Exploitation of archived marine nematodes — a hot lysis DNA extraction protocol for molecular studies

Museums and other research organizations around the world have large numbers of formalin-fixed marine invertebrates in their collections. These have the potential to be a valuable resource for molecular ecological studies, but the development of methodologies for the molecular analysis of formalin-fixed material has been slow. In this study, a hot lysis protocol accompanied by the use of a commercial DNA extraction kit has been employed for DNA recovery from archived marine nematodes, followed by PCR amplification and sequencing. In total, 25 specimens ranging from estuarine to deep sea environments were subjected to molecular analyses. Successful amplification and sequencing of the nuclear small subunit ribosomal RNA (18S rRNA) gene was achieved in all individuals. Additionally, some estuarine nematodes were tentatively identified to genus and species using a phylogenetic approach. In the future, this technique should prove to be profitable for the genetic study of a wide range of formalin-fixed marine invertebrates.     

Lipophilic constituents from aerial and root parts of Mercurialis perennis L.

Introduction – Dog's mercury (Mercurialis perennis L.) is a perennial herb used in remedies for medicinal purposes. The plant is supposed to contain potentially active substances but its constituents have only been rarely studied. Objective – Detailed studies on the phytochemical composition are of great interest to broaden the knowledge on the chemotaxonomy and pharmacognosy of M. perennis. Methodology – Chloroform and hexane extracts from roots and aerial parts were investigated using GC/MS and LC/MS. Results – The whole plant exhihited a broad spectrum of structurally diverse constituents, mainly alkaloids, terpenes, sterols and simple aromatic compounds. Closer inspection of the piperidine alkaloid hermidin revealed its inherent instability towards air oxygen. To obtain quantitative data on these alkaloids the synthesis of the more stable reference compound 4‐methoxy‐1‐methylpyridine‐2,6(1H,3H)‐dione (MMPD) was required. In this study, MMPD was detected for the first time as a genuine compound in Mercurialis. Hermidine quinone and hermidin dimers originating from hermidin via a free anionic radical reaction were also confirmed by GC/MS. Moreover, volatile compounds such as benzylalcohol, 2‐phenylethanol, 4‐methoxy‐ and 3,4‐dimethoxyphenol, (?)‐cis‐ and (+)‐trans‐myrtanol, (?)‐cis‐myrtanal as well as squalene were predominantely present in Mercurialis roots. In contrast, aerial parts mainly contained phytol derivatives, sterols and tocopherols. By changing solvent polarity, lipid and wax‐containing fractions were obtained. LC/MS‐studies on hexane extracts showed the presence of several mixed triglycerides constituted by linolenic, linoleic, oleic, stearic and palmitic acids, as well as lutein, carotenes and pheophytins. Conclusions – The phytochemical data presented complement our knowledge on the rarely studied plant M. perennis and may broaden its use in future phytotherapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Automated analysis of neurite branching in cultured cortical neurons using HCA-Vision.

Manual neuron tracing is a very labor-intensive task. In the drug screening context, the sheer number of images to process means that this approach is unrealistic. Moreover, the lack of reproducibility, objectivity, and auditing capability of manual tracing is limiting even in the context of smaller studies. We have developed fast, sensitive, and reliable algorithms for the purpose of detecting and analyzing neurites in cell cultures, and we have integrated them in software called HCA-Vision, suitable for the research environment. We validate the software on images of cortical neurons by comparing results obtained using HCA-Vision with those obtained using an established semi-automated tracing solution (NeuronJ). The effect of the Sez-6 deletion was characterized in detail. Sez-6 null neurons exhibited a significant increase in neurite branching, although the neurite field area was unchanged due to a reduction in mean branch length. HCA-Vision delivered considerable speed benefits and reliable traces.     

