Identification of differential genes in the ovary relative to the testis and their expression patterns in half-smooth tongue sole (Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is a rare marine flatfish whose mature ovary and testis greatly differ in volume and weight. The length and weight of mature females are over twice greater than those of mature males. To obtain sufficient information on gonad differentiation and the relationship between gonad development and growth in the fish, we compared gene expression between the ovary and testis using suppression subtractive hybridization (SSH). Testis cDNAs are subtracted from...     

Genetic analysis of somatic embryogenesis in carrot cell culture: Initial characterization of six classes of temperature-sensitive variants

Cell cultures of the carrot Daucus carota are a useful experimental system for studying the genetic regulation of plant embryogenesis. A modified filtration-enrichment procedure was used to isolate 21 temperature-sensitive variants in somatic embryogenesis; the variants display normal embryo development at the permissive temperature (24°C) and altered development at the restrictive temperature (33°C). Temperature-shift experiments were performed on these variants to determine the timing of gene action for the putative temperature-sensitive alleles. According to their phenotypes at the restrictive temperature, these variants can be divided into six classes: No Growth, Callus Proliferation, Globularstage Block, Oblong-stage Block, Lateral Growth, and Root Formation. Although many variants exhibit lengthy temperature-sensitive periods, the temperature sensitivity of some variants is restricted to one or two embryonic stages. These results plus those in the literature are incorporated into a preliminary model concerning the genetic regulation of carrot embryogenesis.     

Induction of Expression Instability of the nptII Gene in Transgenic Tobacco Plants

A comparative analysis of the neomycin phosphotransferase (nptII) gene expression was performed in two groups of transformed tobacco plants, one of which included plants with direct and inverted tandem uidA gene repeats in the T-DNA insertion. This insertion of inverted repeats was shown to reduce the level of stable nptII gene expression to 20%, as compared with 65% in the control transformants. The level of unstable expression of this gene substantially increased (up to 71.4% vs. 5.5% in the control group) when homologous sequences were brought together with direct tandem repeats in the genome of hybrid plants.     

Predictivity of an in vitro model for acute and chronic skin irritation (SkinEthic) applied to the testing of topical vehicles

An in vitro human reconstructed epidermis model (SkinEthic) used for screening acute and chronic skin irritation potential was validated against in vivo data from skin tolerability studies. The irritation potential of sodium lauryl sulfate (SLS), calcipotriol and trans-retinoic acid was investigated. The in vitro epidermis-like model consists of cultures of keratinocytes from human foreskin on a polycarbonate filter. The modulation of cell viability, the release and gene expression of proinflammatory cytokines, interleukins 1α and 8, and morphological changes were evaluated during 3 days as endpoints representative for an inflammatory reaction. The cumulative irritation potential of the topical products was evaluated in a human clinical study by visual scoring and biophysical measurement of inflammatory skin reaction after repeated 24 h applications over 3 weeks under Finn chamber patches. All topical products that were nonirritating in the human study were noncytotoxic and did not induce cytokine expression in the in vitro acute model (day 1 exposure). All irritating controls exhibited specific cell viability and cytokine patterns, which were predictive of the in vivo human data. The ranking of mild to moderate skin irritation potential was based on the lack of cytotoxicity and the presence of cytokine patterns including gene expression specific for each irritant, using the chronic in vitro model (up to 3 days exposure). The human reconstructed epidermis model SkinEthic was shown to be a reliable preclinical tool predicting the irritation potential of topical products. Moreover, it is a useful model in a two-step tiered strategy for screening acute and chronic irritation potential for the selection of vehicles for new topical drugs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Overexpression and Purification of an Immunologically Reactive His–BIV Capsid Fusion Protein

The gene of the capsid protein of bovine immunodeficiency virus (BIV) was linked to a sequence encoding for six histidines and expressed as the (His)₆p26 capsid fusion protein. The fusion protein was strongly expressed as both soluble and insoluble forms after induction by isopropylthio-β- -galactoside. Purification was based on interaction of the hexa-histidine polypeptide with metal ions. Expression could represent 11% of the total protein inEscherichia coli,allowing more than 20 mg of highly purified protein to be obtained per liter of bacterial culture. The (His)₆p26 capsid fusion protein purified by immobilized metal affinity chromatography reacted specifically in Western blot with sera from cattle experimentally infected by BIV, as well as with two monoclonal antibodies directed against different epitopes of the Gag protein. The ease of expression, purification, and specificity of this fusion protein should permit a thorough study of prevalence of BIV infection in large-scale serological studies of field samples.     

Gene expression changes associated with cytotoxicity identified using cDNA arrays

In order to investigate gene expression changes associated with cytotoxicity, we used cDNA arrays to monitor the expression of over 5,000 genes in response to toxic stress in the HepG2 liver cell line. Cells were treated with cytotoxic doses of acetaminophen, caffeine or thioacetamide for nine time points ranging from 1 to 24 h. Samples of mRNA from each time point were used to prepare radiolabeled cDNA, which was hybridized to nylon-membrane-based cDNA arrays. High-stringency washes were applied to reduce cross-hybridization. Analysis of spot intensities revealed that each compound led to approximately 150-250 gene expression changes that were sustained over at least three adjacent time points. The affected genes could be classified into clusters based on their temporal patterns of differential expression. A common set of 44 genes showed similar expression changes in response to all three compounds. Of these changes, 90% could be confirmed by quantitative RT-PCR analysis. The results indicate that detailed array-based time-course studies, coupled with a sensitive and highly specific confirmation assay, provide a powerful means of identifying cytotoxicity-associated gene expression changes. Electronic Publication     

Hybridization between the peat mosses, Sphagnum capillifolium and S. quinquefarium (Sphagnaceae, Bryophyta) as inferred by morphological characters and isozyme markers

 Isozyme markers and morphological characters were studied in four populations of Sphagnum capillifolium and S. quinquefarium. Recombinant plants were found in three populations, where the two species occur sympatrically. All recombinants possessed different haplotypes and combinations of morphological characters, which show that they are results of independent hybridization events. Strongly male-biased sex ratios were found for Sphagnum capillifolium in all populations where it grew sympatrically with S. quinquefarium. Most of the recombinants were also male fertile. These observations suggest that S. quinquefarium is the female parent in the primary crosses and in subsequent backcrosses. Received September 3, 2001; accepted March 16, 2002 Published online: November 7, 2002 Addresses of the authors: Nils Cronberg (e-mail: Nils.Cronberg@sysbot.lu.se), Department of Systematic Botany, Lund University, S?lvegatan 37, SE-223 62 Lund, Sweden. Rayna Natcheva (e-mail: renimoss@iph.bio.bas.bg), Institute of Botany, Bulgarian Academy of Sciences, 23 Acad. G. Bonchev Str., 1113 Sofia, Bulgaria.