Optical Properties of a Quantum Dot-Ring System Grown Using Droplet Epitaxy.

Electronic and optical properties of InAs/GaAs nanostructures grown by the droplet epitaxy method are studied. Carrier states were determined by k · p theory including effects of strain and In gradient concentration for a model geometry. Wavefunctions are highly localized in the dots. Coulomb and exchange interactions are studied and we found the system is in the strong confinement regime. Microphotoluminescence spectra and lifetimes were calculated and compared with measurements performed on a set of quantum rings in a single sample. Some features of spectra are in good agreement.

Surface effects of vapour-liquid-solid driven Bi surface droplets formed during molecular-beam-epitaxy of GaAsBi.

Herein we investigate a (001)-oriented GaAs1-xBix/GaAs structure possessing Bi surface droplets capable of catalysing the formation of nanostructures during Bi-rich growth, through the vapour-liquid-solid mechanism. Specifically, self-aligned "nanotracks" are found to exist trailing the Bi droplets on the sample surface. Through cross-sectional high-resolution transmission electron microscopy the nanotracks are revealed to in fact be elevated above surface by the formation of a subsurface planar nanowire, a structure initiated mid-way through the molecular-beam-epitaxy growth and embedded into the epilayer, via epitaxial overgrowth. Electron microscopy studies also yield the morphological, structural, and chemical properties of the nanostructures. Through a combination of Bi determination methods the compositional profile of the film is shown to be graded and inhomogeneous. Furthermore, the coherent and pure zincblende phase property of the film is detailed. Optical characterisation of features on the sample surface is carried out using polarised micro-Raman and micro-photoluminescence spectroscopies. The important light producing properties of the surface nanostructures are investigated through pump intensity-dependent micro-PL measurements, whereby relatively large local inhomogeneities are revealed to exist on the epitaxial surface for important optical parameters. We conclude that such surface effects must be considered when designing and fabricating optical devices based on GaAsBi alloys.

Displacement potency of vitamin D2 analogs in competitive protein-binding assays for 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and 1,25-dihydroxyvitamin D3.

24(R),25-Dihydroxyergocalciferol [24,25-(OH)2-D2] is 1.7 times less potent than 24 (R), 25-(OH)2D3, 25-Hydroxyvitamin D2 (25OHD2), or 25OHD3 in the displacement of (3H)25OHD3 from rat serum binding proteins. 1,25-(OH)2D2 is 1.3 times less potent than 1,25-(OH)2D3 in the displacement of (3H)1,25-(OH)2D3 from a chick intestinal binding receptor. In light of binding affinity and chromatographic differences between vitamin D3 and its D2 analogs, it is our view that methods which purport to measure 1,25-(OH)2D and 24,25-(OH)2D probably understimate the contributions of D2 metabolites. This is particularly important in the case of plasma extracts from patients given large doses of vitamin D2.

Inactivation of the human immunodeficiency virus by hypericin: evidence for photochemical alterations of p24 and a block in uncoating.

Following attachment and entry of human immunodeficiency virus (HIV) into a host cell, the HIV genomic RNA is reverse transcribed to cDNA. This step may be inhibited by hypericin, a compound that induces alterations of the retroviral capsid. Incubation of HIV with hypericin rendered the virus noninfectious. The replication of HIV was blocked early; HIV cDNA could not be detected in cells challenged with hypericin-treated HIV. Hypericin did not inhibit the binding of recombinant gp120 to CD4+ cells, nor did hypericin inhibit syncytium formation. However, reverse transcriptase activity could not be released from hypericin-treated virions. Western blot analysis revealed altered mobility of the HIV major capsid protein (p24) following hypericin treatment. Hypericin-treated recombinant HIV p24 exhibited similar altered mobility. The inactivation of HIV infectivity and the alterations in p24 mobility required hypericin incubations in the presence of visible light. Collectively, these data suggest that photochemical alterations of the HIV capsid may contribute to the hypericin-mediated inactivation of HIV. Such alterations may inhibit the release of RT activity from treated HIV, and prevent uncoating and subsequent reverse transcription of the HIV genome within a target cell.

The structure of hypericin in solution. Searching for hypericin's 1,6 tautomer.

Hypericin in organic solvents displays two types of electronic spectra: one type which shows a distinct solvatochromic effect, the stable form, and the other, the unstable form, which lacks this property. The latter type is formed in dry nonprotic solvents (e.g. tetrahydrofuran, EtOAc) and can be converted to the stable form on addition of protic solvents. In order to establish the tautomeric structure of the unstable form we applied conventional nuclear magnetic resonance techniques as well as two-dimensional gradient-enhanced heteronuclear multiple-quantum correlation, gradient-enhanced ROESY and one-dimensional nuclear Overhauser effect difference experiments. All these techniques pointed to the fact that the unstable form has the 7,14-diketo tautomeric structure, like the stable form, and not the 1,6-diketo structure. Electronic spectroscopy indicated that the unstable form has acidic properties and therefore possesses two free OH groups at C3 and C4 at the bay region of the molecule.