Characterization of human UDP-glucose dehydrogenase. CYS-276 is required for the second of two successive oxidations

UDP-glucose dehydrogenase (UGDH) catalyzes two oxidations of UDP-glucose to yield UDP-glucuronic acid. Pathological overproduction of extracellular matrix components may be linked to the availability of UDP-glucuronic acid; therefore UGDH is an intriguing therapeutic target. Specific inhibition of human UGDH requires detailed knowledge of its catalytic mechanism, which has not been characterized. In this report, we have cloned, expressed, and affinity-purified the human enzyme and determined its steady state kinetic parameters. The human enzyme is active as a hexamer with values for Km and Vmax that agree well with those reported for a bovine homolog. We used crystal coordinates for Streptococcus pyogenes UGDH in complex with NAD+ cofactor and UDP-glucose substrate to generate a model of the enzyme active site. Based on this model, we selected Cys-276 and Lys-279 as likely catalytic residues and converted them to serine and alanine, respectively. Enzymatic activity of C276S and K279A point mutants was not measurable under normal assay conditions. Rate constants measured over several hours demonstrated that K279A continued to turn over, although 250-fold more slowly than wild type enzyme. C276S, however, performed only a single round of oxidation, indicating that it is essential for the second oxidation. This result is consistent with the postulated role of Cys-276 as a catalytic residue and supports its position in the reaction mechanism for the human enzyme. Lys-279 is likely to have a role in positioning active site residues and in maintaining the hexameric quaternary structure.     

Disruption of sonic hedgehog signaling alters growth and patterning of lingual taste papillae

Taste buds on the anterior part of the tongue develop in conjunction with epithelial-mesenchymal specializations in the form of gustatory (taste) papillae. Sonic hedgehog (Shh) and Bone Morphogenetic Protein 4 (BMP4) are expressed in developing taste papillae, but the roles of these signaling molecules in specification of taste bud progenitors and in papillary morphogenesis are unclear. We show here that BMP4 is not expressed in the early tongue, but is precisely coexpressed with Shh in papillary placodes, which serve as a signaling center for both gustatory and papillary development. To elucidate the role of Shh, we used an in vitro model of mouse fungiform papillary development to determine the effects of two functional inhibitors of Shh signaling: anti-Shh (5E1) antibody and cyclopamine. Cultured E11.5 tongue explants express Shh and BMP4(LacZ) in a pattern similar to that of intact embryos, localizing to developing papillary placodes after 2 days in culture. Tongues cultured with 5E1 antibody continue to express these genes in papillary patterns but develop more papillae that are larger and closer together than in controls. Tongues cultured with cyclopamine have a dose-dependent expansion of Shh and BMP4(LacZ) expression domains. Both antibody-treated and cyclopamine-treated tongue explants also are smaller than controls. Taken together, these results suggest that, although Shh is not involved in the initial specification of papillary placodes, Shh does play two key roles during pmcry development: (1) as a morphogen that directs cells toward a nonpapillary fate, and (2) as a mitogen, causing expansion of the interplacodal epithelium and underlying mesenchyme.     

Identification of a mitochondrial glycerol-3-phosphate dehydrogenase from Arabidopsis thaliana: evidence for a mitochondrial glycerol-3-phosphate shuttle in plants

We report molecular characterization of an Arabidopsis gene encoding a mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (FAD-GPDH) that oxidizes glycerol-3-phosphate (G-3-P) to dihydroxyacetone phosphate. We demonstrate through in vitro targeting assays that the encoded gene product can be imported into mitochondrial membrane systems. Enzyme activity of the protein was confirmed through heterologous expression in Escherichia coli. The Arabidopsis gene is expressed throughout plant development, but at the highest level during seed germination. We also show that expression of the Arabidopsis FAD-GPDH gene is coupled to oxygen consumption and affected by ABA and stress conditions. Together with an NAD(+)-dependent GPDH, this enzyme could form a G-3-P shuttle, as previously established in other eukaryotic organisms, and links cytosolic G-3-P metabolism to carbon source utilization and energy metabolism in plants.     

Parasite altered micro-distribution of Gammarus pulex (Crustacea: Amphipoda)

In a river survey, Gammarus pulex amphipods both unparasitised and parasitised with the acanthocephalan Echinorhynchus truttae were distributed similarly with respect to flow regimen, tending to be more abundant in faster, shallower, riffle patches. However, there was a higher prevalence of parasitism in faster, shallower areas than in slower, deeper areas and abundance correlated with macrophyte coverage for unparasitised but not parasitised amphipods, indicating subtle differences in habitat usage. A laboratory 'patch' simulation indicated that parasitism influenced micro-distribution. There were higher proportions of unparasitised amphipods in/under stone substrates and within weed. In contrast, there were higher proportions of parasitised amphipods in the water column and at the water surface. As the experiment progressed, unparasitised but not parasitised amphipod habitat usage shifted from those micro-habitats above the substrate and in the water column to those in/under the substrates. Experiments also demonstrated that parasitised amphipods were more active and had a greater preference for illumination. Previous studies of the effects of acanthocephalan parasitism of amphipod hosts have focussed on how drift behaviour is altered, now we show that subtle differences in micro-habitat usage could translate to greatly increased vulnerability to fish predation. We discuss how aggregation of parasitised individuals within specific habitats could promote parasite transmission.     

