DiffScreen: the merging of image subtraction and molecular genetics for the rapid analysis of differentially screened cDNA libraries.
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UNLABELLED: SymmeTREE implements several tests of differential diversification rates that exploit information on the topological distribution of species diversity throughout entire trees to address two general questions: (1) Has a given tree experienced significant variation in diversification rates among its branches? and (2) If so, along which branches have significant shifts in diversification rate occurred? These explicitly model-based methods are robust to uncertainty in estimates of branch length/duration and can accommodate incompletely resolved trees and other forms of phylogenetic uncertainty. AVAILABILITY: http://www.phylodiversity.net/bmoore/software.html CONTACT: brian.moore@yale.edu.
UNLABELLED: In this article, we combined EST information from the UniGene database and orthologous relationships from the Ensembl database to construct a ZooDDD database. The primary function of ZooDDD is to mine evolutionary conserved, highly expressed, tissue-specific orthologues in model animals. The candidate genes of interest derived from the ZooDDD database will provide biologists with a good step for comparing the expression, functions and evolution of animal genomes. AVAILABILITY: http://bio301.iis.sinica.edu.tw/~ZooDDDNew/main.php.
A failure of T lymphocytes to produce interleukin 2 (IL-2) on restimulation may, in part, account for the specific unresponsiveness that accompanies incomplete activation. The evidence to support this has been derived predominantly from the investigation of the molecular basis of anergy in murine type 1 T cells. In this study, the effects of different tolerogenic signals delivered by specific peptide or Staphylococcus aureus enterotoxin on the ability of antigen-specific human T cells to produce lymphokines, both in the induction phase and in established antigen-specific non-responsiveness, have been examined. Although T cell proliferation was decreased by supraoptimal concentrations of specific peptide in the presence or absence of antigen presenting cells, IL-2, IL-4, and interferon gamma (IFN-gamma) synthesis were comparable to that of activated T cells. The different tolerogenic signals, all capable of inhibiting phase of unresponsiveness. Restimulation of anergic T cells with an antigenic challenge failed to induce lymphokine production, with the exception of allergen-reactive T cells that secreted IFN-gamma. This latter observation is relevant to the desensitization of specific responsiveness in allergic disease.
A method is presented for the positive identification of quinine and quinidine. The method requires extraction of the compounds from an alkaline solution into an organic solvent and concentration of the sample by evaporation of the solvent. The sample is chromatographed without derivatization and may be detected by the flame ionization detector (FID) or mass spectrometry (MS). Limits of sensitivity are less than 5 ng per assay for the underivatized compounds. Analysis of urine extracts is discussed.
Slow progress has been made in discovering plant genes governing the interaction of plant pathogens and their hosts using classical genetic approaches. Extensive studies employing DNA microarray techniques to identify global changes in gene expression during pathogen-host interaction have greatly enhanced discovery of genetic components regulating the plant defence response to pathogen attack. In this study, a complementary approach was used to identify changes in protein abundance during interaction of Arabidopsis cell cultures with a pathogen-derived elicitor. The soluble protein fractions were analysed by two-dimensional difference gel electrophoresis and proteins differentially expressed in response to treatment with fungal elicitor were identified via matrix-assisted laser desorption ionization-time of flight mass spectrometry. Elicitor responsive proteins included molecular chaperones, oxidative stress defence proteins, mitochondrial proteins, and enzymes of a diverse number of metabolic pathways. The findings, in combination with currently available microarray data, will form the basis of a filter to identify pivotal genes whose role in pathogen defence systems will require confirmation using gene knockout mutants.