Solvatochromic effects in the electronic absorption and nuclear magnetic resonance spectra of hypericin in organic solvents and in lipid bilayers.

The natural product hypericin was tested in recent years as a biological photosensitizer with a potential for viral and cellular photodamage. We thus studied extensively its spectroscopy and membrane partitioning. Absorption, fluorescence excitation and emission spectra of the sodium salt (HyNa) were measured in 36 protic and aprotic, polar and apolar, solvents. Electronic transition bands as well as vibrational progressions were identified. Aggregation in some nonpolar solvents and protonation in organic acids were demonstrated. Modeling solvatochromism was done by Lippert equation, by the ET(30) parameter and by the Taft multiparameter approach. In all cases, separation into protic and aprotic solvents gave much better fits to the models. 13C chemical shift data could also be correlated with solvent polarity. They correlated best with Lippert's delta f polarity measure, but tended to fall into two distinct solvent groups--each along different lines--corresponding to protic and aprotic media, respectively. This interesting phenomenon suggests that in the case of the charged and slightly water soluble HyNa, two mechanisms of solvation are involved, each resulting in its own line equation. In aprotic media, dipole-dipole interaction is the predominant solvation mechanism. In protic solvents, the most effective means of solvation is likely to be hydrogen bonding. When intercalated into the liposomal phospholipid bilayer, HyNa is oriented at an angle to the interface, thus experiencing a gradient of solvent polarities: a highly polar environment (similar to methanol) for C-2/5, suggesting that they lie not far from the interface; a moderately polar environment (similar to that of n-propanol) for C-6a/14a, which are somewhat deeper within the bilayer; and a more lipophilic environment (akin to n-hexanol) for C-10/11. The fluorescence excitation peak in liposomes also correlates with an aprotic medium of relatively high polarity, as might be excepted from a molecule in a shallow position in the bilayer.

Hypericin derivatives: substituent effects on radical-anion formation.

The electron-transfer properties of the hypericin derivatives, dibromo-, hexaacetyl-, hexamethyl- and desmethylhypericin, were studied. Cyclovoltammetric measurements revealed that dibromo- and desmethylhypericin have almost the same redox potentials as the parent hypericin. Substitution of the hydroxyl groups by acetoxy leads to less negative E1/2 values, whereas methoxy substitution induces more negative values. Electron paramagnetic resonance (EPR)/electron nuclear double resonance/general TRIPLE spectroscopy and quantum mechanical calculations were used to establish the structure of the one-electron reduced stages of hypericin derivatives. Proton loss in the bay region, already demonstrated for hypericin, was also found for dibromo- and desmethylhypericin. The spin and charge of the radical ions are predominately confined to the central biphenoquinone moiety of the hypericin skeleton. Generation of the radical ions by in situ electrolysis indicates that the redox potentials of hypericin, dibromo- and desmethylhypericin, containing hydroxyls at the 1, 3, 4, 6, 8 and 13 positions, largely depend on the solvent. With phosphate-buffered saline (pH 7.4)/dimethylsulfoxide (DMSO) as the solvent the EPR spectra of the corresponding radical ions appear at markedly lower potentials than in pure DMSO and N,N'-dimethylformamide. However, this effect is not observable for hexaacetyl- and hexamethyl-hypericin-lacking hydroxyl groups. In all cases the EPR data and calculations revealed the presence of 7,14 tautomers.

Strategies for evaluation of enveloped virus inactivation in red cell concentrates using hypericin.

Photodynamically induced virus inactivation appears promising in preventing transmission of enveloped virus infections in transfusible blood products. The potential for utilizing hypericin as a photosensitizer to inactivate key enveloped viruses in packed red cell concentrates (PRC) was evaluated. In addition to inactivating effectively > or = 10(6) TCID50 of human immunodeficiency virus (HIV), inactivation of bovine viral diarrhea virus (BVDV) in PRC was used as a model for hepatitis C virus to overcome the deficiency in reliable experimental systems for hepatitis C virus (HCV) inactivation. BVDV was two orders of magnitude more sensitive to inactivation by hypericin than HIV. As part of the virucidal efficacy analyses, the effects of photosensitization on hemopoietic cell lines carrying quiescent integrated HIV provirus were studied as models for evaluating virus inactivation in latently infected cells. Phorbol ester-induced virus production by these cells was effectively prevented by photosensitization with hypericin. A refinement of the illumination conditions, incorporating a monochromatic sodium light source with an emission spectrum coinciding with the absorption peak of hypericin, was highly virucidal, however, caused unacceptable levels of hemolysis. Red blood cells could be protected from phototoxic cellular damage by complexing hypericin with human serum albumin (albumin-hypericin), but the decrease in hemolysis was at the expense of virucidal efficacy. Thus, excitation of hypericin with a fluorescent source appears to be useful potentially for virus inactivation in PRC.

