胎肝细胞中一个刺激蛋白激酶C的胰岛素介体

经胰岛素处理的人胎肝细胞,经酸性抽提,热处理,活性炭吸附,阴离子AG1×8柱pH梯度洗脱,Sephadex G10脱盐、C18疏水层析。二次薄层层析分离,得到一个对蛋白激酶C有刺激作用的活性物质。组成分析结果显示,该物质含Ser,Ala,Gly,Yal,Gln五种氨基酸和甘露糖、肌醇两种单糖,质谱测出其分子量为841。该活性物质与胰岛素介体有相似的性质、组成和结构,推测它可能为刺激胎肝细胞蛋白激酶C的胰岛素介体。     

苦瓜籽核糖体失活蛋白的理化性质及生物活性

采用硫酸铵分级分离,假配基亲和层析和ＳｅｐｈａｃｒｙｌＳ－１００分子筛层析等方法,从苦瓜籽中获得核糖体失活蛋白（ＲＩＰ）．经ＳＤＳ－ＰＡＧＥ、ＰＡＧＥ、ＩＥＦ和ＰＡＳ方法分析均表明为单一蛋白着色带或单一糖蛋白着色带．根据ＳＤＳ－ＰＡＧＥ和Ｓｅｐｈａｄｅｘ Ｇ－１５０分子筛层析结果计算其相对分子质量为３．０×１０４,经ＩＥＦ－ＰＡＧＥ结果计算其ｐＩ为８．９～９．０．对无细胞系统中蛋白质生物合成抑制活性明显,其ＩＣ５０为５．３×１０－ １０ ｍ ｏｌ／Ｌ左右．体外生物活性试验结果表明其对人肝癌细胞、Ｖｅｒｏ、ＳＰ２／０、３Ｔ３、Ｋｂ、Ｎａｖａｎａ 等肿瘤细胞株均表现有不同程度的抑制作用．而对完整细胞人胚肺二倍体细胞却毒性极小．因此,上述实验结果为该ＲＩＰ的进一步深入研究和有可能开发成免疫毒素的高效弹头药物提供了一定的工作基础．     

Splenic suppressing factor: purification and characterization of a factor suppressing thymidine incorporation into activated lymphocytes.

Suppressing activity upon the mitogen-activated lymphocytes was found in the supernatant (SUP) from the culture of mouse spleen, high-density subpopulation of thymocytes, and peritoneal exudate cells. Suppressing factor was obtained from the non-stimulated lymphocytes cultured for 24 to 36 hr with or without serum. Suppressing activity in the SUP was observed in the incorporation of 3H-thymidine, 3H-uridine, and 3H-leucine into Con A-activated lymphocytes or in the proliferation of L cells. Suppressing factor partially purified by Sephadex G-25 column chromatography was a heat-stable and dialyzable substance(s). Further purification and isolation of this factor by two-dimensional thin layer chromatography revealed that this was thymidine and thymidine monophosphate. The suppression in 3H-thymidine incorporation was attributed to the dilution effect of cold thymidine released from cultured lymphocytes.     

Utilization of Synthetic Compounds with Juvenile Hormone Activity for the Silkworm Rearing

A proteinous inhibitor of lipase was purified from soybean cotyledons by the procedures of ammonium sulfate fractionation, gel-filtration on Sephadex G–150 column, DEAE-cellulose column chromatography and isoelectric focusing. The inhibitor obtained by gelfiltration was separated into three active components, D–1, D–2 and D–3, by DEAE-cellulose column chromatography. The D–3 fraction after isoelectric focusing was homogeneous judged from disc electrophoresis. The inhibitory activity was more stable against treatments of heating and Pronase when the D–3 fraction was preincubated with substrate than without substrate. The extent of inhibition was varied by changing the order of addition of reactants and condition of substrate. From these results, the mode of inhibition is discussed.     

