Core-coat conductor of lipid bilayer and micromachined silicon

We have etched a groove into a (110) plane of silicon and have covered it with a bilayer of glycerol monooleate. We have varied the depth of the groove, the concentration of salt in the electrolyte and the density of gramicidin in the membrane. We have clamped one end of the groove at a constant voltage with respect to the bath keeping the other end sealed or electrically open with respect to the bath. We have measured (i) the voltage at the center of the groove and at the sealed distal end and (ii) the current through the system in sealed and open configuration. We have found that the spread of voltage is in quantitative agreement with the stationary solutions of Kelvin's equation for a homogeneous cable.     

Replica plating of mammalian cells using low melt agarose

We have developed a technique to replica plate mammalian cells grown on plastic dishes using low melt agarose. This method is simpler than previously described methods that use polyester membranes to grow and transfer cells. We have tested the effectiveness of this technique on normal and immortal cell lines and have found that we can transfer cells with an efficiency of 80–90%. We have used this technique to rapidly screen clones for insertion of a lentivirally encoded gene without a selectable marker.     

Stiff polymer adsorption. Onset to pattern recognition

We present the results of extensive off-lattice Monte-Carlo simulations of a stiff polymer chain adsorbing onto a sticky periodic stripe-like pattern of variable width. We have analyzed, in terms of the chain length and rigidity, the adsorption and the pattern recognition process as a function of the stripe width. We have seen that this process is twofold: (i) the chain adsorbs rather isotropically onto the surface at a characteristic temperature T(c) and (ii) a further reduction in the temperature is needed for the chain to reorganize and adjust to the specific pattern. Such polymer reorganization has been studied through the evaluation of the chain degree of stretching and asphericity. We have found an optimal stripe width that maximizes the stretching. We have introduced a criteria to estimate the characteristic temperature at which the pattern recognition takes place T(r)相似文献   

Amino acid compositions and evolutionary relationships with protein families.

We have examined the merits of the three functions based on amino acid compositions which have been proposed to indicate the similarity in amino acid sequences of two proteins; the difference index, the composition divergence and the composition coefficient. We have taken the amino acid compositions and sequences of 41 cytochrome c's and used the 820 values from all possible comparisons in the evaluation. We conclude that the functions do have a limited value in predicting proteins which are closely related in sequence and that the three functions are equivalent in this predictive ability. We have used the composition divergence values obtained from available pyruvate kinase amino acid compositions to generate a phylogenetic tree for this glycolytic enzyme.     

Analysis of the junction between ribosomal RNA genes and single-copy chromosomal sequences in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has a single tandem array of 100 ribosomal RNA (rRNA) genes. We have cloned and characterized a junction between the centromere-distal end of this array and the adjacent single-copy chromosomal sequences. We have shown that the junction occurs within the nontranscribed region of the repeat. By mapping the junction, we have found that the 35S rRNA precursor is transcribed toward the centromere while the 5S rRNA is transcribed away from the centromere. We have also shown that different yeast strains can have different single-copy sequence at the junction.     

Active plant growth at freezing temperatures

As a foundation for speculations in exobiology, we are attempting to understand plant responses to extreme environments on Earth. We have emphasized active plant growth at low temperatures and in response to ultraviolet light. We have studied the winter environment in the mountains near Logan, Utah and have found several plants that grow under the snow. We have measured chlorophyll synthesis, carbohydrate levels, and ion balances in these plants and established field experiments with hardy and nonhardy varieties of wheat. In the laboratory we have studied characteristics of three enzymes in two wheat varieties, finding a number of interesting differences in response to ultraviolet and low-temperature treatments. We have also examined cell ultrastructures of three grass species subjected to a range of temperatures. Chloroplasts were most affected at low temperatures, but other organelles were also influenced. Studies of ion balances substantiate the suggestion from ultrastructure work that membranes may exhibit the primary responses to low temperatures. Cytokinins are also implicated in the cold response. We are presently emphasizing the investigation of membranes. Lunar Science Institute Contribution.     

