Identification of novel genes affecting mesoderm formation and morphogenesis through an enhanced large scale functional screen in Xenopus

The formation of mesoderm is an important developmental process of vertebrate embryos, which can be broken down into several steps; mesoderm induction, patterning, morphogenesis and differentiation. Although mesoderm formation in Xenopus has been intensively studied, much remains to be learned about the molecular events responsible for each of these steps. Furthermore, the interplay between mesoderm induction, patterning and morphogenesis remains obscure. Here, we describe an enhanced functional screen in Xenopus designed for large-scale identification of genes controlling mesoderm formation. In order to improve the efficiency of the screen, we used a Xenopus tropicalis unique set of cDNAs, highly enriched in full-length clones. The screening strategy incorporates two mesodermal markers, Xbra and Xmyf-5, to assay for cell fate specification and patterning, respectively. In addition we looked for phenotypes that would suggest effects in morphogenesis, such as gastrulation defects and shortened anterior-posterior axis. Out of 1728 full-length clones we isolated 82 for their ability to alter the phenotype of tadpoles and/or the expression of Xbra and Xmyf-5. Many of the clones gave rise to similar misexpression phenotypes (synphenotypes) and many of the genes within each synphenotype group appeared to be involved in similar pathways. We determined the expression pattern of the 82 genes and found that most of the genes were regionalized and expressed in mesoderm. We expect that many of the genes identified in this screen will be important in mesoderm formation.     

A high-throughput screen for detection of compound-dependent phosphodiester bond cleavage at abasic sites

We have employed a DNA molecular beacon with a real abasic site, namely a 2-deoxyribose, in a fluorescent high-throughput assay to identify artificial nucleases that cleave at abasic sites. We screened a 1280 compound chemical library and identified a compound that functions as an artificial nuclease. We validated a key structure-activity relationship necessary for abasic site cleavage using available analogs of the identified artificial nuclease. We also addressed the activity of the identified compound with dose titrations in the absence and presence of a source of non-specific DNA. Finally, we characterized the phosphodiester backbone cleavage at the abasic site using denaturing gel electrophoresis. This study provides a useful template for researchers seeking to rapidly identify new artificial nucleases.     

A small scale expression screen identifies tissue specific markers in the Dugesia japonica strain Pek-1

Freshwater planaria has tremendous capacity to reform the missing part of the body and therefore is considered as one of the most important model organism for regeneration study.At present,Schmidtea mediterranea and Dugesia japonica are the two major species utilized for laboratory manipulations.Dugesia japonica flatworms are widely distributed in the Far East including Cherry Valley region in the north-west area of Beijing,China.We reported here the establishment of an asexual Dugesia japonica strain Pek-1,as a suitable system for regeneration study.Using morphological,karyotypical as well as phyiogenetic analyses,we confirmed that these flatworms indeed belonged to Dugesia japonica.We went on to show that the commonly used in situ probes and immunohistochemistry reagents and protocols were applicable to the Pek-1 strain.Using this strain,we carried out small scale analysis on EST,RNAi and gene expression.We identified 193 unique EST sequences and 65 of them had not been reported in planarian.By RNAi analysis,we showed that 48 genes,when down-regulated individually,had no effect on regeneration.Furthermore,we identified 3 groups of tissue specific expressing genes that were useful for cell lineage analysis.We concluded that the Dugesia japonica Pek-1 swain could be another suitable animal model to regeneration research.     

