Phosphorus-based combinatorial libraries: use of amino acid derivatives as synthons.

Phosphorus has been used as a scaffold to prepare combinatorial libraries of phosphoramidates in which one of the diversity elements resulted from derivatives of amino acids. A small library was prepared for analytical and characterization purposes, followed by a larger library of approximately 8800 compounds. Libraries were assembled on solid supports using the conventional pool-and-divide method, followed by cleavage from the supports at the end of the synthesis. Mass spectrometry was used to confirm that library synthesis had been successful. Individual compounds were also prepared to study the stability of compounds of this type.

Laser operator training and competency assessment in the community hospital setting.

At the 279 bed Marymount community hospital, Cleveland, Ohio, the importance of providing a safe laser environment for patients as well as staff was identified. The decision was made to cross-train all full-time RNs, LPNs, and SAs to allow flexibility in staffing all laser procedures with a qualified operator. A competency based training program was implemented, utilizing a combination of techniques to assist the adult learner. Selected video tapes were used to teach laser physics and applications. Comprehension was assessed through written and oral quizzing. Individual time was spent familiarizing trainees with the equipment and accessories. Practice setups were performed until that individual reached a comfort level satisfactory to him/her and the laser coordinator. Actual experience in the various surgical specialties was dependent upon our case load. Initially very close supervision was provided. Each trainee was gradually given more autonomy as his/her skills improved and permitted to operate the laser independently after passing our competency assessment. During the first year, each operator was given competency assessment reviews at regular intervals. This approach was found to have both strengths and weaknesses and the program was modified accordingly.

Circulation of oligonucleotides by disulfide bridge formation.

An effective, convenient method for the circularization of oligonucleotides has been developed. This procedure involved preparation of an oligonucleotide with backbone-linked 5'- and 3'-terminal hexamethylenethiol groups, followed by oxidation of the thiol groups with air of oxygen to produce the corresponding circular sequence bridged via a bis(hexamethylene)-disulfide moiety. The method has been applied to the circularization of oligodeoxynucleotide sequences of varying lengths (5, 10, 15, 20, 30 and 40 bases), and the circularization process was highly efficient as shown by HPLC or gel electrophoresis of the crude reaction mixtures. Competing reactions such as dimerization were not significant except for the longer sequences (30 and 40 bases). The circularization of an eight base RNA sequence was also accomplished, as well as hexa-ethylene glycol bridged poly-T sequences capable of triplex formation.

Structural and functional characterization of potent antithrombotic oligonucleotides possessing both quadruplex and duplex motifs.

We report the results of a selection for single-stranded DNA oligonucleotide ligands to the serine protease thrombin using recently developed methods. This selection yielded a family of DNA sequences that conform to a consensus structure comprised of a unimolecular quadruplex motif and complementary flanking sequences capable of forming an additional Watson-Crick duplex motif. This novel quadruplex/duplex structure was not reported in a previous selection for DNA molecules which bind to thrombin [Bock et al. (1992) Nature 355, 564-566]. All quadruplex/duplex molecules tested bound to thrombin with higher affinity than quadruplex structures lacking the duplex structure. However, binding affinities did not always correlate with inhibitory potency since some molecules with high affinity were not potent inhibitors in vitro. 1H NMR spectroscopy studies demonstrated that the complementarity of bases in the duplex portion of a selected sequence allows it to form multimolecular structures. Constraining these molecules to the unimolecular quadruplex/duplex structure by bridging the 5' and 3' ends of the duplex motif with either triethylene glycol or disulfide bonds improved their thrombin inhibitory activity. All bridged quadruplex/duplex molecules were more potent inhibitors than molecules with only a quadruplex motif. Bridging the ends of these structures not only increased thrombin inhibition but also improved resistance to nucleases in serum more than 40-fold over the unbridged quadruplex. In addition, we have found that both the length and sequence of the duplex motif are important for inhibition.

Inhibition of adenosine kinase and adenosine uptake in guinea-pig CNS tissue by halogenated tubercidin analogues.