Prelife catalysts and replicators

Life is based on replication and evolution. But replication cannot be taken for granted. We must ask what there was prior to replication and evolution. How does evolution begin? We have proposed prelife as a generative system that produces information and diversity in the absence of replication. We model prelife as a binary soup of active monomers that form random polymers. ‘Prevolutionary’ dynamics can have mutation and selection prior to replication. Some sequences might have catalytic activity, thereby enhancing the rates of certain prelife reactions. We study the selection criteria for these prelife catalysts. Their catalytic efficiency must be above certain critical values. We find a maintenance threshold and an initiation threshold. The former is a linear function of sequence length, and the latter is an exponential function of sequence length. Therefore, it is extremely hard to select for prelife catalysts that have long sequences. We compare prelife catalysis with a simple model for replication. Assuming fast template-based elongation reactions, we can show that replicators have selection thresholds that are independent of their sequence length. Our calculation demonstrates the efficiency of replication and provides an explanation of why replication was selected over other forms of prelife catalysis.     

Isolation of Mutations Affecting Neural Circuitry Required for Grooming Behavior in Drosophila Melanogaster

We have developed a screen for the isolation of mutations that produce neural defects in adult Drosophila melanogaster. In this screen, we identify mutants as flies unable to remove a light coating of applied dust in a 2-hr period. We have recovered and characterized six mutations and have found that they produce coordination defects and some have reduced levels of reflex responsiveness to the stimulation of single tactile sensory bristles. The grooming defects produced by all six of the mutations are recessive, and each of the mutations has been genetically mapped. We have also used our assay to test the grooming ability of stocks containing mutations that produce known neural defects.     

Cloning of the riboB locus of Aspergillus nidulans

We have complemented the riboB2 mutation of Aspergillus nidulans by transformation with a plasmid library of wild-type (wt) sequences. We have isolated, by marker rescue from a riboB+ transformant, a plasmid that complements riboB2 efficiently. From this plasmid we have subcloned an A. nidulans sequence that complements riboB2 efficiently and that integrates by homologous recombination at a site closely linked to the riboB locus. We conclude that this sequence contains the wt riboB+ allele.     

A prokaryotic alternative oxidase present in the bacterium Novosphingobium aromaticivorans

The alternative oxidase (AOX) is a terminal oxidase present in the respiratory chain of all plants as well as some yeasts and trypanosomes, but has not previously been found in a prokaryote. We have identified an AOX homologue in Novosphingobium aromaticivorans, the first AOX found in a prokaryote. We have cloned the gene for the N. aromaticivorans AOX and showed it to have a terminal oxidase activity when heterologously expressed in Escherichia coli. We have also shown that this novel AOX is expressed in N. aromaticivorans cells, and that its expression level is greatly influenced by the oxygen level and carbon source of the growth media.     

Arrangement of the genes coding for ribosomal ribonucleic acids in Neurospora crassa.

We have cloned and characterized Neurospora crassa ribosomal deoxyribonucleic acid (rDNA). The rDNA is found as a tandemly repeated 6.0-megadalton sequence. We have mapped a portion of the rDNA repeat unit with respect to its sites for 13 restriction endonucleases and defined those regions coding for the 5. 8S, 17S, and 26S ribosomal ribonucleic acids (rRNA's). We have also isolated several clones containing 5S rRNA sequences. The 5S rRNA coding sequences are not found within the rDNA repeat unit. We found that the sequences surrounding the 5S rRNA coding regions are highly heterogeneous.     

Hydration of DNA bases: analysis of crystallographic data.

We present a systematic analysis of water structure around nucleic acid bases. We have examined 28 crystal structures of oligonucleotides, and have studied the patterns of water around the four bases, guanine, cytosine, adenine, and thymine. The geometries of water positions were calculated up to 4.00 A from base atoms. We have found conformation-dependent differences in both the geometry and extent of hydration of the bases.     

