Development of a novel technique for axenic isolation and culture of thraustochytrids from New Zealand marine environments

Aims: To maintain axenic cultures of commercially important thraustochytrids, a novel procedure was developed for the isolation of zoospores and sporangium from heterotrophic seawater samples and axenic culture on solid media. Methods and Results: Thraustochytrid cultures were isolated from Whangapoua Harbour in North East New Zealand and subjected to two antibiotic and antifungal treatment regimes designed to eliminate bacteria and fungi. Antibiotic trial 1 was designed to determine the appropriate combination of antibiotics (including streptomycin/penicillin, ampicillin, rifampicin, nalidixic acid, tetracycline, gentamicin and the antifungal agent nystatin). Antibiotic trial 2 determined the optimal dosing frequency and concentration of the antibiotics, and antifungal found to be the most promising in trial 1. Axenic cultures were then spread plated onto nutrient agar containing the optimal antibiotic cocktail, and pure thraustochytrid colonies were purified on solid media using standard microbiological techniques. Conclusions: Removal of bacteria and fungi was best accomplished using a mixture of three antibiotics and one antifungal; rifampicin (300 mg l^?1), streptomycin/penicillin (25 mg l^?1) and nystatin (10 mg l^?1) were incorporated in seawater samples and incorporated into cultures every 24 h for a minimum of 2 days. Significance and Impact of the Study: The axenic isolation and culture of marine thraustochytrids from a marine habitat in New Zealand have significant implications for the biotechnological development of these potentially valuable protists. This method has global significance as it is reasonable to assume it could be used throughout the world to obtain axenic thraustochytrid cultures.     

Structure of the yeast vacuolar ATPase

The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.     

Stoichiometry of the peripheral stalk subunits E and G of yeast V1-ATPase determined by mass spectrometry

The stoichiometry of yeast V(1)-ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V(1)-ATPase complex and each of the individual protein components, using native electrospray ionization-MS. The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 and 428,300 Da, respectively. Second, defined amounts of V(1)-ATPase purified from yeast grown on (14)N-containing medium were titrated with defined amounts of (15)N-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, (14)N- and (15)N-containing peptide pairs were used for quantification of subunit stoichiometry using matrix-assisted laser desorption/ionization-time of flight MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V(1)-ATPase is A(3)B(3)DE(3)FG(3)H.     

Soil Bacteria are Differentially Affected by the Resin of the Medicinal Plant Pseudognaphalium vira vira and Its Main Component Kaurenoic Acid

The diterpenoid kaurenoic acid is the main component of the resin from the medicinal plant Pseudognaphalium vira vira. As some diterpenoids have antimicrobial properties, the effect of this resin and the kaurenoic acid on soil bacteria was studied. The resin of P. vira vira and purified kaurenoic acid were two to four times more effective as antibacterial agents with Gram-positive than with Gram-negative soil isolates. The chemical stability of kaurenoic acid and the antibacterial activity of both the resin and the diterpenoid were studied in microcosms containing plant-associated soil. After 15 days of incubation, the diterpenoid was stable, as determined by ¹H nuclear magnetic resonance and thin-layer chromatography, and soil extracts still exhibited antibacterial activity. However, after 30 days of incubation, loss of antibacterial activity of soil extracts correlated with removal or chemical modification of kaurenoic acid. The effect of the resin or this diterpenoid on the soil bacteria community was analyzed by the terminal restriction fragment length polymorphisms technique. After 15 days of incubation, the resin and the pure compound caused significant changes in the soil bacterial community. The relative abundance of specific bacterial groups was differentially affected by the resin components, being the effects with the resin stronger than with the kaurenoic acid. After 30 days of incubation, these changes mostly reverted. These results indicate that a plant resin containing diterpenoid compounds plays a significant role controlling specific groups of microorganisms in the soil associated with the plant.     

Alternative translation initiation in rat brain yields K2P2.1 potassium channels permeable to sodium

K(2P) channels mediate potassium background currents essential to central nervous system function, controlling excitability by stabilizing membrane potential below firing threshold and expediting repolarization. Here, we show that alternative translation initiation (ATI) regulates function of K(2P)2.1 (TREK-1) via an unexpected strategy. Full-length K(2P)2.1 and an isoform lacking the first 56 residues of the intracellular N terminus (K(2P)2.1Delta1-56) are produced differentially in a regional and developmental manner in the rat central nervous system, the latter passing sodium under physiological conditions leading to membrane depolarization. Control of ion selectivity via ATI is proposed to be a natural, epigenetic mechanism for spatial and temporal regulation of neuronal excitability.     

