Ol-Prx 3, a member of an additional class of homeobox genes, is unimodally expressed in several domains of the developing and adult central nervous system of the medaka (Oryzias latipes)

Large-scale genetic screens for mutations affecting early neurogenesis of vertebrates have recently been performed with an aquarium fish, the zebrafish. Later stages of neural morphogenesis have attracted less attention in small fish species, partly because of the lack of molecular markers of developing structures that may facilitate the detection of discrete structural alterations. In this context, we report the characterization of Ol-Prx 3 (Oryzias latipes-Prx 3). This gene was isolated in the course of a large-scale screen for brain cDNAs containing a highly conserved DNA binding region, the homeobox helix-three. Sequence analysis revealed that this gene belongs to another class of homeobox genes, together with a previously isolated mouse ortholog, called OG-12 [Rovescalli, A. C., Asoh, S. & Nirenberg, M. (1996) Proc. Natl. Acad. Sci. USA 93, 10691–10696] and with the human SHOX gene [Rao, E., Weiss, B., Fukami, M., Rump, A., Niesler, B., et al. (1997) Nat. Genet. 16, 54–62], thought to be involved in the short-stature phenotype of Turner syndrome patients. These three genes exhibit a moderate level of identity in the homeobox with the other genes of the paired-related (PRX) gene family. Ol-Prx 3, as well as the PRX genes, are expressed in various cartilaginous structures of head and limbs. These genes might thus be involved in common regulatory pathways during the morphogenesis of these structures. Moreover, this paper reports a complex and monophasic pattern of Ol-Prx 3 expression in the central nervous system, which differs markedly from the patterns reported for the PRX genes, Prx 3 excluded: this gene begins to be expressed in a variety of central nervous system territories at late neurula stage. Strikingly, it remains turned on in some of the derivatives of each territory during the entire life of the fish. We hope this work will thus help identify common features for the PRX 3 family of homeobox genes.

Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: A useful model to monitor germ cells in a live vertebrate

We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange–red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3′ region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20–25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.

Silver toxicity across salinity gradients: the role of dissolved silver chloride species (AgCl x ) in Atlantic killifish (Fundulus heteroclitus) and medaka (Oryzias latipes) early life-stage toxicity.

The influence of salinity on Ag toxicity was investigated in Atlantic killifish (Fundulus heteroclitus) early life-stages. Embryo mortality was significantly reduced as salinity increased and Ag(+) was converted to AgCl(solid). However, as salinity continued to rise (>5 ‰), toxicity increased to a level at least as high as observed for Ag(+) in deionized water. Rather than correlating with Ag(+), Fundulus embryo toxicity was better explained (R(2) = 0.96) by total dissolved Ag (Ag(+), AgCl2 (-), AgCl3 (2-), AgCl4 (3-)). Complementary experiments were conducted with medaka (Oryzias latipes) embryos to determine if this pattern was consistent among evolutionarily divergent euryhaline species. Contrary to Fundulus data, medaka toxicity data were best explained by Ag(+) concentrations (R(2) = 0.94), suggesting that differing ionoregulatory physiology may drive observed differences. Fundulus larvae were also tested, and toxicity did increase at higher salinities, but did not track predicted silver speciation. Alternatively, toxicity began to increase only at salinities above the isosmotic point, suggesting that shifts in osmoregulatory strategy at higher salinities might be an important factor. Na(+) dysregulation was confirmed as the mechanism of toxicity in Ag-exposed Fundulus larvae at both low and high salinities. While Ag uptake was highest at low salinities for both Fundulus embryos and larvae, uptake was not predictive of toxicity.

Developmental toxicity from exposure to various forms of mercury compounds in medaka fish (Oryzias latipes) embryos.