Characterization of Ngef, a novel member of the Dbl family of genes expressed predominantly in the caudate nucleus

We have identified Ngef as a novel member of the family of Dbl genes. Many members of this family have been shown to function as guanine nucleotide exchange factors for the Rho-type GTPases. Ngef is predominantly expressed in brain, with the strongest signal in the caudate nucleus, a region associated with the control of movement. Ngef contains a translated trinucleotide repeat, a polyglutamic acid stretch interrupted by a glycine. We have localized the Ngef gene to mouse chromosome 1 and the human homologue of Ngef to human chromosome 2q37. We have shown in preliminary experiments that Ngef has transforming potential in cell culture and is able to induce tumors in nude mice.     

Developmental changes in upper airway dynamics.

Normal children have a less collapsible upper airway in response to subatmospheric pressure administration (P(NEG)) during sleep than normal adults do, and this upper airway response appears to be modulated by the central ventilatory drive. Children have a greater ventilatory drive than adults. We, therefore, hypothesized that children have increased neuromotor activation of their pharyngeal airway during sleep compared with adults. As infants have few obstructive apneas during sleep, we hypothesized that infants would have an upper airway that was resistant to collapse. We, therefore, compared the upper airway pressure-flow (V) relationship during sleep between normal infants, prepubertal children, and adults. We evaluated the upper airway response to 1). intermittent, acute P(NEG) (infants, children, and adults), and 2). hypercapnia (children and adults). We found that adults had a more collapsible upper airway during sleep than either infants or children. The children exhibited a vigorous response to both P(NEG) and hypercapnia during sleep (P < 0.01), whereas adults had no significant change. Infants had an airway that was resistant to collapse and showed a very rapid response to P(NEG). We conclude that the upper airway is resistant to collapse during sleep in infants and children. Normal children have preservation of upper airway responses to P(NEG) and hypercapnia during sleep, whereas responses are diminished in adults. Infants appear to have a different pattern of upper airway activation than older children. We speculate that the pharyngeal airway responses present in normal children are a compensatory response for a relatively narrow upper airway.     

The Opisthokonta and the Ecdysozoa may not be clades: stronger support for the grouping of plant and animal than for animal and fungi and stronger support for the Coelomata than Ecdysozoa

In considering the best possible solutions for answering phylogenetic questions from genomic sequences, we have chosen a strategy that we suggest is superior to others that have gone previously. We have ignored multigene families and instead have used single-gene families. This minimizes the inadvertent analysis of paralogs. We have employed strict data controls and have reasoned that if a protein is not capable of recovering the uncontroversial parts of a phylogenetic tree, then why should we use it for the more controversial parts? We have sliced and diced the data in as many ways as possible in order to uncover the signals in that data. Using this strategy, we have tested two controversial hypotheses concerning eukaryotic phylogenetic relationships: the placement of arthropoda and nematodes and the relationships of animals, plants, and fungi. We have constructed phylogenetic trees from 780 single-gene families from 10 completed genomes and amalgamated these into a single supertree. We have also carried out a total evidence analysis on the only universally distributed protein families that can accurately reconstruct the uncontroversial parts of the phylogenetic tree: a total of five families. In doing so, we ignore the majority of single-gene families that are universally distributed as they do not have the appropriate signals to recover the uncontroversial parts of the tree. We have also ignored every protein that has ever been used previously to address this issue, simply because none of them meet our strict criteria. Using these data controls, site stripping, and multiple analyses, 24 out of 26 analyses strongly support the grouping of vertebrates with arthropods (Coelomata hypothesis) and plants with animals. In the other two analyses, the data were ambivalent. The latter finding overturns an 11-year theory of Eukaryotic evolution; the first confirms what has already been said by others. In the light of this new tree, we re-analyze the evolution of intron gain and loss in the rpL14 gene and find that it is much more compatible with the hypothesis presented here than with the Opisthokonta hypothesis.     