Two halogenated analogues of tubercidin (7-deazaadenosine) viz. 5-iodotubercidin and 5'-deoxy-5-iodotubercidin, previously were shown to be potent inhibitors of guinea-pig brain adenosine kinase activity and adenosine uptake in guinea-pig cerebral cortex slices. A further series of halogenated tubercidin analogues have been investigated; of the 9 compounds tested, 5'-deoxy-5-iodotubercidin was the most potent adenosine kinase inhibitor while 5-iodotubercidin was the most potent in inhibiting the facilitated uptake of adenosine. These compounds may be useful for elucidating the involvement of adenosine kinase in adenosine uptake, the maintenance of intracellular adenosine levels and in the neuromodulatory actions of adenosine in the CNS.

Stabilization of double-stranded oligonucleotides using backbone-linked disulfide bridges.

A convenient, practical route to the synthesis of disulfide-bridged oligonucleotides has been developed. Aliphatic linkers with terminal thiol groups have been attached to the phosphodiester backbones of partially or fully complementary oligonucleotide sequences and oxidized to yield covalently closed oligonucleotides with disulfide bridges. This procedure has been used to prepare a duplex with disulfide bridges at both ends and stem-loop sequences with single disulfide bridges. Oxidation of a self-complementary duplex possessing terminal thiol groups produced both hairpin and duplex structures with disulfide bridges, the relative proportions of each being dependent upon the reaction conditions. These bridged hairpin and duplex structures were shown to be interconvertible by reduction and re-oxidation. The melting profiles of disulfide-bridged oligonucleotides were compared with the same sequences without bridges and with sequences possessing triethylene glycol bridges, and in all cases the introduction of disulfide bridges resulted in a considerable increase in thermal stability. EcoRI endonuclease was capable of cleaving a disulfide-bridged duplex possessing a recognition site for this enzyme, thus supporting a lack of distortion of the recognition site. The disulfide bridges could be cleaved using a large excess of DTT to regenerate the corresponding sulfhydryl compounds. A study of the serum stabilities of disulfide-bridged oligonucleotides showed that the bridged duplexes were much more stable than their unmodified counterparts, whereas the rate of degradation of the stem-loop structures was more dependent upon the size of the loop than the presence or absence of the disulfide bridge. In summary, we have described a novel methodology, employing commercially available reagents, for the stabilization of oligonucleotide duplexes or stem-loop structures by disulfide bridge formation.

Oligonucleotides with novel, cationic backbone substituents: aminoethylphosphonates.

Oligonucleotide (2-aminoethyl)phosphonates in which the backbone consisted of isomerically pure, alternating (2-aminoethyl)-phosphonate and phosphodiester linkages have been prepared and characterized. One of these single isomer oligonucleotides (Rp) formed a more stable duplex with DNA or RNA than its corresponding natural counterpart. Hybrid stability was more pH-dependent, but less salt-dependent than a natural duplex. The specificity of hybridization was examined by hybridization of an oligonucleotide containing one (2-aminoethyl)phosphonate to oligonucleotides possessing mismatches in the region opposite to the aminoethyl group. In contrast to oligonucleotides containing (aminomethyl)-phosphonate linkages, oligonucleotide (2-aminoethyl)phosphonates were completely stable to hydrolysis in aqueous solution. These oligonucleotides were resistant to nuclease activity but did not induce RNase H mediated cleavage of a complementary RNA strand. Incubation in a serum-containing medium resulted in minimal degradation over 24 hours. Studies of cell uptake by flow cytometry and confocal microscopy demonstrated temperature dependent uptake and intracellular localization. (2-Aminoethyl)phosphonates represent a novel approach to the introduction of positive charges into the backbone of oligonucleotides.

(Aminomethyl)phosphonate derivatives of oligonucleotides.

Oligothymidylate (aminomethyl)phosphonates have been prepared, and their enzymatic and physicochemical properties have been studied. The individual isomers of the protected dimers have been separated, characterized, and incorporated into oligonucleotides in which the backbone consists of alternating (aminomethyl)phosphonate and phosphodiester linkages. One of these net neutral, single isomer oligonucleotides forms a duplex with its complementary sequence which is more stable than the corresponding natural counterpart, whereas the other isomer is considerably less stable. Specificity of hybridization is maintained, as determined by the reduction in melting temperature observed upon the introduction of mismatches into the complementary strand of the duplex. The (aminomethyl)-phosphonate linkage is stable toward enzymatic degradation but can be hydrolyzed in aqueous solution at elevated temperature.

