Ginsenosides enhance the transduction of tat-superoxide dismutase into mammalian cells and skin

We previously reported that Tat-Cu,Zn-superoxide dismutase (Tat-SOD), a major antioxidant enzyme, can be directly transduced into mammalian cells and skin [Kwon et al. (2000); Park et al. (2002)]. To enhance the therapeutic potential of Tat-SOD in the treatment of various disorders, we screened a number of natural products for their ability to increase transduction efficiency. Ginsenosides were effective with cultured HeLa cells and enhanced the penetration of Tat-SOD into both the epidermis and the dermis of the subcutaneous layer when sprayed on mice skin. Although their mechanism of action is not fully understood we believe that ginsenosides may be useful cofactors with this antioxidant enzyme in anti-aging cosmetics or as a therapeutic protein in disorders related to reactive-oxygen species.     

High-throughput enzyme evolution in Saccharomyces cerevisiae using a synthetic RNA switch

Metabolic engineering can produce a wide range of bulk and fine chemicals using renewable resources. These approaches frequently require high levels of activity from multiple heterologous enzymes. Directed evolution techniques have been used to improve the activity of a wide range of enzymes but can be difficult to apply when the enzyme is used in whole cells. To address this limitation, we developed generalizable in vivo biosensors using engineered RNA switches to link metabolite concentrations and GFP expression levels in living cells. Using such a sensor, we quantitatively screened large enzyme libraries in high throughput based on fluorescence, either in clonal cultures or in single cells by fluorescence activated cell sorting (FACS). By iteratively screening libraries of a caffeine demethylase, we identified beneficial mutations that ultimately increased the enzyme activity in vivo by 33 fold and the product selectivity by 22 fold. As aptamer selection strategies allow RNA switches to be readily adapted to recognize new small molecules, these RNA-based screening techniques are applicable to a broad range of enzymes and metabolic pathways.     

Development of a plasmid-mediated reporter system for in vivo monitoring of gene expression in the archaeon Methanosarcina acetivorans

A plasmid-based gene reporter system has been developed to construct lacZ gene fusions for monitoring intrinsic promoter expression in Methanosarcina acetivorans. Constructs transform with high efficiency that can be readily screened by color selection on plates and exhibit a consistent copy number on different substrates negating the need for gene copy normalization. Expression of the CO dehydrogenase-acetyl coenzyme A synthase promoter fusion to lacZ revealed 18- to 54-fold down-regulation in cells grown on methylotrophic substrates compared with acetate-grown cells, which is up to an order of magnitude greater than the range of regulation previously reported by enzyme activity assays. This system complements and expands the current techniques for studying genetics of the methanosarcinal Archaea by providing a rapid method for monitoring and quantifying gene expression.     

A rapid chromogenic microtitre assay of arginine aminopeptidase activity in Mycoplasma strains

Arginine-utilizing strains of Mycoplasma can be screened by assay of their arginine aminopeptidase activity. A standardized chromogenic method is described that enables enzyme detection in small volumes of cell suspension in less than 3 h. Cell suspensions (10 microl) in 96-well microtitre plates are incubated at 37 degrees C, pH 8.0, with 0.1 mM arginyl-beta-naphthylamide (100 microl). This is hydrolysed to release beta-naphthylamine, which gives a coloured product on diazotization with fast garnet. M. alkalescens can be detected in this way with as few as 1.1 x 10(5) viable cells and M. fermentans with 2.3 x 10(6) cells. The method has been shown to enable division of 28 strains into three groups of fermentative and arginine-hydrolysing mycoplasmas. This procedure has potential for routine laboratory use.     

Continuous production of L-phenylalanine by transamination

L-Phenylalanine was produced continuously from L-as-partate and phenylpyruvate by transaminase from a newly screened Pseudomonas putida strain. The process was carried out with an isolated enzyme in homogeneous phase in an enzyme membrane reactor and with immobilized whole cells in a stirred tank reactor, respectively. Due to the difference in transport resistance, the productivity of the free enzyme in homogeneous phase (72 mmol/L h) was about 3 times higher than the productivity achieved using immobilized cells. However, a better stability of the biocatalyst was observed with immobilized cells.     

