Gene expression in precursor cells of prostate cancer associated with activin by combination of subtractive hybridization and microarray technologies

Prostatic intraepithelial neoplasia (PIN) is considered the pre-malignant stage of prostate carcinoma, but little is known of its initiation and evolution. The identification of genes associated with these precursors of prostate cancer may elucidate the pathways of the early oncogenesis of this disease. Previously, we have reported that activin, a member of the TGFbeta superfamily, acted as an inhibitory growth factor in prostate cancer. We used laser capture microdissection, mRNA-library amplification (RNA-PCR), subtractive hybridization, and complementary DNA microarray to examine gene expression profiles in activin-positive PIN, compared with activin-negative PIN. Subtractive hybridization showed that 28 genes were differentially expressed (13 and 15 genes were up- and down-regulated, respectively). Microarray analysis identified 29 and 56 more genes (4 times) up- and down-regulated, respectively, suggesting that DNA microarray is a more effective method in screening gene profiles. We have validated the known genes identified by both subtractive hybridization and microarray technologies, using Northern blot analysis in the mRNA libraries generated from cells microdissected from pathological slides. We have successfully showed that at least 13 genes are involved in activin-associated PIN. The evaluation of candidate genes that emerge from these experiments provides a rational approach to investigate those genes significant in evolution from PIN to prostate carcinoma.     

遗传密码子研究进展

作为生命信息的基本遗传单位,基因组遗传密码的破译对于人们加深对生命本质的认识具有重要的理论价值和现实意义。目前,遗传密码子的研究重心已由遗传密码子的破译及反常密码子的发现转入到遗传密码子的起源与进化及扩张等研究。遗传密码子的起源与进化是当今基因组学研究的热点命题之一,相关的学说、假设层出不穷,但尚未取得实质性突破。另一方面,无义密码子的再定义及遗传密码的扩张等研究却极大的丰富和发展了遗传密码子的科学内涵,推动了生命科学研究的发展。文章综述了遗传密码子的多态性、起源与进化、无义密码子的再定义及遗传密码的扩张等方面的研究进展,并就其应用价值作了评述,期待为其在基因组学、医学等相关领域的应用研究提供参考。     

Activation In Vitro of Mutant MoFe Proteins from Azotobacter vinelandii by Reconstituent Solutions

nifB-MoFe protein （nifB-Av1）, AnifE MoFe protein （△nifE Av1） and AnifZ MoFe protein （△nifZ Av1） were obtained by chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from nifB point-mutated, nifE deleted and nifZ deleted mutant stains （UW45, DJ35 and DJ194） of Azotobacter vinelandii Llpmann, respectively. When complemented with nltrogenase Fe protein （Av2）, AnifZ Av1 had partial activity and both nifB-Avl and △nifE Av1 had hardly any activity, but could be obviously activated by FeMoco extracted from wild-type MoFe protein （OP Av1） or △nifZ Av1. After being Incubated with excess O-phenanthrollne （O-phen） for 150 mln at 30 ℃ and subjected to chromatography on a Sephadex G-25 column In an Ar atmosphere, nifB- Av1C, △nifE Av1C and △nifZ Av1C were obtained, respectively. Based on a calculation of Fe atoms In the Ophen-Fe compound with ε 512nm = 11 100, lost Fe atoms of nifB-Av1, △nifE Av1 and △nifZ Av1 were estimated to be 1.35, 2.89 and 8.44 per molecule of protein, respectively. As a result of the Fe loss, △nifZ Av1 loses Its original activity. In the presence of both MgATP and Av2, these Fe-loslng proteins, but not the original proteins untreated with O-phen, could be significantly activated by reconstltuent solution （RS） composed of dlthlothreltol, ferric homocltrate, Na2S and Na2MoO4, or K2CrO4, or KMnO4. But In the absence of MgATP or Av2, the activation did not occur, with the exception that △nifZ AvlC was partially activated, and the activity was only 17%. These findings Indicate that： （I） △nifZ Avl with half P-cluster content Is somewhat different from FeMoco-deflclent nifB-Avl and ,△nifE Av1 with respect to protein conformation either before or after treatment with O-phen; （11） full activation of these proteins with RS requires pretreatment with O-phen and the simultaneous presence of MgATP and Av2.     