This study examined developmental toxicity of different mercury compounds, including some used in traditional medicines. Medaka (Oryzias latipes) embryos were exposed to 0.001-10 µM concentrations of MeHg, HgCl2, α-HgS (Zhu Sha), and β-HgS (Zuotai) from stage 10 (6-7 hpf) to 10 days post fertilization (dpf). Of the forms of mercury in this study, the organic form (MeHg) proved the most toxic followed by inorganic mercury (HgCl2), both producing embryo developmental toxicity. Altered phenotypes included pericardial edema with elongated or tube heart, reduction of eye pigmentation, and failure of swim bladder inflation. Both α-HgS and β-HgS were less toxic than MeHg and HgCl2. Total RNA was extracted from survivors three days after exposure to MeHg (0.1 µM), HgCl2 (1 µM), α-HgS (10 µM), or β-HgS (10 µM) to examine toxicity-related gene expression. MeHg and HgCl2 markedly induced metallothionein (MT) and heme oxygenase-1 (Ho-1), while α-HgS and β-HgS failed to induce either gene. Chemical forms of mercury compounds proved to be a major determinant in their developmental toxicity.

Systematic Variation in the Pattern of Gene Paralog Retention between the Teleost Superorders Ostariophysi and Acanthopterygii

Teleost fish underwent whole-genome duplication around 450 Ma followed by diploidization and loss of 80-85% of the duplicated genes. To identify a deep signature of this teleost-specific whole-genome duplication (TSGD), we searched for duplicated genes that were systematically and uniquely retained in one or other of the superorders Ostariophysi and Acanthopterygii. TSGD paralogs comprised 17-21% of total gene content. Some 2.6% (510) of TSGD paralogs were present as pairs in the Ostariophysi genomes of Danio rerio (Cypriniformes) and Astyanax mexicanus (Characiformes) but not in species from four orders of Acanthopterygii (Gasterosteiformes, Gasterosteus aculeatus; Tetraodontiformes, Tetraodon nigroviridis; Perciformes, Oreochromis niloticus; and Beloniformes, Oryzias latipes) where a single copy was identified. Similarly, 1.3% (418) of total gene number represented cases where TSGD paralogs pairs were systematically retained in the Acanthopterygian but conserved as a single copy in Ostariophysi genomes. We confirmed the generality of these results by phylogenetic and synteny analysis of 40 randomly selected linage-specific paralogs (LSPs) from each superorder and completed with the transcriptomes of three additional Ostariophysi species (Ictalurus punctatus [Siluriformes], Sinocyclocheilus species [Cypriniformes], and Piaractus mesopotamicus [Characiformes]). No chromosome bias was detected in TSGD paralog retention. Gene ontology (GO) analysis revealed significant enrichment of GO terms relative to the human GO SLIM database for growth, Cell differentiation, and Embryo development in Ostariophysi and for Transport, Signal Transduction, and Vesicle mediated transport in Acanthopterygii. The observed patterns of paralog retention are consistent with different diploidization outcomes having contributed to the evolution/diversification of each superorder.