A large scale screen for neural stem cell markers in Xenopus retina

Neural stem cell research suffers from a lack of molecular markers to specifically assess stem or progenitor cell properties. The organization of the Xenopus ciliary marginal zone (CMZ) in the retina allows the spatial distinction of these two cell types: stem cells are confined to the most peripheral region, while progenitors are more central. Despite this clear advantage, very few genes specifically expressed in retinal stem cells have been discovered so far in this model. To gain insight into the molecular signature of these cells, we performed a large-scale expression screen in the Xenopus CMZ, establishing it as a model system for stem cell gene profiling. Eighteen genes expressed specifically in the CMZ stem cell compartment were retrieved and are discussed here. These encode various types of proteins, including factors associated with proliferation, mitotic spindle organization, DNA/RNA processing, and cell adhesion. In addition, the publication of this work in a special issue on Xenopus prompted us to give a more general illustration of the value of large-scale screens in this model species. Thus, beyond neural stem cell specific genes, we give a broader highlight of our screen outcome, describing in particular other retinal cell markers that we found. Finally, we present how these can all be easily retrieved through a novel module we developed in the web-based annotation tool XenMARK, and illustrate the potential of this powerful searchable database in the context of the retina.     

A new sensitive and fast peptide immunoassay based on enzyme amplification used in the determination of CGRP and the demonstration of its presence in the thyroid

An enzyme amplified immunoassay for rCGRP based on cofactor cycling has been found to be clearly superior to a comparable radioimmunoassay employing the same antiserum in terms of sensitivity, speed and convenience. Correlation between the two methods was very good. With the enzyme amplified immunoassay we have been able to demonstrate the existence of rCGRP in thyroid extract.     

A new generation approach in enzyme immobilization: Organic-inorganic hybrid nanoflowers with enhanced catalytic activity and stability

Many different micro and nano sized materials have been used for enzymes immobilization in order to increase their catalytic activity and stability. Generally, immobilized enzymes with conventional immobilization techniques exhibit improved stability while their activity is lowered compared to free enzymes. Recently, an elegant immobilization approach was discovered in synthesis of flower-like organic-inorganic hybrid nanostructures with extraordinary catalytic activity and stability. In this novel immobilization strategy, proteins (enzymes) and metal ions acted as organic and inorganic components, respectively to form hybrid nanoflowers (hNFs). It is demonstrated that the hNFs highly enhanced catalytic activities and stability in a wide range of experimental conditions (pHs, temperatures and salt concentration, etc.) compared to free and conventionally immobilized enzymes. This review mainly discussed the synthesis, characterization, development and applications of organic-inorganic hybrid nanoflowers formed of various enzymes and metal ions and explained potential mechanism underlying enhanced catalytic activity and stability.     

Cellcube: A new system for large scale growth of adherent cells

A new disposable cell culture unit for adherent cell lines, the CellCube, was used to grow a variety of mammalian cell lines. A small unit with 2m² growth surface area generated up to 4·10⁹ cells. The disposable system consists of a series of polystyrene plates, mounted into a cubical container. A simple construction consisting of a spinner system for medium conditioning, rotameters for gas mixing and a peristaltic pump for medium circulation provided conditions for culture growth.Abbreviations COS M6 Green Monkey Kidney Cells - SKNMC Human Neuroblastoma Cell Line - BHK Baby Hamster Kidney Cells - CHO Chinese Hamster Ovary Cells - EDTA Ethtylenediaminetetraacetic Acid - PBS Phosphate Buffered Saline - FCS Fetal Calf Serum - MEM Minimum Essential Medium, Alpha Modification     

南美斑潜蝇在温室蔬菜上种群动态的初步观察

系统观察结果表明,南美斑潜蝇Liriomyza huidobrensis(Blanchard)在平凉市日光温室蔬菜全生产期内的种群消长明显呈“两头高中间低”的态势,成虫发生量以春夏生产期最大,可达2.1头/叶;深冬生产期最小,仅为0.1~0.2头/叶;秋冬生产期较大,保持在1.0~1.2头/叶之间。温室内温、湿度条件的变化是引起该虫种群数量波动的主要原因。根据有效积温计算结果,该虫在平凉市日光温室蔬菜全生产期发生9.1代,世代平均发育速率0.030,不同生产期的发生代数和发育速率差异较大。生产上宜采取“前控后治中间松”的防治策略。     

Engineering dicamba selectivity in crops: a search for appropriate degradative enzyme(s)

