Towards Sixfold Functionalization of Buckminsterfullerene (C(60)) at Fully Addressable Octahedral Sites.

Selective C(60)-functionalizations that provide access to unusual multifunctional molecules are of interest in the construction of highly organized three-dimensional assemblies. The temporary "masking" of three of the most reactive sites on C(60) by a bisdiene tether has allowed the facile and high-yielding formation of the fully differentiated trisadduct 1 and the interesting hexaadduct 2.

Formation of an Effective Opening within the Fullerene Core of C(60) by an Unusual Reaction Sequence.

A large hole in a fullerene: The addition of dioxygen to the highly reactive 1,4-diaminobutadiene moiety of 1, formed from the reaction of C(60) with a rigid diazidobutadiene, results in the very efficient formation of an open fullerene (see the space-filling model) with the largest orifice created so far on a fullerene. The opening may be large enough to allow the smallest atoms, molecules, or ions to pass through.

Optimizing the binding of fullerene inhibitors of the HIV-1 protease through predicted increases in hydrophobic desolvation.

We have developed and applied a computational strategy to increase the affinity of fullerene-based inhibitors of the HIV protease. The result is a approximately 50-fold increase in affinity from previously tested fullerene compounds. The strategy is based on the design of derivatives which may potentially increase hydrophobic desolvation upon complex formation, followed by the docking of the hypothetical derivatives into the HIV protease active site and assessment of the model complexes so formed. The model complexes are generated by the program DOCK and then analyzed for desolvated hydrophobic surface. The amount of hydrophobic surface desolvated was compared with a previously tested compound, and if this amount was significantly greater, it was selected as a target. Using this approach, two targets were identified and synthesized, using two different synthetic approaches: a diphenyl C60 alcohol (5) based on a cyclopropyl derivative of Bingel (Chem.Ber. 1993, 126, 1957-1959) and a diisopropyl cyclohexyl C60 alcohol (4a) as synthesized by Ganapathi et al. (J. Org.Chem. 1995, 60, 2954-2955). Both showed tighter binding than the originally tested compound (diphenethylaminosuccinate methano-C60, Ki = 5 microM) with Ki values of 103 and 150 nM, respectively. In addition to demonstrating the utility of this approach, it shows that simple modification of fullerenes can result in high-affinity ligands of the HIV protease, for which they are highly complementary in structure and chemical nature.

High-resolution 1H NMR spectroscopy following experimental brain trauma.

We investigated acute metabolic changes following parasagittal fluid-percussion brain injury in the rat, using high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy. Sixty minutes following brain injury or sham (surgery, no injury) treatment, brains were rapidly removed and the injured and control cortices were isolated (n = 5/group). Isolates of brain cortices were then placed in buffer and studied in a 400-MHz spectrometer with measurements taken every 15 min over a 145-min period. At the initial NMR evaluation (immediately following dissection), we observed significantly lower levels of N-acetyl aspartic acid (NAA) in the injured group compared to the sham group. Surprisingly, a reciprocal increase in the concentration of acetate, a major metabolic product of NAA, was not observed at this timepoint. At subsequent timepoints, a progressive loss of NAA was observed in both injured and sham cortices, presumably due to ischemic conditions of the ex vivo samples. However, this progressive loss of NAA was now accompanied by a commensurate accumulation of acetate. These results suggest that (1) a decrease in the concentration of NAA occurs by 1 h following experimental brain trauma, potentially marking traumatic neural injury; (2) the initial absence of an expected reciprocal increase in acetate concentration may signify rapid utilization of acetate following trauma, potentially for reparative processes; and (3) in contrast to trauma alone, post mortem ischemic conditions may induce an increase in acetate concentrations.

Benzoyl peroxide cytotoxicity evaluated in vitro with the human keratinocyte cell line, RHEK-1.

