3-fold. NDEA treatment at 2.1 μg/ mL induced a 1.9-fold increase in CYP3A1 mRNA. Understanding the cumulative effects in target cells during precarcinogenesis is crucial to understanding the mode of action of potential carcinogens and in order to develop comprehensive chemical toxicity profiles.     

Effect of time and sex on tissue selenium concentrations in chicks fed practical diets supplemented with sodium selenite or calcium selenite

An experiment was conducted with 384 1-d-old male and female broiler-chicks. The basal corn-soybean meal diet (.07 ppm Se DM basis) was supplemented with 0, .1, .2, or .3 ppm added Se as either sodium selenite (Na2SeO3) or calcium selenite (CaSeO3), and fed for 1, 3, or 5 wk. There was no effect of Se source or level on feed intake or gain, but males consumed more (P less than .01) feed than females. There was no effect (P greater than .10) of sex or Se source on plasma, liver, or kidney Se concentration. The Se concentration of all tissues increased (P less than .01) with time and increasing dietary Se concentration. Based on multiple regression slope ratios of liver, kidney, and plasma Se concentrations, Se from CaSeO3 was as available (103%) as Se from Na2SeO3.     

In vivo effect of selenium on the mutagenic activity of aflatoxin B1

Experiments were carried out to verify the effect of selenium on the mutagenic activity of AFB1. After 14 days of selenium administration to experimental animals (Chinese hamsters, Cricetulus griseus) in the form of 2 ppm Na2SeO3 solution available ad libitum the incidence of chromosomal aberrations in bone marrow cells due to a single p.o. administration of 5 mg AFB1 per 1 kg body weight was significantly reduced. The incidence of chromosomal aberrations was monitored till day 32 after AFB1 administration. A significant decrease in the frequency of aberrant cells, breaks and gaps was observed at almost any time during the investigation. 2 ppm Na2SeO3 solution itself did not enhance the frequency of chromosomal aberrations. The mechanism of the protective effect of selenium vis-a-vis the mutagenic and carcinogenic action of AFB1 remains obscure.     

Microcalorimetric studies of the action of Na2SeO3 on the growth of Halobacterium halobium R1

The effect of Na₂SeO₃ on the growth of Halobacterium halobium R1 was investigated by means of microcalorimetry at 37°C. The biological response to toxicants is observed as the inhibition of the rate constant of growth of living cells. A low concentration of Na₂SeO₃ stimulated the growth of H. halobium R1, and a high concentration of Na₂SeO₃ inhibited the growth of H. halobium R1. Toxicity may be expressed as the half-inhibition concentration (IC₅₀). The rate constants of growth (k) and the concentrations of Na₂SeO₃ (c) shows a linear relationship: k=1.790 × 10⁻⁶ − 2.27 × 10⁻³ c. The value of IC₅₀ obtained from the accompanying figure of I-c is 679 μg/mL.     

Effects of inorganic selenium compounds on oxidative DNA damage

Exposure of Escherichia coli or mammalian cells to H2O2 results in cell death due to iron-mediated DNA damage. Since selenium compounds have been examined for their ability to act as antioxidants to neutralize radical species, and inorganic selenium compounds are used to supplement protein mixes, infant formula, and animal feed, determining the effect of these compounds on DNA damage under conditions of oxidative stress is crucial. In the presence of Fe(II) and H2O2, the effects of Na2SeO4, Na2SeO3, SeO2 (0.5-5000 microM), and Na2Se (0.5-200 microM) on DNA damage were quantified using gel electrophoresis. Both Na2SeO4 and Na2Se have no effect on DNA damage, whereas SeO2 inhibits DNA damage and Na2SeO3 shows antioxidant or pro-oxidant activity depending on H2O2 concentration. Similar electrophoresis experiments with [Fe(EDTA)](2-) (400 microM) and Na2SeO3 or SeO2 show that metal coordination by the selenium compound is required for antioxidant activity. In light of these results, Na2SeO4 may be safer than Na2SeO3 for nutritional supplements.     