表达新城疫病毒HN基因的重组火鸡疱疹病毒的构建

应用聚合酶链反应技术,用设计带有限制性酶切位点的引物,特异地扩增从起始密码子开始的１ ．８ ｋｂ 新城疫病毒（ Ｎ Ｄ Ｖ） 血凝素神经氨酸酶（ Ｈ Ｎ） 基因开放式阅读框,然后插入ｐ Ｔ Ｋ２ Ｂ 的 Ｎｈｅ Ⅰ位点,构建了含 Ｎ Ｄ Ｖ Ｈ Ｎ 基因的插入载体ｐ Ｔ Ｋ Ｈ Ｎ１ 和ｐ Ｔ Ｋ Ｈ Ｎ２ ,再与感染火鸡疱疹病毒（ Ｈ Ｖ Ｔ） 细胞的总 Ｄ Ｎ Ａ 共转染鸡胚成纤维细胞（ Ｃ Ｅ Ｆ） ,经有限稀释法和 Ｄｏｔｂｌｏｔ 筛选,得到含有 Ｈ Ｎ 基因的重组体ｒ Ｈ Ｖ Ｔ１ 和ｒ Ｈ Ｖ Ｔ２ 。经组织培养传代和 Ｗｅｓｔｅｒｎ ｂｌｏｔ 分析,表明重组体在感染细胞中表达了 Ｈ Ｎ 蛋白。重组体在 Ｃ Ｅ Ｆ 上的生长特性与亲本病毒相同,且在连续传代过程中保持稳定。为国内新城疫基因工程疫苗研制奠定了基础。     

Development of a Plasmid-Mediated Reporter System for In Vivo Monitoring of Gene Expression in the Archaeon Methanosarcina acetivorans

A plasmid-based gene reporter system has been developed to construct lacZ gene fusions for monitoring intrinsic promoter expression in Methanosarcina acetivorans. Constructs transform with high efficiency that can be readily screened by color selection on plates and exhibit a consistent copy number on different substrates negating the need for gene copy normalization. Expression of the CO dehydrogenase-acetyl coenzyme A synthase promoter fusion to lacZ revealed 18- to 54-fold down-regulation in cells grown on methylotrophic substrates compared with acetate-grown cells, which is up to an order of magnitude greater than the range of regulation previously reported by enzyme activity assays. This system complements and expands the current techniques for studying genetics of the methanosarcinal Archaea by providing a rapid method for monitoring and quantifying gene expression.     

Expression of a Streptococcus mutans glucosyltransferase gene in Escherichia coli.

Chromosomal DNA from Streptococcus mutans strain UAB90 (serotype c) was cloned into Escherichia coli K-12. The clone bank was screened for any sucrose-hydrolyzing activity by selection for growth on raffinose in the presence of isopropyl-beta-D-thiogalactoside. A clone expressing an S. mutans glucosyltransferase was identified. The S. mutans DNA encoding this enzyme is a 1.73-kilobase fragment cloned into the HindIII site of plasmid pBR322. We designated the gene gtfA. The plasmid-encoded gtfA enzyme, a 55,000-molecular-weight protein, is synthesized at 40% the level of pBR322-encoded beta-lactamase in E. coli minicells. Using sucrose as substrate, the gtfA enzyme catalyzes the formation of fructose and a glucan with an apparent molecular weight of 1,500. We detected the gtfA protein in S. mutans cells with antibody raised against the cloned gtfA enzyme. Immunologically identical gtfA protein appears to be present in S. mutans cells of serotypes c, e, and f, and a cross-reacting protein was made by serotype b cells. Proteins from serotype a, g, and d S. mutans cells did not react with antibody to gtfA enzyme. The gtfA activity was present in the periplasmic space of E. coli clones, since 15% of the total gtfA activity was released by cold osmotic shock and the clones were able to grow on sucrose as sole carbon source.     

Definitive prenatal diagnosis for type III glycogen storage disease.

Prenatal diagnosis for type III glycogen storage disease was performed by using (1) immunoblot analysis with a polyclonal antibody prepared against purified porcine-muscle debranching enzyme and (2) a qualitative assay for debranching-enzyme activity. Cultured amniotic fluid cells from three pregnancies (three families in which the proband had absence of debrancher protein) were subjected to immunoblot analysis. Two unaffected and one affected fetus were predicted. In addition, cultured amniotic fluid cells from nine pregnancies (eight families) were screened with a qualitative assay based on the persistence of a polysaccharide that has a structure approaching that of a phosphorylase limit dextrin when the cells were exposed to a glucose-free medium. This qualitative assay predicted six unaffected and three affected fetuses. All predictions by either method were confirmed postnatally except for one spontaneously aborted fetus. Our data indicate that a definitive diagnosis of type III glycogen storage disease can be made prenatally by these methods.     

