Characterization of transducin from bovine retinal rod outer segments. Participation of the amino-terminal region of T alpha in subunit interaction

The GTP-induced dissociation of T alpha from T beta gamma initiates the release of transducin from photolyzed rhodopsin and the subsequent activation of the cGMP phosphodiesterase. In this study, site-specific proteolysis and immunoprecipitation were used to map the domain of T alpha that interacts with T beta gamma. We found that Staphylococcus aureus V8 protease rapidly removes a small fragment from T alpha under native conditions, resulting in the formation of a single 38-kDa polypeptide (T alpha'). Under the same conditions, T beta gamma remains intact. A 4.5-fold decrease in the rate of T alpha cleavage by S. aureus protease was observed in the presence of T beta gamma, suggesting T beta gamma binding blocks the protease-sensitive site on T alpha. Amino acid sequence analysis indicated that T alpha' is derived from the cleavage of T alpha at Glu-21. The ability of T alpha' to interact with and activate the retinal phosphodiesterase is not diminished. However, T alpha' is unable to participate in T beta gamma-dependent activities such as the light-stimulated binding of guanine nucleotides, binding to photoexcited rhodopsin, and ADP-ribosylation catalyzed by pertussis toxin. Moreover, the anti-T alpha monoclonal antibody TF16 was able to precipitate T beta gamma in the presence of T alpha, but not with either T alpha' or T alpha-guanosine 5'-O-(3-thiotriphosphate). We conclude that the amino-terminal region of T alpha participates in T beta gamma interaction and discuss our results with respect to the known structure and function of transducin.     

The determination of intracellular water space by NMR

A new method for the determination of intracellular water space using NMR spectroscopy is described. The method is based on the measurement of 59Co NMR signal intensity of an inert, stable and membrane-impermeable cobalt(III) compound such as Co(CN)3-6 or Co(imidazole)3+6 and the 2H or 1H NMR signal intensities of the freely permeable water. As an example of the method, the variation of the intracellular water space of human erythrocytes as a function of osmolality was measured.     

Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae.

High-resolution phosphorus-31 nuclear magnetic resonance (31P NMR) spectra of wild-type and mutant strains of Saccharomyces cerevisiae were observed at a frequency of 145.7 MHz. Levels of various phosphorus metabolites were investigated upon addition of glucose under both aerobic and anaerobic conditions. Three mutant strains were isolated and their biochemical defects characterized: pfk lacked phosphofructokinase activity; pgi lacked phosphoglucose isomerase activity; and cif had no glucose catabolite repression of the fructose bisphosphatase activity. Each mutant strain was found to accumulate characteristic sugar phosphates when glucose was added to the cell suspension. In the case of the phosphofructokinase deficient mutant, the appearance of a pentose shunt metabolite was observed. 31P NMR peak assignments were made by a pH titration of the acid extract of the cells. Separate signals for terminal, penultimate, and central phosphorus atoms in intracellular polyphosphates allowed the estimation of their average molecular weight. Signals for glycero(3)phosphochline, glycero(3)phosphoserine, and glycero(3) phosphoethanolamine as well as three types of nucleotide diphosphate sugars could be observed. The intracellular pH in resting and anaerobic cells was in the range 6.5--6.8 and the level of adenosine 5'-triphosphate (ATP) low. Upon introduction of oxygen, the ATP level increased considerably and the intracellular pH reached a value of pH 7.2--7.3, irrespective of the external medium pH, indicating active proton transport in these cells. A new peak representing the inorganic phosphate of one of the cellular organelles, whose pH differed from the cytoplasmic pH, could be detected under appropriate conditions.     

Enhancing single-molecule fluorescence with nanophotonics

Single-molecule fluorescence spectroscopy has become an important research tool in the life sciences but a number of limitations hinder the widespread use as a standard technique. The limited dynamic concentration range is one of the major hurdles. Recent developments in the nanophotonic field promise to alleviate these restrictions to an extent that even low affinity biomolecular interactions can be studied. After motivating the need for nanophotonics we introduce the basic concepts of nanophotonic devices such as zero mode waveguides and nanoantennas. We highlight current applications and the future potential of nanophotonic approaches when combined with biological systems and single-molecule spectroscopy.     

High-school students in university research labs? Implementing an outreach model based on the ‘science as inquiry’ approach

In this study we designed, implemented, and evaluated an outreach programme for high-school biology students rooted in the ‘science as inquiry’ approach. Accordingly, students learn about science from experts in the field, as well as through in-class exposure to the history and philosophy of science. Our sample consisted of 11th graders (n?=?497), ages 16–17, attending advanced biology classes. Our goal was to determine whether this programme had a significant effect on students’ understanding of the ‘nature of science’ (NOS) and on their attitudes towards science. Using a controlled pre-post research design, we asked participants to complete a Likert-like questionnaire. Also, we conducted post-programme semi-structured interviews with 35 of the participants. Results show that completion of the programme significantly enhanced participants’ NOS understanding and improved their attitudes towards science. Participants expressed a deep level of NOS understanding and explicitly stated that the field visits to experts’ labs had changed their attitude towards science. We believe that our outreach programme can be adapted for teaching other sciences and for societies worldwide, as long as there is access to university laboratories and researchers willing to interact with young citizens and potential future scientists.     

Mechanisms of interrelations in a system Globodera rostochiensis ‐ host plant”;

In researching the function of the system of Globodera rostochiensis resistant plants, we observed the development of G. rostochiensis (pathotype Ro 1) in glasshouse experiments. For a period of two months, we determined the qualities (the presence of juveniles stages) and quantities (the ratio of ages and the duration of periods of development of the juveniles stages) of parameters of nematodes development on potato wild and cultured species as well as hybrids. Comparative analysis of the rate and specifics of ontogenetic changes in G. rostochensis (Ro 1) during parasitism in the boundaries of specific plant groups, allowed us to assert the following types of resistance to G. rostochiensis: antixenosis, antibiosis, hypersensitive response.     

Spectroscopic and biochemical analysis of regions of the cell wall of the unicellular 'mannan weed', Acetabularia acetabulum

Although the Dasycladalean alga Acetabularia acetabulum has long been known to contain mannan-rich walls, it is not known to what extent wall composition varies as a function of the elaborate cellular differentiation of this cell, nor has it been determined what other polysaccharides accompany the mannans. Cell walls were prepared from rhizoids, stalks, hairs, hair scars, apical septa, gametophores and gametangia, subjected to nuclear magnetic resonance and Fourier transform infrared spectroscopy, and analyzed for monosaccharide composition and linkage, although material limitations prevented some cell regions from being analyzed by some of the methods. In diplophase, walls contain a para-crystalline mannan, with other polysaccharides accounting for 10-20% of the wall mass; in haplophase, gametangia have a cellulosic wall, with mannans and other polymers representing about a quarter of the mass. In the walls of the diplophase, the mannan appears less crystalline than typical of cellulose. The walls of both diploid and haploid phases contain little if any xyloglucan or pectic polysaccharides, but appear to contain small amounts of a homorhamnan, galactomannans and glucogalactomannans, and branched xylans. These ancillary polysaccharides are approximately as abundant in the cellulose-rich gametangia as in the mannan-rich diplophase. In the diplophase, different regions of the cell differ modestly but reproducibly in the composition of the cell wall. These results suggest unique cell wall architecture for the mannan-rich cell walls of the Dasycladales.