Comutagenic and coclastogenic effects of selenium in vitro and in vivo

The comutagenic activity of selenium was investigated using in vitro and in vivo techniques, including the liquid suspension modification of the standard Salmonella/microsome mutagenicity assay, the metaphase analysis of chromosome aberrations in CHO cells and in mouse bone marrow as well as the micronucleus assay in mouse bone marrow. 4 h growth of S. typhimurium TA1535 in a nutrient broth containing 2.9 x 10(-5) M but not 1.16 x 10(-5) M Na2SeO3 caused an up to 10-fold increase of the number of N-methylnitrosourea (MNU, 2.0-2.5 mM)-induced his+ revertants and an up to 2-fold elevation of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 1.48 x 10(-5))-induced mutation rate. Pretreatment of bacteria with Na2SeO3 alone had no effect on the spontaneous mutation level. The combined treatment of CHO cells with MNNG (1.25 x 10(-5) M) or tobacco smoke (TS, 2-3 puffs generated by a cigarette inhalation machine) plus Na2SeO3 (0.58-1.16 x 10(-5) M) starting 2 h and 4 h before the MNNG or TS treatment respectively resulted in a 2-3-fold increase in the percent of metaphases with chromosome aberrations. Furthermore, treatment for 7-14 days of male BDF1 (C57Bl x DBA2) or CC57W mice with Na2SeO3, added to the drinking water at a concentration of 10 ppm, potentiated by 2-3 times the chromosome-damaging activity of urethane (0.5-1.0 g/kg, i.p.) in mouse bone marrow, as measured by the formation of micronuclei or chromosome aberrations. In addition, Na2SeO3 increased up to 43.8% the number of micronucleated polychromatic erythrocytes (MNPCE) induced by mitomycin C (MMC, 1.5 mg/kg, i.p.) in BDF1 mouse bone marrow. Treatment of mice with Na2SeO3 alone had no effect on the spontaneous level of MNPCE. All these findings are consistent with a comutagenic and coclastogenic activity of selenium both in prokaryotes and in eukaryotes, in vitro as well as in vivo after pretreatment of target cells with the trace element.     

Effects of intraperitoneally injected selenium and vitamin E in rats anesthetized with halothane.

Halothane, commonly used for anesthetizing humans and animals, is one of the most important volatile anesthetics and may cause the formation of free radicals during its biotransformation. Free radicals may lead to degeneration of liver cells. Vitamin E and glutathione peroxidase (GSH-Px) containing selenium are two natural antioxidants, and these may protect the cellular lipid and lipoproteins against oxidative damage caused by free radicals. Therefore, the purposes of the present study were to investigate the probable protective effects of intraperitoneally administered Se and vitamin E on liver enzymes and to determine some other hematological parameters in the halothane anesthesia of rats. All rats were randomly divided into five groups. The first group was used as a control, and physiological saline (0.9%) was intraperitoneally injected into these animals as a placebo. The second group was used as an anesthesia control group and was only anesthetized with halothane for two hours. The third group received intraperitoneally administered Se (Na2SeO3, 0.3 mg/200 g body weight), the fourth group vitamin E (dl-alpha-tocopheryl acetate, 100 mg/kg body weight), and the fifth group a Se plus vitamin E combination (Na2SeO3, 0.3 mg/200 g body weight + dl-alpha-tocopheryl acetate, 100 mg/kg body weight). The activities of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, triglycerides, erythrocyte counts, the packet-cell volume, hemoglobin concentrations and neutrophyle rates significantly increased (p < 0.05 to p < 0.01) after halothane anesthesia and returned to near control levels after Se, vitamin E and Se plus vitamin E injections. The values of cholesterol, total protein, white blood cell counts and lymphocyte rates significantly decreased (p < 0.05 to p < 0.01) in the anesthesia control group. However, the levels of albumin, total bilirubin, creatinine, the mean corpuscular volume, the mean corpuscular hemoglobin, and the mean corpuscular hemoglobin concentration were not statistically influenced. In conclusion, we have determined that halothane anesthesia affected some liver enzymes and some other biochemical and hematological parameters. Se, vitamin E and their combination may prevent the increase of liver enzymes after halothane anesthesia. Based upon these results, Se and vitamin E may play an important role in the indication of hepatic cellular injury produced by halothane.