The biotechnologial approaches to conferring crop selectivity to herbicides have been demonstrated for a number of compounds such as glyphosate, glufosinate, imidazolinones and cyclohexanediones. Imidazolinone-resistant and cyclohexanedione-resistant maize lines are already in the market. There are several other effective and environmentally benign herbicides such as dicamba, for which engineering crop selectivity is desirable, to broaden the product utility in different crops and provide new solutions for weed control. One of the most effective approaches to conferring dicamba selectivity in crops is to incorporate a gene for its rapid metabolism. It is advantageous to have different dicamba-metabolizing enzymes in order to maximize the chances of at least one functioning optimally in a plant environment. Three different metabolizing enzymes are currently available to engineer crop selectivity. The first one is the folate-dependent O-demethylase from Clostridium thermoaceticum, that converts dicamba to herbicidally inactive 3,6-dichlorosalicylate. The second enzyme is the NADH-dependent, multi-component monooxygenase from Pseudomonas maltophilia DI-6 that also converts dicamba to 3,6-dichlorosalicylate. The third enzyme is from corn endosperm cultures that catalyzes the 5-hydroxylation of dicamba. The merits of these three enzymes are discussed with respect to conferring crop selectivity to dicamba. In addition, a rapid microbial screen was conceived for discovery of new dicamba-degrading bacteria, which resulted in identification of Pseudomonas orvilla. This bacteria degraded dicamba by the same pathway, perhaps using a similar enzyme system as Pseudomonas maltophilia DI-6. However, the microbial screen has the potential to identify novel bacteria that degrade dicamba by a different pathway, providing more options for metabolizing enzymes to confer herbicide selectivity in crops. Received 13 February 1997/ Accepted in revised form 26 June 1997     

The validity of in vitro screening methods in the search for fungal antagonists of Sclerotinia sclerotiorum causing wilt of sunflower

A multi‐test screening system identified 63 fungal isolates with high in vitro biocontrol activity against Sclerotinia sclerotiorum. A bioassay method was developed, using sunflower seedlings growing in an unsterilized loam mixture. Twenty‐six isolates were tested in a series of five bioassay tests and a significant correlation (P < 0.01) was found between sclerotial infection in vitro and the number of healthy plants in vivo. Conversely, activity in an in vitro mycelial overgrowth test was not significantly correlated with activity in vivo. However, some isolates showing only mycelial activity still exerted significant disease control in both the bioassays at Littlehampton and in three additional bioassays at Sittingbourne. Only one isolate not previously reported showed significant activity in both sets of bioassays and the lack of consistency in disease control activity by all other isolates, and biocontrol agents in general, was deemed a major barrier to their use.     

Nitrification and denitrification in soil: A comparison of enzyme assay,incubation and enumeration methods

Summary Three methods for assaying nitrification and denitrification were compared in agricultural field plots in the Southeastern USA. Nitrification was measured using the chlorate inhibition enzyme assay, by measuring (NO ₃ ⁻ −N) production in controlled incubations and by most probable number (MPN) determinations. Denitrification was measured by the phase I enzyme assay, by incubation of soil cores and by MPN determinations. The methods were compared in terms of their representation of seasonal patterns and treatment differences. The enzyme assays were most effective for showing treatment differences because they measure maximum potential enzyme biomass activity which is an integrated product of treatment effects. The incubation methods required minimal alteration ofin situ soil conditions but were confounded by other biological processes and by high spatial and temporal variabiltiy. MPN determinations were time consuming and were least effective for illustrating treatment differences and seasonal patterns.     

Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity

Heparosan synthase catalyzes the polymerization of heparosan (-4GlcUAβ1-4GlcNAcα1-)(n) by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site-directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA(+)/PmHS2-GlcNAc(+) showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence, not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars, UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars.     