Triton's Geyser-Like Plumes: Discovery and Basic Characterization.

At least four active geyser-like eruptions were discovered in Voyager 2 images of Triton, Neptune's large satellite. The two best documented eruptions occur as columns of dark material rising to an altitude of about 8 kilometers where dark clouds of material are left suspended to drift downwind over 100 kilometers. The radii of the rising columns appear to be in the range of several tens of meters to a kilometer. One model for the mechanism to drive the plumes involves heating of nitrogen ice in a subsurface greenhouse environment; nitrogen gas pressurized by the solar heating explosively vents to the surface carrying clouds of ice and dark partides into the atmosphere. A temperature increase of less than 4 kelvins above the ambient surface value of 38 +/- 3 kelvins is more than adequate to drive the plumes to an 8-kilometer altitude. The mass flux in the trailing clouds is estimated to consist of up to 10 kilograms of fine dark particles per second or twice as much nitrogen ice and perhaps several hundred or more kilograms of nitrogen gas per second. Each eruption may last a year or more, during which on the order of a tenth of a cubic kilometer of ice is sublimed.

Voyager 2 at neptune: imaging science results.

Voyager 2 images of Neptune reveal a windy planet characterized by bright clouds of methane ice suspended in an exceptionally clear atmosphere above a lower deck of hydrogen sulfide or ammonia ices. Neptune's atmosphere is dominated by a large anticyclonic storm system that has been named the Great Dark Spot (GDS). About the same size as Earth in extent, the GDS bears both many similarities and some differences to the Great Red Spot of Jupiter. Neptune's zonal wind profile is remarkably similar to that of Uranus. Neptune has three major rings at radii of 42,000, 53,000, and 63,000 kilometers. The outer ring contains three higher density arc-like segments that were apparently responsible for most of the ground-based occultation events observed during the current decade. Like the rings of Uranus, the Neptune rings are composed of very dark material; unlike that of Uranus, the Neptune system is very dusty. Six new regular satellites were found, with dark surfaces and radii ranging from 200 to 25 kilometers. All lie inside the orbit of Triton and the inner four are located within the ring system. Triton is seen to be a differentiated body, with a radius of 1350 kilometers and a density of 2.1 grams per cubic centimeter; it exhibits clear evidence of early episodes of surface melting. A now rigid crust of what is probably water ice is overlain with a brilliant coating of nitrogen frost, slightly darkened and reddened with organic polymer material. Streaks of organic polymer suggest seasonal winds strong enough to move particles of micrometer size or larger, once they become airborne. At least two active plumes were seen, carrying dark material 8 kilometers above the surface before being transported downstream by high level winds. The plumes may be driven by solar heating and the subsequent violent vaporization of subsurface nitrogen.

The relationship of the extrinsic blood supply to regeneration in graft reconstructed peripheral nerves.

Median nerves of rats were reconstructed with conventional or vascular nerve grafts. After 2, 3 and 4 weeks, Allen Video-Enhanced Contrast, Differential Interference Contrast (AVEC-DIC) microscopy revealed axonal transport in most preparations, with varying degrees of myelination. Radio-isotope tracer was measured in the nerve. Two and 4 week measurements revealed no difference between the graft types. At 3 weeks the vascular graft group exhibited transport along the entire length of the nerve in contrast to a relatively abbreviated path length in the conventional graft group. Nerve conduction velocities (NCV) were measured proximal to, within and distal to the grafts. Three week NCV showed no difference between the graft types. The 4 week NCV revealed normal values in the vascular graft group at points distal to and within the graft. Significant slowing was seen in the conventional grafts at both points.

Synthesis and hybridization of a series of biotinylated oligonucleotides.