The human keratinocyte cell line, RHEK-1, was used to evaluate the cytotoxicity of benzoyl peroxide (BZP). As determined with the neutral red (NR) cytotoxicity assay, the 24-h midpoint (NR50) toxicity values, in mM, were 0.11 for BZP and 29.5 for benzoic acid, the stable metabolite of BZP. Irreversible cytotoxicity occurred after a 1-h exposure to 0.15 mM BZP and greater. When exposed to BZP for 7 days, a lag in growth kinetics was first observed at 0.06 mM BZP. Damage to the integrity of the plasma membrane was evident, as leakage of lactic acid dehydrogenase occurred during a 4-h exposure to BZP at 0.05 mM and greater. Intracellular membranes were also affected, as extensive vacuolization, initially perinuclear but then spreading throughout the cytoplasm, was noted in BZP-stressed cells. The generation of reactive free radicals from BZP was suggested by the following: the intracellular content of glutathione was lowered in cells exposed to BZP; cells pretreated with the glutathione-depleting agent, chlorodinitrobenzene, were hypersensitive to a subsequent challenge with BZP; lipid peroxidation by BZP was inducible in the presence of Fe2+; and cells previously maintained in a medium amended with vitamin E, an antioxidant, were more resistant to BZP, showed less lipid peroxidation in the presence of BZP+Fe2+ and did not develop the extensive intracellular vacuolization as compared to non-vitamin E maintained cells.

N-acetylaspartate complexes with calcium and lanthanide ions.

N-acetylaspartate (NAA) is the one of the most prominent resonances observed in the solvent-suppressed NMR spectrum of the human brain. Although it is present in the brain at about 10 mM, its precise metabolic function is still unclear, We have examined the NAA as a potential chelator for divalent metal ions such as Ca2+. We have employed the perturbations induced by Ln3+ ions in the 1H and 13C NMR spectrum of NAA to monitor formation of NAA complexes. 1H NMR measurements showed that the dissociation constants for the formation of Eu(3+)-NAA, Yb(3+)-NAA, and Ca(2+)-NAA complexes were 0.07, 0.13, and 0.86 mM, respectively. Scatchard analysis of the results indicates the formation of a 1:1 metal-ligand complex. We also inferred the structure of the NAA-metal ion complex from an analysis of paramagnetic perturbations induced in the 1H NMR and 13C NMR spectra of NAA. The structural analysis of the NAA-metal ion complex indicates that the two carboxylic groups participate in chelating the metal ion, forming the binding site for the metal ion.

The effect of N-acetylaspartate on the intracellular free calcium concentration in NTera2-neurons.

We have studied the effect of relatively high concentrations of extracellular N-acetylaspartate (NAA) on the intracellular free calcium concentration [Ca2+]i in NTera2-neurons. While low concentrations of extracellular NAA (0.1, 1 mM) had no effect on the [Ca2+]i, high concentrations of extracellular NAA (3, 10 mM) elicited sharp and statistically significant elevations of [Ca2+]i. Different classes of antagonists of the N-methyl-D-aspartate (NMDA) receptor abolished the NAA induced elevations of the [Ca2+]i, indicating the involvement of the NMDA receptor in NAA-induced elevations of [Ca2+]i.

The effect of furosemide on calcium ion concentration in myocardial cells.

The effect of furosemide and ouabain on the intracellular concentration of Ca2+ was studies in myocardial cell cultures using Fura-2, a fluorescent agent, as an intracellular Ca2+ indicator. Introduction of 200 microM ouabain to the cultured cells increased the intracellular calcium concentration from an average of 236 nM up to an average of 833 nM. Introduction of 100 microM furosemide, prior to the administration of ouabain, decreased the ouabain induced Ca2+ elevation to an average of only 473 nM. Introduction of 2.5 mM EGTA prior to the administration of ouabain abolished the ouabain induced Ca2+ increase.

Improved hypothermic preservation of rat hearts by furosemide.

The effect of furosemide, a blocker of the Na+/K+/Cl- cotransporter, on hypothermic preservation of rat hearts was studied with use of the Langendorff perfusion system and electron microscopy. Furosemide significantly improved the mechanical recovery and the coronary flow of the hearts preserved for 8 hours in St. Thomas' Hospital cardioplegic solution at a temperature of 4 degrees C. Furosemide at the concentration of 100 mumol/L was found to have an optimal effect, whereas at high concentrations (1000 mumol/L) it was found to have toxic effects. In addition, furosemide reduces the time elapsed between the end of the preservation time and the resumption of myocardial contractions. Ultrastructural evaluations were done in which the presence of swollen mitochondria was chosen as a criterion of hypothermic ischemic damage to the myocardium. Morphometric analysis indicated that the mitochondrial volume of hearts stored for 8 hours in St. Thomas' Hospital cardioplegic solution increased by 72% as compared with the mitochondrial volume of hearts that were not exposed to the hypothermic ischemic conditions (control group). The addition of 100 mumol/L furosemide to the cardioplegic solution resulted in a significant reduction of mitochondrial swelling during the period of 8 hours' storage, which amounted only to 28% as compared with the figure for the control group. The reduction of mitochondrial swelling by furosemide and the improved mechanical and coronary flow recoveries are thought to be related to the blocking of the sarcolemmal Na+/K+/Cl- cotransporter and consequently the reduction of the Na+ influx during hypothermic ischemic storage.