Incorporation of flexibility into rigid-body docking: applications in rounds 3-5 of CAPRI

We have submitted models for all 9 targets in Rounds 3-5 of CAPRI and have predicted at least 30% of the correct contacts for 4 of the targets and at least 10% of the correct contacts for another 4 targets. We have employed a variety of techniques but have had the greatest success by combining established rigid-body docking with a variety of initial conformations generated by molecular dynamics.     

Native quaternary structure of bovine alpha-crystallin

Alpha-crystallin is the most important soluble protein in the eye lens. It is responsible for creating a high refractive index and is known to be a small heat-shock protein. We have used static and dynamic light scattering to study its quaternary structure as a function of isolation conditions, temperature, time, and concentration. We have used tryptophan fluorescence to study the temperature dependence of the tertiary structure and its reversibility. Gel filtration, analytical ultracentrifugation, polyacrylamide gel electrophoretic analysis, and absorption measurements were used to study the chaperone-like activity of alpha-crystallin in the presence of destabilized lysozyme. We have demonstrated that the molecular mass of the in vivo alpha-crystallin oligomer is about 700 kDa (alpha(native)) while the 550 kDa molecule (alpha(37 degrees C),diluted), which is often found in vitro, is a product of prolonged storage at 37 degrees C of low concentrated alpha-crystallin solutions. We have proven that the molecular mass of the alpha-crystallin oligomer is concentration dependent at 37 degrees C. We have found strong indications that, during chaperoning, the alpha-crystallin oligomer undergoes a drastic rearrangement of its peptides during the process of complex formation with destabilized lysozyme. We propose the hypothesis that all these processes are governed by the phenomenon of subunit exchange, which is well-known to be strongly temperature-dependent.     

Molecular mechanism underlying impaired platelet responsiveness in liver cirrhosis

We have studied platelet function in 10 patients with severe liver cirrhosis, compared to healthy subjects. Using washed platelets, we have investigated the molecular mechanism underlying the defect in platelet aggregation frequently observed in these patients. We have found that platelets from cirrhotic patients have a reduced responsiveness to thrombin and collagen in terms of aggregation, and receptor-dependent activation of phospholipase C, A2 and cyclooxygenase/thromboxane synthetase. We thus suggest that this impairment in transmembrane signalling is responsible for the defective platelet function observed in cirrhosis.     

Identification of microRNAs as potential prognostic markers in ependymoma

Introduction

We have examined expression of microRNAs (miRNAs) in ependymomas to identify molecular markers of value for clinical management. miRNAs are non-coding RNAs that can block mRNA translation and affect mRNA stability. Changes in the expression of miRNAs have been correlated with many human cancers.

Materials and Methods

We have utilized TaqMan Low Density Arrays to evaluate the expression of 365 miRNAs in ependymomas and normal brain tissue. We first demonstrated the similarity of expression profiles of paired frozen tissue (FT) and paraffin-embedded specimens (FFPE). We compared the miRNA expression profiles of 34 FFPE ependymoma samples with 8 microdissected normal brain tissue specimens enriched for ependymal cells. miRNA expression profiles were then correlated with tumor location, histology and other clinicopathological features.

Results

We have identified miRNAs that are over-expressed in ependymomas, such as miR-135a and miR-17-5p, and down-regulated, such as miR-383 and miR-485-5p. We have also uncovered associations between expression of specific miRNAs which portend a worse prognosis. For example, we have identified a cluster of miRNAs on human chromosome 14q32 that is associated with time to relapse. We also found that miR-203 is an independent marker for relapse compared to the parameters that are currently used. Additionally, we have identified three miRNAs (let-7d, miR-596 and miR-367) that strongly correlate to overall survival.

Conclusion

We have identified miRNAs that are differentially expressed in ependymomas compared with normal ependymal tissue. We have also uncovered significant associations of miRNAs with clinical behavior. This is the first report of clinically relevant miRNAs in ependymomas.