A convenient source of CFU-s inhibitors: the fetal calf liver

The presence of a bone-marrow stem-cell inhibitor able to prevent CFU-s entry into DNA synthesis after cytosine arabinoside (Ara-C) treatment has been detected in 7-month-old fetal calf liver. The inhibitory fraction was obtained through ultrafiltration of a delipidated tissue extract powder and purified by BioGel-P-2 chromatography. The elution pattern on Sephadex G10 is similar to that of the bone-marrow inhibitory extract previously obtained from fetal calf bone marrow. It corresponds to a low-molecular-weight molecule (MW less than 2000), devoid of species specificity and having no inhibitory effect on GM-CFC proliferation.     

Purification and Properties of Rape Alcohol Dehydrogenase

Germinating seeds with the highest specific activity (24 hour germination) were used for isolation of alcohol dehydrogenase, ADH, from rape (Brassica napus L. cv. T?ebi?ská). The rape ADH was purified by fractionation with ammonium sulphate, desalting on Sephadex G 25, chromatography on DEAE cellulose and gel filtration on Sephadex G 150. Using this isolation procedure, enzyme with a specific activity 85.6 times higher than that of the crude extract was obtained. The molecular weight of the enzyme obtained is 66.000. The enzyme is a metallo-enzyme containing sulfhydryl groups as evidence by the inhibitory effect of chelating compounds and thiol reagents. The optimum pH for the oxidation of ethanol is 8.5 and for reduction of acetaldehyde 7.0. The enzyme exhibits a relatively wide substrate specificity towards alcohols. Dimethyl-sulphoxide (DMSO), some amides and oximes and some intermediates of the carbohydrate metabolism act as ADH inhibitors, ATP as analogue of NAD also exhibits an inhibitory effect. The inhibitory effect of heterocyclic substances (pyrazol, imidazol, pyridine) is similar to the effect on liver alcohol dehydrogenase.     

Cytostatic peptide isolation from culture media of mouse peritoneal exudate cells

It was found that the supernatant of mouse PEC culture medium (MCM) (both resident and casein-elicited cells) has an inhibitory effect in vitro on the incorporation of [3H]TdR into DNA of mouse spleen cells. The inhibitory effect in the MCM appears in the first 24 hr and also reaches its maximum value within this time. The inhibitory effect of this factor could not be demonstrated in the extract of freshly harvested M phi cells. The factors responsible for inhibition proved to be heat stable at 80 degrees C for longer than 30 min. Following heat treatment, the crude extract was separated into four fractions absorbing uv light at 280 nm using Sephadex G-25 column chromatography, and the most potent biologically active inhibitory factor was eluted in the last fraction. This fraction could also be obtained with a more effective permeation volume using Trysacryl GF 05 gel chromatography, and the active B fraction from this chromatography could be separated into four subfractions by isotachophoresis (ITP). The active fraction, which was obtained by Trysacryl GF 05 gel chromatography and further separated by ITP, was found to be highly inhibitory. It contained a peptide-like substance with a molecular mass of approximately 2.0 kDa and had an anionic character at pH 4.0. The inhibitory effect of MCM cannot be influenced either by inhibitory compounds of protein synthesis or by proteolysis blocking agents. Furthermore, the inhibitory effect is shown to be reversible and is more pronounced on B cells than on T lymphocytes.     

Specificity and structural analysis of a guinea pig transfer factor-like activity.

A transfer factor-like activity was prepared by Sephadex G-25 chromatography of immune guinea pig leukocyte lysates. This isolated material leads to antigen-dependent migration inhibition and thymidine uptake by nonimmune lymphoid cells. Tests of the "transfer factor" from guinea pigs immunized to either ovalbumin or bovine gamma-globulin demonstrated the donor specificity of the in vitro activity. The activity is susceptible to heat (56 degrees C), alkali (0.5 M sodium hydroxide), pronase, and phosphodiesterase. The pronase susceptibility is blocked by traysylol, a protease inhibitor; the phosphodiesterase susceptibility is not bocked by traysylol. The guinea pig factor was purified further by alkaline phosphatase treatment. Sephadex G-25 chromatography, and DEAE-cellulose chromatography. The final product, active in vitro, represents about 0.03% of the cellular material absorbing 260 nm light, and contains polymerized amines and phosphate. Gel electrophoresis of the fluram-reactive components suggests a limited heterogeneity of the DEAE-cellulose-purified material. These data are consistent with the active "transfer factor" molecule including both peptide and phosphate-containing components.     