Hydrolysis of pectin: an enzymatic approach and its application in banana fiber processing

Sediment samples were collected from different estuarine and marine areas along the West coast of India. Eighteen actinomycete cultures were isolated using starch casein agar and were screened for polygalacturonase activity by growing them on pectin-agar plates. Clear zones were visualized using 1% cetrimide. Out of the 18 strains screened ten cultures could effect hydrolysis of pectin. The above cultures were subjected to secondary screening under submerged fermentation. The actinomycete strain of Streptomyces lydicus was found to be a potent producer of polygalacturonase. Different growth media were screened for enzyme production and the best medium was selected for further studies. The crude enzyme was used for the treatment of raw banana fibers.     

N—乙酰氨基己糖苷酶产生菌的筛选与产酶条件

About 1200 strains of microorganisms were screened including fungi, actinomyces, and bacteria, in which 237 strains producing the enzyme desired. The results showed that the beta-GlcNAcase and beta-GalNAcase always co-existed in one strain, though may be in different ratio. From strains mentioned above the authors screened out a potent beta-N-acetylhexosaminidase producing strain, Aspergillus tamarii S215, from the soil sample. The optimal conditions for enzyme production were as follows: the microorganisms was inoculated in a 5% wheat bran suspension, cultured at 28-30 degrees C on shaker for 5-6 days. The productivity can be moderately enhanced by the addition of cellobiose or glucosamine or galactosamine or by the extra supplement of (NH4)2SO4 and NH4NO3 as N sources. In the culture filtrate of Asp. tamarii, the alpha, (beta)-galactosidase, beta-glucosidase, alpha-mannosidase and beta-fucosidase were also found.     

Screening of large protein libraries by the cell immobilized on adsorbed bead approach

Screening of mutant libraries for in vitro enzyme evolution is carried out primarily by physical separation of the cells, followed by growth of individual clones and screening of biocatalytic activity on the basis of color or fluorescence signal development. Currently, most frequently employed methods are labor-intensive or require robotic equipment, resulting in screening limited to a relatively small fraction of the potential inherent in a given library. In this study we present a design, development, and feasibility demonstration of a new screening approach, providing convenient handling of large libraries consisting of 106 to 107 clones and screening based on a simultaneous enzymatic assay with commercially available substrates. This new screening method is based on the "cell immobilized on adsorbed bead" approach: the cell population to be screened is mixed with an excess of medium pre-equilibrated polyacrylamide beads, chemically derivatized to affect quantitative cell immobilization by adsorption. The resulting bead population, comprising of single cell on a bead or blank beads, is then immobilized on a solid glass support. After removal of the freely flowing liquid, the cells immobilized on the adsorbed beads are allowed to grow into microcolonies, utilizing the medium retained within the supporting hydrogel matrix. These colonies are subsequently equilibrated with chromogenic or fluorogenic substrate and screening is affected under a stereomicroscope, resulting in readily retrieved of the most active colonies. This technique may be particularly useful when the screened mutants are expressed and displayed on the cell surface, providing an active and homogeneous "naturally immobilized" enzyme population with minimal substrate diffusion limitations.     

温特曲霉转富马酸为L-苹果酸的初步研究*

从大量霉菌中选育到一株具有较高富马酸酶活性的温特曲霉(Aspergillus wentii) A5-61。在摇瓶培养条件下,32℃ 96小时,产L-苹果酸达10.49g/100ml,对富马酸的转化率达90.80%。利用菌体细胞,进行酶转化试验,结果表明：1.6g湿菌体接入25ml含富马酸10.0%（用NaOH中和至pH7.0）的转化液中,35℃16～24小时,连续转化三次,分别产生L—苹果酸9.61g/100ml、9.73g/100ml、6.93g/100ml。对菌体整体细胞酶学性质的研究表明,其最适反应温度35℃,最适反应pH7.0,Cu2＋对该酶有明显的抑制作用,该酶的Km＝0.154mol/L,Vmax＝0.0571mol/L·h。     

Branching enzyme activity of cultured amniocytes and chorionic villi: prenatal testing for type IV glycogen storage disease.