An in vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells.

Chlorhexidine digluconate is the active ingredient in mouthrinses used to prevent dental plaque and gingivitis. The in vitro cytotoxicity of chlorhexidine was evaluated with the Smulow-Glickman (S-G) gingival epithelial cell line. The potency of chlorhexidine was dependent on the length of exposure and composition of the exposure medium. The midpoint cytotoxicity values for 1-, 24-, and 72-h exposures were 0.106, 0.011, and 0.0045 mmol/L, respectively. S-G cells exposed for 2 h to chlorhexidine and then maintained for 48 h in chlorhexidine-free medium were unable to recover from the initial insult. The adverse effects of chlorhexidine on the plasma membrane were suggested by the leakage of lactic acid dehydrogenase from chlorhexidine-treated S-G cells and by the increased permeability of chlorhexidine-treated liposomes to Ca2+. The toxicity of a 24-h exposure to chlorhexidine to the S-G cells was progressively lessened as the content of fetal bovine serum (FBS) in the exposure medium was increased from 2% to 8%. The potency of a 1-h exposure to chlorhexidine was reduced in medium amended with albumin, lecithin, and heat-killed Escherichia coli. These reductions in toxicity were presumably due to the binding of the cationic chlorhexidine to the negatively charged chemical moieties of the components of FBS and of albumin and lecithin and of sites on the surfaces of bacteria. Combinations of chlorhexidine and carbamide peroxide were additive in their cytotoxicities.

6-Thioguanine luminescence probe to study DNA and low-molecular-weight systems.

6-Thioguanine, an antitumor drug, has been tested as a luminescence probe to study DNA and cryoprotector solutions at temperatures between 4.2 and 273 K. The electronic structure of the tautomeric and ionic forms of 6-thioguanine is studied comprehensively both theoretically and experimentally. An excited-state diagram of 6-thioguanine N9H tautomer is proposed. The temperature behavior of 6-thioguanosine is examined in different cryoprotector solutions and with different aggregate states of solvents. Structure and phase transitions in low-molecular-weight cryoprotectors (glycerol, ethanol, propanediol, DMSO) and their water solution are investigated in the 4.2-273 K temperature range. New structural transitions in propanediol-water solutions are found in the temperature interval 10-180 K. DNA solutions are investigated by using 6-thioguanine incorporated in DNA by the method of biosynthesis. Phosphorescence intensity curves for 6-thioguanosine in native DNA manifest peculiarities at 21, 64, 87, 140, 180, and 268 K.

Inhibition of sodium influx and improved preservation of rat hearts during hypothermic ischemia by furosemide and bumetanide: a 23Na- and 31P-NMR study.

The importance of the Na+/K+/Cl- co-transport system of the rat myocardial sarcolemma was studied under hypothermic ischemia by investigating the effect of the co-transport blockers furosemide and bumetanide on the sodium influx into the myocardium. The intracellular Na+ accumulation during hypothermic ischemia was followed by 23Na-NMR. For this purpose the shift reagent [Dy(TTHA)3-] (SR) was added to the Krebs-Henseleit (KH) perfusion solution. The same solution was also present during the hypothermic preservation. A significant reduction in the intracellular Na+ accumulation after 12 h was found when 100 microM furosemide was present during the perfusion and preservation periods. The intracellular Na+ levels returned to the pre-ischemic values after 1 h of reperfusion with KH in both the treated and control groups. Dose-response studies have indicated that 1-100 microM furosemide or 0.1 microM bumetanide added to the KH-SR solution reduced the Na+ influx significantly over 4 h of hypothermic ischemia. No statistically significant effect was found with furosemide concentration of 0.1 microM or with bumetanide concentrations higher or lower than 0.1 microM. 31P-NMR measurements showed no effect of the 100 microM furosemide on the intracellular ATP, the sum of inorganic phosphate and phosphomonoester, or pH levels over 4 h or after 12 h of hypothermic ischemia. Hearts treated with KH containing 100 microM furosemide showed, significantly higher functional recovery after 12 h of hypothermic ischemia than hearts treated only with KH. This study strongly indicates the existence of the Na+/K+/Cl- co-transport system in the intact rat heart sarcolemma, and its major role in sodium influx during hypothermic ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular haemodynamics of oriental hornet venom sac extract.