A note on some valence bond calculations for the ground-state of the hydrogen molecule

The results of ab initio valence bond (VB) calculations are reported for H₂, with up to 30 nuclear centred (15 at each H atom) 1s, 2s, 2p, 3s, 3p, 3d and 4s (cc-pVTZ+4s) atomic orbitals (AOs) included in them. A 30 AO complete active space VB calculation (CASVB) with 465 S = 0 spin structures gives an cc-pVTZ+4s energy of −1.17245 a.u. for d(H-H) = 1.4 a.u., which is identical to the CISD/cc-pVTZ+4s estimate obtained from a molecular orbital (MO) configuration interaction (CI) calculation with all single and double (CISD) substitutions.     

Characterization of benzaldehyde lyase from Pseudomonas fluorescens: A versatile enzyme for asymmetric C-C bond formation

The thiamin-diphosphate-dependent enzyme benzaldehyde lyase is a very import catalyst for chemoenzymatic synthesis catalyzing the formation and cleavage of (R)-hydroxy ketones. We have studied the stability of the recombinant enzyme and some enzyme variants with respect to pH, temperature, buffer salt, cofactors and organic cosolvents. Stability of BAL in chemoenzymatic synthesis requires the addition of cofactors to the buffer. Reaction temperature should not exceed 37 degrees C. The enzyme is stable between pH 6 and 8, with pH 8 being the pH-optimum of both the lyase and the ligase reaction. Potassium phosphate and Tris were identified as optimal reaction buffers and the addition of 20 vol% DMSO is useful to enhance both the solubility of aromatic substrates and products and the stability of BAL. The initial broad product range of BAL-catalyzed reactions has been enlarged to include highly substituted hydroxybutyrophenones and aliphatic acyloins.     

The requirement for DsbA in pullulanase secretion is independent of disulphide bond formation in the enzyme

Results from previous studies have suggested that an intramolecular disulphide bond in the exoprotein pullulanase is needed for its recognition and transport across the outer membrane. This interpretation of the data is shown here to be incorrect: pullulanase devoid of all potential disulphide bonds is secreted with apparently the same efficiency as the wild-type protein. Furthermore, the periplasmic disulphide bond, oxidoreductase DsbA, previously shown to catalyse the formation of a disulphide bond in pullulanase and to decrease its transit time in the periplasm, is shown here to be required for the rapid secretion of pullulanase devoid of disulphide bonds. Several possible explanations for the role of DsbA in pullulanase secretion are discussed.     

A high-throughput screen utilizing the fluorescence of riboflavin for identification of lumazine synthase inhibitors

A high-throughput screening method based on the competitive binding of a lumazine synthase inhibitor and riboflavin to the active site of Schizosaccharomyces pombe lumazine synthase was developed. This assay is sensitive, simple, and robust. During assay development, all of the known active inhibitors tested were positively identified. Preliminary high-throughput screening in 384-well format resulted in a Z factor of 0.7. The approach utilizes a thermodynamic assay to bypass the problems associated with the instabilities of both lumazine synthase substrates that complicate the use of a kinetic assay in a high-throughput format, and it removes the time element from the assay, thus simplifying the procedure.     

Evaluation of cardosin A as a proteolytic probe in the presence of organic solvents

This investigation showed that cardosin A not only is active in media with organic solvents, cleaving the β-chain of oxidised insulin at three susceptible peptide bonds, but also maintains its specificity in all media tested. Additionally, the presence of organic solvents in the reaction media led to modifications of enzyme selectivity, which enabled the detection of intermediate products. While solvents like ethyl acetate induced a decrease in enzymatic activity, both by reducing the amount of active enzyme and presumably due to an inhibiting effect of ethyl acetate (which might compete with the substrate for the active site of the enzyme), n-hexane caused an increase in the hydrolysis velocity of one peptide bond. In view of the activity and specificity of cardosin A (which shows high preference for hydrophobic residues), it is proposed as a reliable probe for limited proteolysis in the presence of organic solvents. This may become particularly useful for structural characterisation of membrane proteins.     

A cell-based screen for inhibitors of protein folding and degradation

Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.