A Convenient Source of Cfu-S Inhibitors: the Fetal Calf Liver*

Abstract The presence of a bone-marrow stem-cell inhibitor able to prevent CFU-s entry into DNA synthesis after cytosine arabinoside (Ara-C) treatment has been detected in 7-month-old fetal calf liver. the inhibitory fraction was obtained through ultrafiltration of a delipidated tissue extract powder and purified by BioGel-P-2 chromatography. the elution pattern on Sephadex G10 is similar to that of the bone-marrow inhibitory extract previously obtained from fetal calf bone marrow. It corresponds to a low-molecular-weight molecule (MW > 2000), devoid of species specificity and having no inhibitory effect on GM-CFC proliferation.     

Secretin-like bioactivity in extracts of porcine brain

Porcine brain extracts were found to stimulate exocrine pancreatic secretion in a secretin-like fashion. The active material may be secretin since it behaved as secretin through several purification steps including chromatography on Sephadex G-25 and carboxymethyl cellulose, and on high performance liquid chromatography. Furthermore, the highly purified preparation contained, like secretin, C-terminal valine amide, and one of its tryptic fragments had the same mobility as the C-terminal tryptic fragment of secretin on thin layer chromatography.     

鸭儿芹的化学成分及对Hep G2细胞毒性

该研究采用大孔树脂(D101)、硅胶、羟丙基葡聚糖凝胶(Sephadex LH-20)和十八烷基硅烷键合硅胶(ODS)等色谱方法,对鸭儿芹的化学成分进行了分离纯化,根据理化性质、质谱和核磁共振波谱数据,并参考相关文献综合分析化合物结构,进而采用噻唑蓝(MTT)法,对鸭儿芹化合物抗Hep G2细胞活性进行筛选。结果表明:共从鸭儿芹中分离鉴定了7个化合物,分别为p-(acetylamino)phenol(1),辛酸甲酯(2),丁酸异戊酯(3),N,N-二甲基-苯并咪唑-2胺(4),5-羟基-1-(4-羟基-3-甲氧苯基)庚3酮(5),3,5二丁基六氢吡咯里嗪(6),(S)-4-(1-hydroxyallyl)phenyl acetate(7)。其中,化合物6对细胞具有抑制作用,抑制率达到89.1%。该研究结果表明化合物1-7均为首次从鸭儿芹中分离得到,其中化合物6对Hep G2细胞的生长具有抑制作用,且具有剂量依赖性。     

条斑紫菜多糖的分离纯化与抗肿瘤活性的初探

目的：本文主要应用生化提纯技术从条斑紫菜（ Porphyra yezoensis ）中提取不同纯度的多糖,并观察各多糖组分对人乳腺癌细胞 MCF-7 生长的影响。方法：通过 DEAE-52 柱层析和 Sephadex G-200 柱层析,对条斑紫菜粗多糖热水提取物进行纯化;经HPLC,紫外和红外光谱对多糖各组分的纯度及结构进行初步鉴定;并用 MTT 法和流式细胞仪检测多糖对人乳腺癌细胞 MCF-7 生长的影响。结果：分离纯化出的各组分条斑紫菜粗多糖对人乳腺癌细胞 MCF-7生长均有不同程度的抑制作用,其中PY-D2可诱导MCF-7细胞凋亡。结论：条斑紫菜多糖具有杀伤肿瘤细胞MCF-7的作用。     

Isolation and Identification of a Novel Antifungal Protein from a Rhizobacterium Bacillus subtilis Strain F3

