Proteomic study of a model causative agent of harmful red tide,Prorocentrum triestinum I: Optimization of sample preparation methodologies for analyzing with two-dimensional electrophoresis

A comprehensive study to find the optimal sample preparation conditions for two-dimensional electrophoresis (2-DE) analysis of Prorocentrum triestinum, a model causative agent of harmful algal blooms (HABs) was carried out. The four major sample preparation steps for 2-DE: (a) cell disruption: i.e. sonication and homogenization with glass beads; (b) protein extraction : i.e. sequential and independent extraction procedures; (c) pre-electrophoretic treatment: these included (i) treatment with RNAase/DNAase or benzonase; (ii) ultracentrifugation to sediment large macromolecules such as DNA; (iii) desalting and concentration by ultrafiltration through a Microcon centrifugal filter device (MWCO: 3000 daltons); and (iv) desalting by a micro BioSpin chromatography column (MWCO: 6000 daltons); and (d) rehydration buffers, reducing agents and sample application in the first dimension isoelectric focussing were studied. Our results showed that sonication is easy to perform and resulted in a higher protein yield. Among the four extraction buffers, the urea containing buffers resulted in the extraction of the highest amount of protein while tris(hydroxymethyl)aminomethane buffers and trichloroacetic acid (TCA)/acetone precipitation allowed detection of a higher number of protein species (i.e. protein spots). Desalting by BioSpin and ultrafiltration have improved the 2-DE resolution of the water soluble fraction but have less effect on urea containing fractions. TCA/acetone precipitation was able to desalt all protein fractions independent of the extraction media, however extended exposure to this low pH medium has caused protein modification. Introduction of either DNase/RNase or benzonase treatment did not improve the discriminatory power of the 2-DE but this treatment did yield 2-DE with the clearest background. Proteolytic digestion was inhibited by addition of a protease inhibitor cocktail. Taken overall, a combination of sequential extraction and desalting by BioSpin chromatography for sample treatment before first dimension of 2-DE gave best results based on its simplicity and minimal protein loss. Finally, triscarboxyethylphosphine (TCEP) has performed well as a reducing agent in both the rehydration and equilibration buffers. The rehydration buffer found to be best in this study was 8.0 M urea, 2% 3-[(3-cholamidoprphyldimethylamino]-1-propanesulfonate, 4 mM TCEP and 1% immobilized pH gradient buffer. Subsequently, we applied this finding and performed 2-DE analysis on the soluble protein fractions extracted from light-starved cultured algal cells (nonblooming) and cultured cells grown under optimal conditions (blooming). 2-DE maps of these algal cultures were visibly different and many differentially expressed proteins were found.     

The influence of sampling method on the classification of wetland macroinvertebrate communities

Macroinvertebrate communities sampled by a corer, plankton net and sweep net from five wetlands on the Swan Coastal Plain were compared. The composition of the fauna collected in sweeps and tows was generally similar and differed from that collected in the cores. Cores caught fewer species than tows and sweeps at all wetlands and did not capture fast swimming hemipterans or less abundant taxa. The highest species richness was recorded in sweep samples in four out of the five wetlands. Classification (TWIN-SPAN) and ordination (SSH) of the samples collected in sweeps and tows gave good separation of the wetlands, whereas classification of core samples did not. Coring appeared to be the least suitable sampling method for describing the major components of the macroinvertebrate communities of these wetlands. Plankton tows were useful if the time available for sorting was limited as these samples were free of sediments and generally gave similar results to those obtained with sweeps. Sweeps appeared to be the most useful method for a large classification study as they collected more species and resulted in the best discrimination amongst wetlands.     

Establishing the trochlear motor axon trajectory: role of the isthmic organiser and Fgf8

Formation of the trochlear nerve within the anterior hindbrain provides a model system to study a simple axonal projection within the vertebrate central nervous system. We show that trochlear motor neurons are born within the isthmic organiser and also immediately posterior to it in anterior rhombomere 1. Axons of the most anterior cells follow a dorsal projection, which circumnavigates the isthmus, while those of more posterior trochlear neurons project anterodorsally to enter the isthmus. Once within the isthmus, axons form large fascicles that extend to a dorsal exit point. We investigated the possibility that the projection of trochlear axons towards the isthmus and their subsequent growth within that tissue might depend upon chemoattraction. We demonstrate that both isthmic tissue and Fgf8 protein are attractants for trochlear axons in vitro, while ectopic Fgf8 causes turning of these axons away from their normal routes in vivo. Both inhibition of FGF receptor activation and inhibition of Fgf8 function in vitro affect formation of the trochlear projection within explants in a manner consistent with a guidance function of Fgf8 during trochlear axon navigation.     