Although type IV glycogen storage disease (Andersen disease; McKusick 23250) is considered to be a rare, autosomally recessive disorder, of the more than 600 patients with glycogenosis identified in our laboratory by enzymatic assays, 6% have been shown to be deficient in the glycogen branching enzyme. Most of the 38 patients with type IV glycogen storage disease who are known to us have succumbed at a very early age, with the exception of one male teenager, an apparently healthy 7-year-old male, and several 5-year-old patients. Fourteen pregnancies at risk for branching enzyme deficiency have been monitored using cultured amniotic fluid cells, and four additional pregnancies have been screened using cultured chorionic villi. Essentially no branching enzyme activity was detectable in eight samples (amniocytes); activities within the control range were found in five samples (three amniocyte and two chorionic villi samples); and five samples appeared to have been derived from carriers. In two of the cases lacking branching enzyme activity, in which the pregnancies were terminated and fibroblasts were successfully cultured from the aborted fetuses, no branching enzyme activity was found. Another fetus, which was predicted by antenatal assay to be affected, was carried to term. Skin fibroblasts from this baby were deficient in branching enzyme. Pregnancies at risk for glycogen storage disease due to the deficiency of branching enzyme can be successfully monitored using either cultured chorionic villi or amniocytes.     

Purification and characterization of L-pyrrolidonecarboxylate peptidase from Bacillus amyloiliquefaciens.

Microorganisms capable of producing L-pyrrolidonecarboxylate peptidase [L-pyrrolidonyl peptidase, EC 3.4.11.8] were screened and a strain of Bacillus amyloliquefaciens was chosen as one of the most potent producers of the enzyme. The enzyme was purified from lysozyme-lysate of the bacterial cells by salting out with ammonium sulfate, adsorption on DEAE-cellulose, covalent chromatography on PCMB-Sepharose and by gel filtration on Sephadex G-150. By these procedures, the enzyme was purified about 800-fold with an activity recovery of 9%, and the preparation was electrophoretically homogenous. The enzyme was most active and stable at pH 7-8. The presence of 2-mercaptoethanol and EDTA was effective for stabilizing the enzyme. The molecular weight was estimated to be 72,000 by the gel filtration method and to be 24,000 by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a subunit oligomer, presumably trimer. The enzyme was inactivated by the addition of PCMB, sodium tetrathionate, Hg2+ and Cu2+, but the activity lost was restored by the addition of 2-mercaptoethanol and EDTA. The purified enzyme split amide and ester linkages in L-pyroglutamyl derivatives of L-alanine, beta-naphthylamine, alpha-naphthol, and 4-methylumbelliferone, but was completely inert towards various peptides and esters used as substrates for usual amino- and carboxy-peptidases, and for endopeptidases such as trypsin, subtilisin and alpha-chymotrypsin.     

CHD5基因RNAi慢病毒载体的构建及其对结直肠癌细胞生物学功能的影响

目的:研究人类肿瘤相关基因CHD5基因miR-shRNA慢病毒对结直肠癌Lovo细胞的影响。方法:利用软件设计对CHD5基因干扰有效的序列,合成靶序列,退火形成双链DNA,酶切后与载体相连接。将重组慢病毒载体pPRIME-TET-GFP-CHD5与慢病毒包装质粒pCMV-VSV-G,pRSV-Rev,pMDLg-pRRE共转染293FT细胞,将包装重组慢病毒感染人类结直肠癌Lovo细胞。通过荧光定量PCR和Western blot验证CHD5基因在细胞中的表达情况,应用MTT检测CHD5低表达对Lovo细胞增殖的影响。结果:成功构建pPRIME-TET-GFP-CHD5重组质粒,经酶切及序列测定正确,包装的病毒滴度为3.1×106TU/ml。用制备的病毒上清感染Lovo细胞后,荧光定量PCR和Western blot分析结果显示该慢病毒可分别在转录和蛋白质水平上抑制Lovo细胞CHD5基因的表达,并使得Lovo细胞增殖失控。结论:成功构建CHD5慢病毒表达载体,表达的慢病毒可有效的感染Lovo细胞,提高Lovo细胞的增殖能力。     