A series of oligonucleotides containing biotin-11-dUMP at various positions were synthesized and compared in quantitative, colorimetric hybridization-detection studies. A deoxyuridine phosphoramidite containing a protected allylamino sidearm was synthesized and used in standard, automated synthesis cycles to prepare oligonucleotides with allylamino residues at various positions within a standard 17-base sequence. Biotin substituents were subsequently attached to the allylamino sidearms by reaction with N-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester. These oligomers were hybridized to target DNA immobilized on microtiter wells (ELISA plates), and were detected with a streptavidin-biotinylated horseradish peroxidase complex using hydrogen peroxide as substrate and o-phenylenediamine as chromogen. We found that the sensitivity of detection of target DNA by biotin-labeled oligonucleotide probes was strongly dependent upon the position of the biotin label. Oligonucleotides containing biotin labels near or off the ends of the hybridizing sequence were more effective probes than oligonucleotides containing internal biotin labels. An additive effect of increasing numbers of biotin-dUMP residues was found for some labeling configurations.

Voyager 2 in the uranian system: imaging science results.

Voyager 2 images of the southern hemisphere of Uranus indicate that submicrometersize haze particles and particles of a methane condensation cloud produce faint patterns in the atmosphere. The alignment of the cloud bands is similar to that of bands on Jupiter and Saturn, but the zonal winds are nearly opposite. At mid-latitudes (-70 degrees to -27 degrees ), where winds were measured, the atmosphere rotates faster than the magnetic field; however, the rotation rate of the atmosphere decreases toward the equator, so that the two probably corotate at about -20 degrees . Voyager images confirm the extremely low albedo of the ring particles. High phase angle images reveal on the order of 10(2) new ringlike features of very low optical depth and relatively high dust abundance interspersed within the main rings, as well as a broad, diffuse, low optical depth ring just inside the main rings system. Nine of the newly discovered small satellites (40 to 165 kilometers in diameter) orbit between the rings and Miranda; the tenth is within the ring system. Two of these small objects may gravitationally confine the e ring. Oberon and Umbriel have heavily cratered surfaces resembling the ancient cratered highlands of Earth's moon, although Umbriel is almost completely covered with uniform dark material, which perhaps indicates some ongoing process. Titania and Ariel show crater populations different from those on Oberon and Umbriel; these were probably generated by collisions with debris confined to their orbits. Titania and Ariel also show many extensional fault systems; Ariel shows strong evidence for the presence of extrusive material. About halfof Miranda's surface is relatively bland, old, cratered terrain. The remainder comprises three large regions of younger terrain, each rectangular to ovoid in plan, that display complex sets of parallel and intersecting scarps and ridges as well as numerous outcrops of bright and dark materials, perhaps suggesting some exotic composition.

Influences of catecholamines on growth hormone release in female goldfish, Carassius auratus.

The influence of catecholamines on growth hormone (GH) release in female goldfish was investigated by monitoring serum GH levels following injections of drugs known to alter catecholamine synthesis and neural activities. Intraperitoneal (i.p.) injection of 6-hydroxydopamine, a catecholaminergic neurotoxin, or alpha-methyl-p-tyrosine, a catecholamine synthesis inhibitor, decreased serum GH levels. Intraperitoneal injection of L-beta-dihydroxyphenylalanine (L-dopa) increased serum GH concentrations in a dose-dependent manner. The L-dopa-induced increase in serum GH was potentiated by i.p. injection of carbidopa, which would increase the availability of L-dopa to brain tissues by blocking the peripheral conversion of L-dopa to dopamine (DA). These results suggest that L-dopa or one of its catecholamine metabolites acts centrally to increase GH release. Intraventricular (i.v.t.) injection of DA and i.p. injection of apomorphine, a DA agonist that crosses the blood-brain barrier, increased serum GH. Intraperitoneal injection of DA did not alter circulating GH levels in normal fish or fish bearing preoptic lesions that abolish an inhibitory hypothalamic influence on GH release; however, DA increased serum GH in fish which had their blood-brain barrier destroyed by sham operation procedures. These results indicate that DA acts centrally to stimulate GH secretion, possibly by inhibiting the release and/or synthesis of GH release-inhibitory factor. Serum GH concentrations were decreased in a dose-dependent manner by i.p. injection of norepinephrine (NE), whereas i.v.t. injection of NE did not alter serum GH levels. These results indicate that NE acts outside of the blood-brain barrier to decrease serum GH levels in the goldfish, possibly by directly influencing pituitary GH cells.(ABSTRACT TRUNCATED AT 250 WORDS)