Bioremediation of PAHs - Limitations and soultions

Introduction 1.1 Occurrence of polycyclic aromatic hydrocarbons (PAH) in the environment Worldwide industrial and agricultural developments have released a large number of natural and synthetic hazardous compounds into the environment due to careless waste disposal, illegal waste dumping and accidental spills. As a result, there are numerous sites in the world that require cleanup of soils and groundwater. Polycyclic aromatic hydrocarbons (PAHs) are one of the major groups of these contaminants (Da Silva et al., 2003). PAHs constitute a diverse class of organic compounds consisting of two or more aromatic rings with various structural configurations (Prabhu and Phale, 2003). Being a derivative of benzene, PAHs are thermodynamically stable. In addition, these chemicals tend to adhere to particle surfaces, such as soils, because of their low water solubility and strong hydrophobicity, and this results in greater persistence under natural conditions. This persistence coupled with their potential carcinogenicity makes PAHs problematic environmental contaminants (Cerniglia, 1992; Sutherland, 1992). PAHs are widely found in high concentrations at many industrial sites, particularly those associated with petroleum, gas production and wood preserving industries (Wilson and Jones, 1993). 1.2 Remediation technologies Conventional techniques used for the remediation of soil polluted with organic contaminants include excavation of the contaminated soil and disposal to a landfill or capping - containment - of the contaminated areas of a site. These methods have some drawbacks. The first method simply moves the contamination elsewhere and may create significant risks in the excavation, handling and transport of hazardous material. Additionally, it is very difficult and increasingly expensive to find new landfill sites for the final disposal of the material. The cap and containment method is only an interim solution since the contamination remains on site, requiring monitoring and maintenance of the isolation barriers long into the future, with all the associated costs and potential liability. A better approach than these traditional methods is to completely destroy the pollutants, if possible, or transform them into harmless substances. Some technologies that have been used are high-temperature incineration and various types of chemical decomposition (for example, base-catalyzed dechlorination, UV oxidation). However, these methods have significant disadvantages, principally their technological complexity, high cost , and the lack of public acceptance. Bioremediation, on the contrast, is a promising option for the complete removal and destruction of contaminants. 1.3 Bioremediation of PAH contaminated soil & groundwater Bioremediation is the use of living organisms, primarily microorganisms, to degrade or detoxify hazardous wastes into harmless substances such as carbon dioxide, water and cell biomass Most PAHs are biodegradable unter natural conditions (Da Silva et al., 2003; Meysami and Baheri, 2003) and bioremediation for cleanup of PAH wastes has been extensively studied at both laboratory and commercial levels- It has been implemented at a number of contaminated sites, including the cleanup of the Exxon Valdez oil spill in Prince William Sound, Alaska in 1989, the Mega Borg spill off the Texas coast in 1990 and the Burgan Oil Field, Kuwait in 1994 (Purwaningsih, 2002). Different strategies for PAH bioremediation, such as in situ , ex situ or on site bioremediation were developed in recent years. In situ bioremediation is a technique that is applied to soil and groundwater at the site without removing the contaminated soil or groundwater, based on the provision of optimum conditions for microbiological contaminant breakdown.. Ex situ bioremediation of PAHs, on the other hand, is a technique applied to soil and groundwater which has been removed from the site via excavation (soil) or pumping (water). Hazardous contaminants are converted in controlled bioreactors into harmless compounds in an efficient manner. 1.4 Bioavailability of PAH in the subsurface Frequently, PAH contamination in the environment is occurs as contaminants that are sorbed onto soilparticles rather than in phase (NAPL, non aqueous phase liquids). It is known that the biodegradation rate of most PAHs sorbed onto soil is far lower than rates measured in solution cultures of microorganisms with pure solid pollutants (Alexander and Scow, 1989; Hamaker, 1972). It is generally believed that only that fraction of PAHs dissolved in the solution can be metabolized by microorganisms in soil. The amount of contaminant that can be readily taken up and degraded by microorganisms is defined as bioavailability (Bosma et al., 1997; Maier, 2000). Two phenomena have been suggested to cause the low bioavailability of PAHs in soil (Danielsson, 2000). The first one is strong adsorption of the contaminants to the soil constituents which then leads to very slow release rates of contaminants to the aqueous phase. Sorption is often well correlated with soil organic matter content (Means, 1980) and significantly reduces biodegradation (Manilal and Alexander, 1991). The second phenomenon is slow mass transfer of pollutants, such as pore diffusion in the soil aggregates or diffusion in the organic matter in the soil. The complex set of these physical, chemical and biological processes is schematically illustrated in Figure 1. As shown in Figure 1, biodegradation processes are taking place in the soil solution while diffusion processes occur in the narrow pores in and between soil aggregates (Danielsson, 2000). Seemingly contradictory studies can be found in the literature that indicate the rate and final extent of metabolism may be either lower or higher for sorbed PAHs by soil than those for pure PAHs (Van Loosdrecht et al., 1990). These contrasting results demonstrate that the bioavailability of organic contaminants sorbed onto soil is far from being well understood. Besides bioavailability, there are several other factors influencing the rate and extent of biodegradation of PAHs in soil including microbial population characteristics, physical and chemical properties of PAHs and environmental factors (temperature, moisture, pH, degree of contamination). Figure 1: Schematic diagram showing possible rate-limiting processes during bioremediation of hydrophobic organic contaminants in a contaminated soil-water system (not to scale) (Danielsson, 2000). 1.5 Increasing the bioavailability of PAH in soil Attempts to improve the biodegradation of PAHs in soil by increasing their bioavailability include the use of surfactants , solvents or solubility enhancers.. However, introduction of synthetic surfactant may result in the addition of one more pollutant. (Wang and Brusseau, 1993).A study conducted by Mulder et al. showed that the introduction of hydropropyl-ß-cyclodextrin (HPCD), a well-known PAH solubility enhancer, significantly increased the solubilization of PAHs although it did not improve the biodegradation rate of PAHs (Mulder et al., 1998), indicating that further research is required in order to develop a feasible and efficient remediation method. Enhancing the extent of PAHs mass transfer from the soil phase to the liquid might prove an efficient and environmentally low-risk alternative way of addressing the problem of slow PAH biodegradation in soil.