The effects of hornet venom sac extract on the functioning of the cardiovascular system was assessed in dogs by concomitant examination of the following parameters: Systemic blood pressure, blood pressure and the dp/dt derivative of the left ventricle of the heart, heart rate, direct measurement of the left ventricular wall thickness, cardiac output, and body temperature. The effect of adult hornet venom sac extract on the canine cardiovascular system was rapid and led to a sharp drop in the systemic blood pressure, a similar drop in the pressure and dp/dt derivative in the left ventricle, to bradycardia, immediate thickening of the left ventricular wall and to an immediate rise in the cardiac output. As for the canine temperature, this dropped immediately following the administration of venom sac extract and remained at the new low level for at least 30 min. The smaller molecular weight components (less than 10 kilodalton) in venom sac extract have been found to be responsible for the immediate effects of venom sac extract on the cardiovascular system. On the other hand the high-molecular weight components exert more delayed effects which lead to a cumulative disturbance of cardiac function.

Performance under dichoptic versus binocular viewing conditions: effects of attention and task requirements.

Three experiments investigated subjects' ability to allocate attention and cope with task requirements under dichoptic versus binocular viewing conditions. Experiments 1 and 2 employed a target detection task in compound and noncompound stimuli, and Experiment 3 employed a relative-proximity judgment task. The tasks were performed in a focused attention condition in which subjects had to attend to the stimulus presented to one eye or field (under dichoptic and binocular viewing conditions, respectively) while ignoring the stimulus presented to the other eye or field, and in a divided attention condition in which subjects had to attend the stimuli presented to both eyes or fields. Subjects' performance was affected by the interaction of attention conditions with task requirements, but it was generally the same under dichoptic and binocular viewing conditions. The more dependent the task was on finer discrimination, the more performance was impaired by divided attention. These results suggest that at least with discrete tasks and relatively short exposure durations, performance when each eye is presented with a separate stimulus is the same as when the entire field of stimulation is viewed by both eyes.

Atomic Force Microscope Studies of Fullerene Films: Highly Stable C60 fcc (311) Free Surfaces.

Atomic force microscopy and x-ray diffractometry were used to study 1500 A-thick films of pure C(60) grown by sublimation in ultrahigh vacuum onto a CaF(2) (111) substrate. Topographs of the films did not reveal the expected close-packed structures, but they showed instead large regions that correspond to a face-centered cubic (311) surface and distortions of this surface. The open (311) structure may have a relatively low free energy because the low packing density contributes to a high entropy of the exposed surface.

The Higher Fullerenes: Isolation and Characterization of C76, C84, C90, C94, and C70O, an Oxide of D5h-C70.

The toluene extract of the fluffy carbon material produced by resistive heating of graphite contains a variety of molecules larger than C(60) and C(70) in a total amount of 3 to 4% by weight. Repeated chromatography of this material on neutral alumina has led to the isolation of stable solid samples of C(76), C(84), C(90), and C(94). The characterization, which includes mass spectrometry, (13)C nuclear magnetic resonance, electronic absorption (ultraviolet/visible) and vibrational (infrared) spectroscopy identifies these all-carbon molecules as higher fullerenes. In addition, C(70)O, a stable oxide, has been isolated that is structurally and electronically closely related to D5h-C(70). This compound forms during the resistive heating process and probably has an oxygen atom inserted between two carbon atoms on the convex external surface of the C(70) skeleton.

All-Carbon Molecules: Evidence for the Generation of Cyclo[18]carbon from a Stable Organic Precursor.

The unambiguous structural characterization of a single-sized all-carbon molecule requires its chemical synthesis. For cyclo[18]carbon, ab initio calculations predict a relatively stable, cyclic D9h ground state geometry with alternating C-C (1.36 angstroms) and C identical withC (1.20 angstroms) bonds. The synthesis and x-ray crystal structure of a direct precursor to C(18) are described. The analysis of laser flash heating experiments on this precursor by time-of-flight mass spectroscopy shows a sequence of retro-Diels-Alder reactions leading to C(18) as the predominant fragmentation pattern. Structural evidence is provided for the generation of an all-carbon molecule from a well-characterized organic precursor.