Science and the Concept of Evolution: From the Big Bang to the Origin and Evolution of Life

The common thread of evolution runs through all science disciplines, and the concept of evolution enables students to better understand the nature of the universe and our origins. “Science and the Concept of Evolution” is one of two interdisciplinary science Core courses taken by Dowling College undergraduates as part of their General Education requirements. The course examines basic principles and methods of science by following the concept of evolution from the big bang to the origin and evolution of life. Case studies of leading scientists illustrate how their ideas developed and contributed to the evolution of our understanding of the world. Evidences for physical, chemical, and biological evolution are explored, and students learn to view the evolution of matter and of ideas as a natural process of change over space and time.     

Neutrale Mutationen und evolutionäre Fortentwicklung

Neutral mutation and evolutionary progress The process and causes of regressive evolution are still under debate. Contrary to DARWIN'S original assumption, Neo-Darwinian proponents make selection responsible for reduction. Biologically functionless structures like eye and pigmentation in cave animals deliver excellent material to study this problem. Comparison of regressive (eye, pigmentation, aggression, dorsal light reaction) and constructive traits (gustatory equipment, egg yolk content, feeding behavior) in epigean and cave fish (Astyanax fasciatus, Characidae) reveal a high variability of the regressive features in the cave forms. Contrary to this, the constructive traits are characterized by a low variability in epigean and cave fish. This difference is attributed to the lack of selection on regressive structures. The existence of an intermediate cave population between epigean and true cave fish of A. fasciatus makes possible the study of evolutionary rates. It is shown that the regressive traits do not evolve more quickly than the constructive ones do. On the contrary, constructive traits like egg yolk content are even more rapid because they are of great biological value in the cave biotope. Especially energy economy is claimed by Neo-Darwinists to play a decisive role as a selective force. Comparison of the development of epi- and hypogean larvae of A. fasciatus shows that the formation of a smaller and less differentiated eye in the cave specimens has no effect on body growth. Furthermore, even behavioral traits like aggressiveness, schooling, dorsal light reaction, or negative phototaxis, which all are not performed in darkness by the epigean ancestor, become genetically reduced in the cave fish. The principles of regressive evolution, loss of selection and increase in variability, play a central role in evolution in general. When biota with empty niches are colonized, stabilizing selection relaxes from the special adaptations to the niche inhabited before by the invading species. Variability may arise in these and is permitted as long as fitness is guaranteed. Such processes characterize adaptive radiation. Examples are given by the species flocks on isolated islands or in chemically abnormal lakes like those of the East African Rift Valley. Only secondarily, on the basis of the arisen variability, does directional selection promote the newly developing species into different niches. The loss of stabilizing selection is an important factor for the evolutionary process to be open for evolutionary progress.     

Resource availability and the trichome defenses of tomato plants

We conducted two experiments to determine how resource availability influenced allocation by tomato (Lycopersicon esculentum) to trichomes, and how different patterns of trichome allocation by plants grown in different resource environments might then influence the behavior of tobacco hornworm (Manduca sexta) caterpillars. In the first experiment we used high and low levels of light and water, and then, using scanning electron microscopy, determined trichome densities on the leaves and stems. We sampled leaves and stems at several places throughout the plant to determine whether there were within-plant differences in allocation to trichomes. The results of the first experiment showed that resource availability influenced allocation to trichome growth. Patterns in high and low-light supported both the growth-differentiation balance hypothesis (GDBH) and the carbon-nutrient balance hypothesis (CNBH). However, the GDBH was not supported by differences among water treatments. Contrary, to predictions of the GDBH, plants with intermediate growth did not have the highest trichome densities, and plants with similar growth differed in trichome density. Possible biological and artifactual explanations are discussed. The first experiment also showed that there was within-plant variation in allocation to trichomes, and that plant resource availability may influence within-plant variation in allocation to trichomes. In the second experiment, we grew plants in high and low-light, and then monitored the behavior of tobacco hornworms on the stems of these plants in the laboratory. This experiment demonstrated that the light environment that tomato plants were grown in influenced the resting behavior of caterpillars. Furthermore, it demonstrated that both glandular and non-glandular trichomes impeded caterpillars from searching for food. Overall, this study indicated that plant resource availability can influence allocation to trichome defenses, and that these differences may affect insect herbivores.