Bacillus subtilis strain F3, isolated from peach rhizosphere soil, is an antifungal bacterium against many plant pathogens. In this study, the antifungal protein was isolated and purified by ammonium sulphate and chromatography, then identified by mass spectrum analysis. By sequential chromatography of Sephadex G‐50, DEAE‐Sephadex A‐25 anion exchange and Sephadex G‐100, a fraction designated as F3A was isolated to show a single protein band in SDS‐PAGE and be antagonistic towards Monilinia fructicola. The peptide mass fingerprinting of the protein band of F3A had high similarity with the amino acid sequences of several flagellin protein of B. subtilis. There were seven amino acid fragments matched with the protein having the highest score, and sequence coverage was 33%. F3A showed a strongly inhibitory effect to the growth and sporulation of M. fructicola. There were little aerial hyphae and conidia at the antifungal zone, and the hyphae were abnormal with some cell wall collapse and several vacuoles in cells.     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

Some properties of 3-phosphoglycerate phosphatase from developing rice grain

Some properties of 3-P-glycerate phosphatase from developing caryopsis of rice (Oryza sativa L., variety IR26) were studied. The enzyme was found to be soluble and not bound to starch, and concentrated mainly in the pericarp-aleurone layer; its maximum activity was at 12 to 14 days after flowering. Contents of 3-P-glycerate and chlorophyll were highest in the grain at 7 to 8 days after flowering when starch synthesis was at a maximum. The enzyme was purified about 100-fold by precipitation with 50 to 80% ammonium sulfate, followed by chromatography through Sephadex G-200 and CM-Sephadex C-50. The pH optimum was from 5.7 to 6 and no cation was required for activity. The purified preparation had an apparent Km of 2.85 mm and was inhibited by Cu(2+), Hg(2+), Zn(2+), Fe(3+), molybdate, and F(-). The enzyme also exhibited high activity toward UTP, ATP, and p-nitrophenyl phosphate; moderate activity toward other phosphates; but no activity toward phytate. A molecular weight of about 23,000 was obtained for the 3-P-glycerate peak during gel filtration on Sephadex G-200, which corresponded to a value of 26,000 for the major protein fraction by thin layer gel filtration on Sephadex G-150. Zymograms of the whole extract and semipurified preparations showed two phosphatase bands with 3-P-glycerate as substrate.     

Chemical differences between bovine pineal antigonadotropin and arginine vasotocin

Studies were conducted in order to characterize chemically a partially purified antigonadotropic factor extracted from bovine pineal glands (PAG). Because several reports have appeared recently suggesting a role for arginine vasotocin (AVT) as a pineal antigonadotropin, our experiments were designed to determine the presence or absence of this nonapeptide in our material. A comparison of biologically active PAG and synthetic AVT revealed dissimilar UV absorption and fluorescence maxima, different mobilities on thin layer chromatography and paper electrophoresis, as well as different elution patterns on DEAE Sephadex ion-exchange chromatography. Amino acid analysis using 2 different methods revealed dissimilar amino acid compositions. On the basis of these and other chemical data, it is concluded that our preparations of PAG do not contain AVT.     

Characterisation of cathepsin B and collagenolytic cathepsin from human placenta

1. Human placental cathepsin B and collagenolytic cathepsin were separated by chromatography on columns of Amberlite CG-50. Collagenolytic cathepsin was partially purified by chromatography on DEAE-Sephadex (A-50) and Sephadex G-100. Cathepsin B was purified by chromatography on CM-cellulose and Sephadex G-100. 2. Both enzymes required activation by thiol compounds and were bound to organomercurial-Sepharose-4B. Sulphydryl-blocking reagents were inhibitory, which confirmed an essential thiol group to be present. 3. The enzymes degraded soluble calf skin collagen and insoluble bovine tendon collagen in the telopeptide region at pH 3.5 and 28 degrees C to yield mainly alpha-chain components. 4. In contrast to cathepsin B, collagenolytic cathepsin was found not to hydrolyse any of the low-molecular-weight synthetic substrates that were tested. 5. Leupeptin, a structural analogue of arginine-containing synthetic substrates, and antipain, an inhibitor of papain, were strongly inhibitory to both enzymes. 6. The isoelectric points of the enzymes were similar, being 5.4 for cathepsin B and 5.1 for collagenolytic cathepsin. 7. From chromatography on Sephadex G-100 the molecular weight of cathepsin B was calculated to be 24 500 and that of collagenolytic cathepsin to be 34 600.     

