Proteomic analysis of the eyespot of Chlamydomonas reinhardtii provides novel insights into its components and tactic movements

Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. To further understand the molecular organization of the eyespot apparatus and the phototactic movement that is controlled by light and the circadian clock, a detailed understanding of all components of the eyespot apparatus is needed. We developed a procedure to purify the eyespot apparatus from the green model alga Chlamydomonas reinhardtii. Its proteomic analysis resulted in the identification of 202 different proteins with at least two different peptides (984 in total). These data provide new insights into structural components of the eyespot apparatus, photoreceptors, retina(l)-related proteins, members of putative signaling pathways for phototaxis and chemotaxis, and metabolic pathways within an algal visual system. In addition, we have performed a functional analysis of one of the identified putative components of the phototactic signaling pathway, casein kinase 1 (CK1). CK1 is also present in the flagella and thus is a promising candidate for controlling behavioral responses to light. We demonstrate that silencing CK1 by RNA interference reduces its level in both flagella and eyespot. In addition, we show that silencing of CK1 results in severe disturbances in hatching, flagellum formation, and circadian control of phototaxis.     

Enhancer detection in the zebrafish using pseudotyped murine retroviruses

Vectors based on murine retroviruses are among the most efficient means to insert reporter constructs into the context of a vertebrate chromosome with the aim to visualize cis-regulatory information available to a basal promoter at the site of insertion. In combination with using the zebrafish embryo as a readout for the activity of regulatory elements, enhancer detection becomes a powerful technique for gene discovery and for the mapping of the extent of regulatory domains in a vertebrate genome. Our laboratory has performed the only large-scale enhancer detection screen to date in any vertebrate and we describe in this paper the methods we developed to generate viral particles, to insert reporter constructs into the zebrafish germ line, the screening of detection events in heterozygous F1 embryos, and the isolation of genomic sequence flanking the inserted vector for the purpose of genomic mapping. Given sufficient scale, the technology described here can be used to obtain cis-regulatory information across the entire zebrafish genome for any given basal promoter.     

In vitro and in vivo complementation of the Helicobacter pylori arginase mutant using an intergenic chromosomal site

BACKGROUND: Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects. MATERIALS AND METHODS: A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203-hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF). RESULTS: A rocF mutant unable to hydrolyze or consume l-arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3' ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice. CONCLUSIONS: This complementation strategy should greatly facilitate genetic experiments in H. pylori.     

Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72

Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agrobiotechnological interest because it supplies biologically fixed nitrogen to its host and colonizes plants in remarkably high numbers without eliciting disease symptoms. The complete genome sequence is 4,376,040-bp long and contains 3,992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. Coding sequences involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant-associated bacteria. Strain BH72 appears to be 'disarmed' compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactones-based communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment.     

Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems

Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.     

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.