Detection of sequence-specific tyrosine nitration of manganese SOD and SERCA in cardiovascular disease and aging

Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies toward site-specific nY-modified proteins and to use histochemistry and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies toward peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with ANG II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite, which chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.     

Isolation of targeted AAV2 vectors from novel virus display libraries

Random peptide ligands displayed on viral capsids are emerging tools for selection of targeted gene transfer vectors even without prior knowledge of the potential target cell receptor. We have previously introduced adeno-associated viral (AAV)-displayed peptide libraries that ensure encoding of displayed peptides by the packaged AAV genome. A major limitation of these libraries is their contamination with wild-type (wt) AAV. Here we describe a novel and improved library production system that reliably avoids generation of wt AAV by use of a synthetic cap gene. Selection of targeted AAV vectors from wt-containing and the novel wt-free libraries on cell types with different permissivity for wt AAV2 replication suggested the superiority of the wt-free library. However, from both libraries highly specific peptide sequence motifs were selected which improved transduction of cells with moderate or low permissivity for AAV2 replication. Strong reduction of HeLa cell transduction compared to wt AAV2 and only low level transduction of non-target cells by some selected clones showed that not only the efficiency but also the specificity of gene transfer was improved. In conclusion, our study validates and improves the unique potential of virus display libraries for the development of targeted gene transfer vectors.     

Medium components and culture conditions affect the thermotolerance of aerial conidia of fungal biocontrol agent Beauveria bassiana

AIMS: To produce more thermotolerable conidia of Beauveria bassiana, a well-known fungal biocontrol agent, by optimizing the medium components and culture conditions. METHODS AND RESULTS: The conidia produced on media including 0.5-6% glucose, sucrose or starch as carbon source and 50-300-microg ml(-1) Cu2+, Zn2+, Mn2+ or Fe3+ as additive to Sabouraud dextrose medium at 15-30 degrees C, pH 4-8 or KCl-adjusted water availabilities were exposed to 30-min wet heat stress at 48 degrees C. The medium components for conidial production with greatly enhanced thermotolerance included 4% glucose as optimum or 1% starch as alternative for the carbon source and < or =50-microg ml(-1) Mn2+ for the metal additive. The culture conditions were optimized as 25 degrees C and pH 5-6. Conidial thermotolerance decreased remarkably when sucrose and Fe3+ or Cu2+ were used in the cultures, but altered slightly when 50-200-microg ml(-1) Zn2+ were included. CONCLUSIONS: The tolerance of B. bassiana conidia to the thermal stress was significantly affected by the medium composition and culture conditions under which the conidia were produced. SIGNIFICANCE AND IMPACT OF THE STUDY: Proper treatment of small grains as mass production substrates for more glucose release and supplement of glucose or 50-microg ml(-1) Mn2+ are possible means to enhancing conidial thermotolerance and field persistence for improved insect control.     

Purification and characterization of hydrosoluble components from the sap of Chinese lacquer tree Rhus vernicifera

Continuous gradient elution chromatography (CGEC) was employed to purify and separate enzymes and polysaccharides from the sap of Rhus vernicifera Chinese lacquer tree. There are three different molecules with laccase enzyme activity. Two are enzymes of each other (L1, and L2), whereas the third (RL) is an entirely separate entity. Two polysaccharides (GP1 and GP2) were also found. The Rhus laccase (RL), and isoenzymes L1 and L2, have peak molecular masses of 109,100, 120,000, 103,000 respectively; each has four copper atoms per molecule, and the pI values were 8.2, 8.6, and 9.1, respectively. The structure of the laccases was studied by Fourier-transform infrared (FT-IR) and Matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry. The typical amide I (1646 cm⁻¹) and amide II (1545 cm⁻¹) bands were observed. The results from MALDI-TOF were similar to those from CGEC, but the molecular mass from the MALDI-TOF was significantly different from that obtained from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).     