斜链拟青霉多糖活性组分的分离及其清除自由基活性

对斜链拟青霉胞外多糖进行体外清除自由基活性研究。利用醇沉法从斜链拟青霉发酵液中醇沉获得斜链拟青霉胞外多糖,经DEAE-纤维素-52柱层析后得到1个主峰P3和2个小峰P1与P2,对主峰P3进行Sephadex G-100柱层析,表明P3为纯化物; 应用DPPH-酶标法和化学发光法分别对P3进行清除DPPH·、·OH和O-2·能力测试。结果表明：P3对DPPH·、·OH和O-2·均具有明显的清除能力,且与浓度呈量效关系,IC50分别为0.083、0.121和0.214 mg/mL。     

Further purification of bovine spleen inhibitors of lymphocyte DNA synthesis (chalones).

Partially purified lymphocytic factors were obtained from boving spleen; these factors are non-cytotoxic and biologically active in vitro and in vivo: [3H]Thymidine incorporation into DNA of mitogen-stimulated mouse spleen cells in culture is inhibited; similar results are obtained with phytohaemagglutinin-stimulated peripheral human lymphocytes, where blast cells transformation is blocked. [3H]Thymidine incorporation into DNA of mitogen stimulated lymphocytes withdrawn from in vivo treated mice, is also reduced. The two factors in vitro and in vivo seem to act preferentially on mouse spleen cells compared to their action on liver, kidney and testicle cells in cluture, as far as thymidine incorporation into DNA is concerned. The following techniques were applied for their purification: 1. Homogenization of the fresh tissue in water, centrifugation, dialysis of the supernatant, centrifugation, fractionation of the supernatant by alcoholic precipitation and finally concentration in vacuo and lyophilization of the material soluble at 75% of alcohol yielded fraction F. 2. Preparation of an acetone powder from the spleen, extraction of the dry powder with water, then high speed centrifugation, followed by lyophilization of the supernatant produced fraction F'. Both fractions F and F' were further fractionated by chromatography on a Sephadex G75 column: 7 peaks were obtained (F1--F7). Biological activity was found in fraction F1, corresponding to high molecular weight material, and in fraction F6, corresponding to low molecular weight substances. By rechromatography on Sephadex G 75, it is easy to dissociate from F1 a small molecular weight fraction which might be similar to F6 as far as elution volume and biological properties are concerned.     

从蛹虫草小麦培养基残渣分离虫草素及提取物对肝癌细胞的抑制效果

从蛹虫草Cordyceps militaris小麦培养基残渣中分离和纯化虫草素,并进行了提取物体外人肝癌细胞Hep G2 (human hepatoma cell line,Hep G2)的抑制作用研究。以蛹虫草小麦培养基残渣为原料,热超声水提取虫草素,探究pH值、温度、浸提时间和超声功率对虫草素的提取效果,用苯乙烯型弱极性大孔吸附树脂(AB-8)柱层析分离、重结晶纯化虫草素,并用高效液相色谱法(high performance liquid chromatography,HPLC)、红外光谱法(infrared spectroscopy,IR)鉴定。虫草素处理肝癌细胞Hep G2后,倒置相差显微镜下观察肝癌细胞形态变化,并用四甲基偶氮唑蓝(methylthiazolyltetrazolium,MTT)法测定细胞生长情况。结果表明：固液比1:50、pH值6.0、温度60 ℃、浸提时间3 h和超声功率300 W时提取效果好。经鉴定,分离提取物的虫草素纯度达到99%以上。不同浓度的虫草素对肝癌细胞Hep G2有明显的抑制作用,且与虫草素浓度和作用时间呈正相关,被抑制的细胞凝集、变圆、碎裂增加。本提取工艺得到的虫草素纯度高,工艺简单易扩大,且提取物对肝癌细胞增殖作用具有明显的抑制作用,为其在功能性食品添加剂方向的应用提供了数据基础。