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OBJECTIVES: To explore the expressions of metastasis-related proteins between metastatic LS174T and non-metastatic SW480 human colorectal carcinoma cell lines. METHODS: Two-dimensional gel electrophoresis (2-DE) was applied to separate the total proteins of cells. The silver-stained gels were analysed by 2-DE software Image Master 2D Elite. Selected differential protein spots were identified with peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searching. RESULTS: The protein endothelial cell growth factor 1 (platelet-derived), rhotekin protein (RTKN), septin 1, cyclin-dependent kinase 1, sialic acid binding Ig-like lectin 11, tyrosinase-related protein-2, translin-like protein, and DNA directed RNA polymerase II polypeptide J-related gene isoform 2 appeared in metastatic but were not detected in non-metastatic cell lines, whereas integrin-linked kinase-associated protein phosphatase 2C isoform 2, MHC class I promoter binding protein, protein phosphatase 2A regulatory subunit B' (PR 53), carboxypeptidase A5, paired box transcription factor, zinc finger protein 79, and apolipoprotein B-48 were detected in non-metastatic but were absent in metastatic cell lines. In addition, cyclin fold protein 1 variant A and pre-B-cell leukemia transcription factor 1 were lowly expressed in the non-metastatic cell line and were significantly upregulated in the metastatic cell line. These identified proteins were involved in cell growth, motility, invasion, adhesion, apoptosis and tumour immunity, which is associated with distinct aspects of tumour metastasis. CONCLUSIONS: These data are valuable for the identification of differentially expressed proteins involved in human colorectal carcinoma carcinogenesis and metastasis.
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I have developed promoter expression binary vectors based on the tumor-inducing plasmid of Agrobacterium tumefaciens to facilitate elucidation of plant gene regulation. Promoter activity can be determined by inserting DNA fragments into the multiple cloning sites of the vectors forming transcriptional and/or translational fusions between the cat structural gene and an inserted promoter region. The activity of the nopaline synthase (nos) promoter was demonstrated with the vector. However, three animal promoters tested with this system showed no measurable activity in plant cells. Examination of 40 independently derived transformed tissues revealed a 200-fold difference in the nos promoter activity. Furthermore, there is no apparent correlation between the neomycin phosphotransferase and chloramphenicol acetyltransferase activities, although both genes are closely linked and under control of identical nos promoters. These results indicate that vast differences in promoter activity of transferred genes can occur within the same cell, as well as in independently derived cell lines.
A Random Amplified Polymorphic DNA (RAPD) fingerprinting method was developed to differentiate isolates of Salmonella serotype typhi (S. typhi) and other Salmonella isolates. A panel of five primers was used to examine 63 isolates of Salm. typhi, including 56 strains isolated in Taiwan and seven strains obtained abroad. Twenty-one RAPD types were revealed using the RAPD fingerprinting method. An RAPD with primer 6032 yielded a polymorphism in a 350 bp fragment that differentiated the attenuated vaccine strain Salm. typhi Ty21a from the rest of the Salm. typhi strains. Strains of Salm. typhi were divided into five types with primer D14307. Primer D14307 also proved capable of discrimination among 65 other Salmonella isolates representing 42 different serotypes. The bacterial DNA used in this RAPD protocol was obtained using a commercially available DNA extraction kit (GeneReleaser). The DNA of various strains of Salmonella from this simple extraction procedure could be discriminated within a few hours using the RAPD technique.
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Bovine lens epithelial cells, in vivo, are known to perform two determined functions. First, they synthesize the lens capsule and subsequently, in the germinal region, they differentiate in fiber cells with massive production of crystallin proteins, inactivation and pyknosis of the nucleus. Bovine lens epithelial cells from adult origin can be cultured but so far no massive crystallin production has been demonstrated in vitro. We have studied the growth and differentiation of these cells and shown that in long term culture they acquire spontaneously many characteristics of transformation: unlimited growth potential, abnormal karyotype, multilayering. Viral particles were scarcely detected. However, they retain their epithelioid character and the ability to synthesize lens capsule material. Kinetic characteristics of those cells have been determined. When injected into nude mice, they actively proliferate and form tumors in which synthesis of alpha-crystallin can be demonstrated. These results show that in vitro transformation of lens epithelial cells does not affect their potential for terminal differentiation.
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