Incorporation of3H-thymidine into DNA and the activity of alkaline phosphatase in zinc-deficient fetal rat brains

The incorporation of³H-thymidine into DNA in the brains of the 17-day and 20-day old rat fetuses was significantly reduced by maternal zinc restriction during pregnancy. The activity of the enzyme thymidine kinase (EC 2.7.1.21) was similarly reduced in the zine-deprived fetal brains on days 14 and 20 of gestation, but not on day 17. Fetal brain alkaline phosphatase (EC 3.1.3.1) was significantly depressed by maternal zinc deprivation on days 17 and 20 of pregnancy. The data suggest an association between thymidine kinase and the reduced incorporation of³H-thymidine into DNA in the brains of 20-day old fetuses but not in animals on day 17. Alkaline phosphatase was however depressed at this stage. The suggestion is made that because of the complexity of brain development, future biochemical studies in this area should concern specific structures in the brain at particular critical stages during neurogenesis.     

Comprehensive DNA signature discovery and validation

DNA signatures are nucleotide sequences that can be used to detect the presence of an organism and to distinguish that organism from all other species. Here we describe Insignia, a new, comprehensive system for the rapid identification of signatures in the genomes of bacteria and viruses. With the availability of hundreds of complete bacterial and viral genome sequences, it is now possible to use computational methods to identify signature sequences in all of these species, and to use these signatures as the basis for diagnostic assays to detect and genotype microbes in both environmental and clinical samples. The success of such assays critically depends on the methods used to identify signatures that properly differentiate between the target genomes and the sample background. We have used Insignia to compute accurate signatures for most bacterial genomes and made them available through our Web site. A sample of these signatures has been successfully tested on a set of 46 Vibrio cholerae strains, and the results indicate that the signatures are highly sensitive for detection as well as specific for discrimination between these strains and their near relatives. Our approach, whereby the entire genomic complement of organisms are compared to identify probe targets, is a promising method for diagnostic assay development, and it provides assay designers with the flexibility to choose probes from the most relevant genes or genomic regions. The Insignia system is freely accessible via a Web interface and has been released as open source software at: http://insignia.cbcb.umd.edu.     

Mitochondrial ribosomes in a trypanosome

The nature, and even the existence, of trypanosome mitochondrial ribosomes has been the subject of some debate. We investigated this further in the insect trypanosome, Crithidia fasciculata. In sucrose gradients of parasite lysates, mitochondrial ribosomal RNA co-sediments at approximately 35S with nascent peptides synthesized in the presence of the cytosolic translational inhibitor, cycloheximide. Co-sedimenting peptides in this peak are much reduced when the parasites are treated with the bacterial translational inhibitor, chloramphenicol. In CsCl gradients this peak resolves at a buoyant density of 1.42 g/cm(3), a value typical for mito-ribosomes. Electron microscopy of peak material shows particles smaller than cytosolic ribosomes, but with characteristic ribosomal shapes. We propose that these particles represent the parasite's mitochondrial ribosomes.     

Vindija cave and the modern human peopling of Europe

Vindija cave in Croatia has yielded the youngest securely dated Neandertal skeletal remains in Central/Eastern Europe. In addition, these remains have been found in association with archaeological material exhibiting Upper Paleolithic elements. Due to its geographic location and date, the Vindija remains are particularly crucial for the understanding of initial modern human peopling of Europe and the nature of the Neandertal demise. The significance of archaeological and paleontological finds and hominin fossils from this site is discussed in the light of new finds at Vindija and recent developments in the fields of paleoanthropology and prehistoric archaeology. Furthermore, the impact of revised chronology for several crucial specimens and sites throughout Europe, including Vindija, is discussed.