Optimization of the cell seeding density and modeling of cell growth and metabolism using the modified Gompertz model for microencapsulated animal cell culture

Cell microencapsulation is one of the promising strategies for the in vitro production of proteins or in vivo delivery of therapeutic products. In order to design and fabricate the optimized microencapsulated cell system, the Gompertz model was applied and modified to describe the growth and metabolism of microencapsulated cell, including substrate consumption and product formation. The Gompertz model successfully described the cell growth kinetics and the modified Gompertz models fitted the substrate consumption and product formation well. It was demonstrated that the optimal initial cell seeding density was about 4-5 x 10(6) cells/mL of microcapsule, in terms of the maximum specific growth rate, the glucose consumption potential and the product formation potential calculated by the Gompertz and modified Gompertz models. Modeling of cell growth and metabolism in microcapsules provides a guideline for optimizing the culture of microencapsulated cells.     

SIRT2 activity is required for the survival of C6 glioma cells

SIRT2 is a tubulin deacetylase, which can play either detrimental or beneficial roles in cell survival under different conditions. While it has been suggested that reduced SIRT2 expression in human gliomas may contribute to development of gliomas, there has been no study that directly determines the effects of decreased SIRT2 activity on the survival of glioma cells. In this study we applied both pharmacological and molecular approaches to determine the roles of SIRT2 in the survival of glioma cells. Our studies, by conducting such assays as flow cytometry-based Annexin V assay and caspase-3 immunostaining, have indicated that decreased SIRT2 activity leads to apoptosis of C6 glioma cells by caspase-3-dependent pathway. Our experiments have further shown that reduced SIRT2 activity produces necrosis of C6 glioma cells. Moreover, our study applying SIRT2 siRNA has also shown that decreased SIRT2 leads to both necrosis and apoptotic changes of C6 glioma cells. Collectively, our study has provided novel evidence indicating that SIRT2 activity plays a key role in maintaining the survival of glioma cells, and that reduced SIRT2 activity can induce both necrosis and caspase-3-dependent apoptosis of C6 glioma cells. These results have also suggested that inhibition of SIRT2 might become a novel therapeutic strategy for gliomas.     

Simultaneous determination of ascorbic acid, dopamine and uric acid using high-performance screen-printed graphene electrode

A disposable and sensitive screen-printed electrode using an ink containing graphene was developed. This electrode combined the advantages of graphene and the disposable characteristic of electrode, which possessed wide potential window, low background current and fast electron transfer kinetics. Compared with the electrodes made from other inks, screen-printed graphene electrode (SPGNE) showed excellent electrocatalytic activity for the oxidation of ascorbic acid (AA), dopamine (DA), and uric acid (UA). Three well-defined sharp and fully resolved anodic peaks were found at the developed electrode. Differential pulse voltammetry was used to simultaneous determination of AA, DA, and UA in their ternary mixture. In the co-existence system of these three species, the linear response ranges for the determination of AA, DA, and UA were 4.0-4500 μM, 0.5-2000 μM, and 0.8-2500 μM, respectively. The detection limits (S/N=3) were found to be 0.95 μM, 0.12 μM, and 0.20 μM for the determination of AA, DA, and UA, respectively. Furthermore, the SPGNE displayed high reproducibility and stability for these species determination. The feasibility of the developed electrode for real sample analysis was investigated. Results showed that the SPGNE could be used as a sensitive and selective sensor for simultaneous determination of AA, DA, and UA in biological samples, which may provide a promising alternative in routine sensing applications.