Possible ecological risks of transgenic organism release when transgenes affect mating success: Sexual selection and the Trojan gene hypothesis

Widespread interest in producing transgenic organisms is balanced by concern over ecological hazards, such as species extinction if such organisms were to be released into nature. An ecological risk associated with the introduction of a transgenic organism is that the transgene, though rare, can spread in a natural population. An increase in transgene frequency is often assumed to be unlikely because transgenic organisms typically have some viability disadvantage. Reduced viability is assumed to be common because transgenic individuals are best viewed as macromutants that lack any history of selection that could reduce negative fitness effects. However, these arguments ignore the potential advantageous effects of transgenes on some aspect of fitness such as mating success. Here, we examine the risk to a natural population after release of a few transgenic individuals when the transgene trait simultaneously increases transgenic male mating success and lowers the viability of transgenic offspring. We obtained relevant life history data by using the small cyprinodont fish, Japanese medaka (Oryzias latipes) as a model. Our deterministic equations predict that a transgene introduced into a natural population by a small number of transgenic fish will spread as a result of enhanced mating advantage, but the reduced viability of offspring will cause eventual local extinction of both populations. Such risks should be evaluated with each new transgenic animal before release.

Zebrafish KLF4 is essential for anterior mesendoderm/pre-polster differentiation and hatching

Gene knockout studies of Kruppel-like factors (KLFs) in mice have shown essential roles in organogenesis. A screen for KLF family members in zebrafish identified many KLFs. One of these, zebrafish KLF4 (zKLF4) is the homologue of neptune, a Xenopus laevis KLF. zKLF4 is expressed from approximately 80% epiboly a patch of dorsal/anterior mesendodermal cells called the pre-polster and, subsequently, in the polster and hatching gland. Here we investigate the function of zKLF4 using morpholino-based antisense oligonucleotides. Knockdown of zKLF4 resulted in complete absence of hatching gland formation and subsequent hatching in zebrafish. In addition, there was early knockdown of expression of the pre-polster/anterior mesendoderm markers CatL, cap1, and BMP4. These results indicate zKLF4 is expressed within the pre-polster, an early mesendodermal site, and that it plays a critical role in the differentiation of these cells into hatching gland cells.

Expression of Bf/C2 Gene(S) in the Japanese Medaka Fish

Complement factor B and C2 are two central serine proteases of the alternative and classical complement pathways, respectively, that serve as the catalytic subunits of the C3 convertase. Research has been completed using a female Japanese medaka fish, (Oryzias latipes), and other teleost and elasmobrach species in order to isolate eDNA clones and perform linkage analysis of the Bf/C2 gene(s). To further analyze the evolution of the complement system in teleosts, different tissues than the ones from previous studies of medaka fish were analyzed for the constitutive gene expression of factor B and C2. Bf/C2 sequences were amplified by reverse transcription-polymerase chain reaction with primers corresponding to the common amino acid sequences shared by mammalian Bf and C2. Agarose gel electrophoresis was used to visualize sample bands and to calculate the concentration of gene expression of the Bf/C2 gene(s) in each tissue. All five tissue types, kidney, liver, muscle, testis, and spleen from a male medaka fish demonstrated Bf/C2 gene(s) expression, confirming that the messages of Bf/C2 gene(s) are distributed throughout the medaka fish. Tissues of the spleen, liver, and kidney contained the highest concentrations of expression of Bf/C2 gene( s ), while tissues of the muscle and testis contained the lowest concentrations. This research also determined that RT-PCR allowed for more sensitive analysis of gene expression than other molecular biology techniques such as Northern blotting analysis.

PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.

Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit, we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays.

Anti-Helicobacter pylori activity and oxidative burst inhibition by the naphthoquinone 5-methoxy-3,4-dehydroxanthomegnin from Paepalanthus latipes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A new cytotoxic naphthoquinone from Paepalanthus latipes

Quinones constitute an important class of naturally occurring compounds. They are found in plants, fungi and bacteria. Large number of quinones has been associated with antitumor, antibacterial, antimalarial and antifungal activities. In this work we describe the isolation, structure determination and the cytotoxic index of a new 1,4-naphthoquinone isolated from the capitula of Paepalanthus latipes.

Avaliação da atividade mutagênica do extrato etanólico bruto de Paepalanthus latipes (Eriocaulaceae) e dos compostos flavonoídicos 7-metoxilados relacionados

Paepalanthus latipes (Silveira) é uma das plantas brasileiras nativas da Serra do Cipó, Minas Gerais, conhecida como sempre-vivas e muito exportada para os Estados Unidos, Europa e Japão, por seu valor ornamental. Poucos resultados são relatados quanto à atividade biológica destas plantas. Este trabalho teve como objetivo avaliar a atividade mutagênica do extrato etanólico bruto de P. latipes e de dois flavonóides 7-metoxilados dele isolados (7-metilquercetagetina e 7-metilquercetagetina-4'-O-β-D-glicopiranosídeo), através do teste de Ames, utilizando linhagens mutantes de Salmonella typhimurium TA98 e TA100. A atividade mutagênica das amostras testadas, em concentrações variando de 62,5 a 375 μg/placa, foi determinada com ativação metabólica (utilizando-se a fração microssomal S9) e na ausência dessa. Os resultados demonstraram que, tanto o extrato etanólico bruto de P. latipes quanto os flavonóides 7-metoxilados testados, não induziram atividade mutagênica nas linhagens TA98 e TA100.

Quercetagetin 7-methyl ether glycosides from Paepalanthus vellozioides and Paepalanthus latipes

Several new quercetagetin 7-methyl ether glycosides were characterized from the ethanolic extracts of scapes and leaves of Paepalanthus vellozioides and P. latipes. Their structures were determined mainly by 600 MHz NMR spectroscopy. (C) 1999 Elsevier B.V. Ltd. All rights reserved.

New naphthopyranone glycosides from Paepalanthus vellozioides and Paepalanthus latipes

Three new compounds-3,4-dihydro-10-hydroxy-7-methoxy-3-(R)-methyl-1H-3,4-dihydronaphtho-]2,3c]pyran-1-one-9-O-beta-D-glucopy ranoside (1), 3,4-dihydro-10-hydroxy-7-methoxy-3-(R)-methyl-1H-3,4-dihydronaphtho-[2,3c]-pyran-1-one-9-O-beta-D-glucopyranosyl-(1-->6)-glucopyranoside (2), and 3,4-dihydro-10-dihydroxy-7-methoxy-3-(R)-methyl-1H-3,4-dihydronaphtho-[2,3c]-pyran-1-one-9-O-beta-D-allopyranosyl (1-->6)-glucopyranoside (3)-were isolated from the leaves of Paepalanthus vellozioides and Paepalanthus latipes and characterized by spectrometric methods, mainly electrospray mass spectrometry and 1D and 2D NMR experiments. These unusual glycosylated dihydronaphthopyranones may serve as taxonomic markers of the genus Paepalanthus, since these compounds were not detected in other genera belonging to the Eriocaulaceae family.