人ABCB6表达载体的构建及稳定表达细胞株A375的筛选

目的构建表达载体pIRES2-ZsGreen1-ABCB6,在转染的人黑素瘤细胞系株A375中筛选其稳定表达的细胞株。方法抽取健康人外周血,分离外周血单个核细胞,提取总RNA,逆转录获取cDNA序列,加入特异性引物经PCR扩增获得ABCB6cDNA双链,再经过BglII、EcoRI双酶切PCR产物及质粒载体pIRES2-ZsGreen1,酶切产物经回收、T4DNA连接酶连接,产物转化到大肠杆菌DH5α,挑取阳性克隆经菌落PCR鉴定、酶切鉴定和测序分析,以确定构建质粒正确。转染人黑素瘤细胞株A375,G418筛选稳定表达ABCB6的单克隆细胞株,应用荧光显微镜鉴定ABCB6蛋白的表达情况。结果 pIRES2-ZsGreen1-ABCB6质粒经菌落PCR、酶切、测序鉴定正确,经过G418筛选后获得稳定细胞株,在荧光显微镜下可观察到绿色荧光蛋白在A375细胞中的表达。结论表达载体pIRES2-ZsGreen1-ABCB6构建正确,并成功筛选出稳定表达ABCB6的A375细胞株,为进一步研究ABCB6的生物学功能奠定了良好基础。     

D-泛解酸内酯水解酶产生菌的筛选及产酶条件研究

筛选到一株产D-泛解酸内酯水解酶的菌株,经鉴定为串珠镶孢霉菌（Fusarium monili-forme)SW-902。产酶条件研究表明,用甘油作碳源,蛋白胨作氮源,初始pH8.0,温度26℃,摇瓶培养3d,产酶量最高,在60L发酵罐中通风发酵45h,产菌丝体生物量7.18g干菌体/L,D-泛解酸内酯水解酶酶活力达到0.92IU/g干菌体。     

Electrophoretically detectable mutations induced in CHO cells by varying doses of ultraviolet radiation

Chinese hamster ovary (CHO) cells were subjected to ultraviolet radiation (UV) at doses resulting in 100% (no irradiation), 50–30%, 20–10% and ≈1% survival. 2 divisions after UV exposure surviving cells were cloned and clones expanded for electrophoretic analysis of the products of ≈40 enzyme loci. 4 different classes of variants (electrophoretic shifts, nulls, enzyme re-expression and enzyme modification) were detected in 29 of 1329 clones analyzed and proven mutants by subclone analysis. The frequency of mutants in the irradiated groups (28/38391 loci screened or 7.3 × 10^?4) was significantly higher than controls. The frequency of shift mutants at 10–20% survival was higher than shifts at 30–50% survival and was significantly higher than shifts at ≈1% survival. The frequency of nulls increased with dose. 12 of the 28 mutants obtained in the irradiated groups were at only 3 of the mean 41 loci screened/clone. The results indicated that shift mutants could be detected more efficiently than nulls at lower dose and that loci varied widely with respect to their susceptibility to UV mutagenesis. Multiple null mutants at 2 loci, isocitrate dehydrogenase 2 and hexokinase 2, indicated they may be hemizygous in CHO cells.     

Evaluation of production of some antifungal metabolites by fluorescent pseudomonads in vitro and their inhibitory effect on Penicillium digitatum in semi-commercial condition

Epiphytic fluorescent Pseudomonads isolated from the surfaces of citrus leaves and fruits collected from the citrus orchards located in the north of Iran were screened for antagonistic activity against Penicillium digitatum caused agent of green mold of citrus fruit. 9 isolates revealed antagonistic effect in dual culture and then evaluated for production of antimicrobial metabolites such as production hydrogen cyanide on King's B medium containing glycine, production of siderophore on blue CAS-agar medium and production protease enzyme on Skim Milk Agar (SMA) medium. About 28% strains produced hydrogen cyanide, 83% strains produced siderophore and 70% of them produced protease. Antagonistic effects of the screened 9 isolates were studied by inoculating of the infected orange fruits with the bacterium at the concentration of 10(8) cells/ml under sterilized condition at 20 degrees C. The three final screened antagonist isolates were more studied in semi commercial trails in cold storage with dipping of the orange fruits in the bacterial suspension at the concentration of 10(8) cells/ml and then 5 weeks storage at 4 degrees C. The isolate P39 showed to be the most effective for controlling of this disease and decreased the damage by 77.28% respectively.