Parasite transmission and cannibalism in an amphipod (Crustacea)

In its freshwater amphipod host Gammarus duebeni celticus, the microsporidian parasite Pleistophora mulleri showed 23% transmission efficiency when uninfected individuals were fed infected tissue, but 0% transmission by water-borne and coprophagous routes. Cannibalism between unparasitised and parasitised individuals was significantly in favour of the former (37% compared to 0%). In addition, cannibalism between parasitised individuals was significantly higher than between unparasitised individuals (27% compared to 0%). Thus, parasitised individuals were more likely to be cannibalised by both unparasitised and parasitised individuals. We discuss the conflicting selective forces within this host/parasite relationship, the implications of parasite mediated cannibalism for host population structure and the impacts this may have on the wider aquatic community.     

The signal sequence of exported protein-1 directs the green fluorescent protein to the parasitophorous vacuole of transfected malaria parasites

The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the erythrocytes of its human host. In the mature stages of intraerythrocytic growth, the parasite undertakes extensive remodeling of its adopted cellular home by exporting proteins beyond the confines of its own plasma membrane. To examine the signals involved in export of parasite proteins, we have prepared transfected parasites expressing a chimeric protein comprising the N-terminal region of the Plasmodium falciparum exported protein-1 appended to green fluorescent protein. The majority of the population of the chimeric protein appears to be correctly processed and trafficked to the parasitophorous vacuole, indicating that this is the default destination for protein secretion. Some of the protein is redirected to the parasite food vacuole and further degraded. Photobleaching studies reveal that the parasitophorous vacuole contains subcompartments that are only partially interconnected. Dual labeling with the lipid probe, BODIPY-TR-ceramide, reveals the presence of membrane-bound extensions that can bleb from the parasitophorous vacuole to produce double membrane-bound compartments. We also observed regions and extensions of the parasitophorous vacuole, where there is segregation of the lumenal chimera from the lipid components. These regions may represent sites for the sorting of proteins destined for the trafficking to sites beyond the parasitophorous vacuole membrane.     

Replication-competent bacterial artificial chromosomes of Marek's disease virus: novel tools for generation of molecularly defined herpesvirus vaccines

Marek's disease (MD), a highly infectious disease caused by an oncogenic herpesvirus, is one of the few herpesvirus diseases against which live attenuated vaccines are used as the main strategy for control. We have constructed bacterial artificial chromosomes (BACs) of the CVI988 (Rispens) strain of the virus, the most widely used and effective vaccine against MD. Viruses derived from the BAC clones were stable after in vitro and in vivo passages and showed characteristics and growth kinetics similar to those of the parental virus. Molecular analysis of the individual BAC clones showed differences in the structure of the meq gene, indicating that the commercial vaccine contains virus populations with distinct genomic structures. We also demonstrate that, contrary to the published data, the sequence of the L-meq of the BAC clone did not show any frameshift. Virus stocks derived from one of the BAC clones (clone 10) induced 100 percent protection against infection by the virulent strain RB1B, indicating that BAC-derived viruses could be used with efficacies similar to those of the parental CVI988 vaccines. As a DNA vaccine, this BAC clone was also able to induce protection in 6 of 20 birds. Isolation of CVI988 virus from all of these six birds suggested that immunity against challenge was probably dependent on the reconstitution of the virus in vivo and that such viruses are also as immunogenic as the in vitro-grown BAC-derived or parental vaccine viruses. Although the reasons for the induction of protection only in a proportion of birds (33.3%) that received the DNA vaccine are not clear, this is most likely to be related to the suboptimal method of DNA delivery. The construction of the CVI988 BAC is a major step towards understanding the superior immunogenic features of CVI988 and provides the opportunity to exploit the power of BAC technology for generation of novel molecularly defined vaccines.