Generation of free radicals by emodic acid and its [D-Lys6]GnRH-conjugate.

In an attempt to develop an efficient chemotherapeutic agent targeted at malignant cells that express receptors to gonadotropin releasing hormone (GnRH) we coupled [D-Lys6]GnRH covalently to an emodin derivative, i.e. emodic acid (Emo) to yield [D-Lys6(Emo)]GnRH. Emodin is a naturally occurring anthraquinone which is widely used as a laxative and has other versatile biological activities. Physico-chemical studies employing electron paramagnetic resonance and electrochemistry of the conjugate as well as the (Emo) moiety showed that these compounds could be easily reduced either chemically, photochemically or enzymatically to their corresponding semiquinones. In the presence of oxygen the semiquinones generated reactive oxygen species (ROS), mainly superoxide and hydroxyl radicals, which were detected by the spin trapping method. Moreover, upon irradiation with visible light these compounds produced ROS and a highly reactive excited triplet state of Emo, which by itself may cause the oxidation of certain electron acceptors such as amino acids and bases of nucleic acids. Thus, [D-Lys6]GnRH-photosensitizer conjugates may be potentially used for targeted photodynamic chemotherapy aimed at treating cancer cells that carry GnRH receptors. These conjugates may also induce cytotoxicity in the dark similar to common conventional chemotherapeutic agents. The peptidic moiety, [D-Lys6]GnRH, was found to be stable toward highly reactive ROS generated either from enzymatic reduction or upon photoirradiation. The physico-chemical properties of Emo were only marginally influenced by the peptidic [D-Lys6]GnRH carrier.

Liposome binding constants and singlet oxygen quantum yields of hypericin, tetrahydroxy helianthrone and their derivatives: studies in organic solutions and in liposomes.

The spectroscopy and photophysics of several hypericin and helianthrone derivatives were studied in methanol and when bound to liposomes. The singlet oxygen quantum yields (phi(delta)) were measured indirectly relative to Rose Bengal and hematoporphyrin IX, employing 9,10-dimethylanthracene as a singlet oxygen trap. Hypericin was found to have a phi(delta) of 0.39+/-0.01 in methanol, and 0.35+/-0.05 in lecithin vesicles, in agreement with literature values. A heavy atom effect was evident upon bromination, resulting in phi(delta) for tetrabromohypericin of 0.72+/-0.02, presumably due to enhanced intersystem crossing. Elimination of the anionic hydroxyls by methylation also enhanced phi(delta) to 0.81+/-0.01. Conversely, addition of anionic sulfate groups drastically reduced phi(delta) resulting in phi(delta)'s of 0.12+/-0.01, 0.052+/-0.003 and 0.40+/-0.01 for hypericin disulfonate, hypericin tetrasulfonate and hexamethyl hypericin tetrasulfonate, respectively. The non-sulfonated helianthrones exhibited low phi(delta)'s in solution. The liposome binding constants, Kb, were measured using a spectroscopic assay. Except for hexamethyl hypericin, all non-sulfonated compounds bound well with Kb's ranging from 15.5+/-0.1 to 48.7+/-3.9 (mg/ml)(-1). None of the tetrasulfonated compounds bound, however the hypericin disulfonate had a Kb of 4.1+/-0.2 (mg/ml)(-1). The phi(delta)'s of the compounds capable of binding were measured and, in the case of the hypericin derivatives, were found not to vary dramatically from those in the free state. Liposome-bound helianthrone and dimethyl tetrahydroxy helianthrone both exhibited high phi(delta)'s, i.e. >0.5. The variations in binding constant and sensitization efficiencies are explained in conjunction with the molecular structure. The relevance of the above data to photodynamic therapy is briefly discussed.

Use of 1alpha-hydroxycholecalciferol in the prevention of bovine parturient paresis.

A synthetic biologically active derivative of vitamin D (350 microgram of 1alpha-hydroxycholecalciferol [1alpha(OH)CC]) was injected into 2 nonlactating 7-year-old Israeli-Friesian cows. Plasma calcium values increased after 24 hours, peaked at 48 hours, and returned to base-line values 120 hours after injection. An injection of 350 microgram of 1alpha(OH)CC was given to 23 parturient-paresis-prone Israeli-Friesian cows from 7 days to 6 hours prepartum; 13 cows were injected once, 6 were injected twice, and 4 were injected 3 times, all at 48-hour intervals. Parturient-paresis-prone cows (n = 23) of the same breed were used as controls. Within 0 to 36 hours postpartum, plasma calcium concentrations were found to be higher in cows injected with 1alpha(OH)CC than in the control cows. The increase was highly significant (P less than 0.01) in cows injected at least twice. None of the cows injected with 1alpha(OH)CC, within 72 to 24 hours prior to calving developed parturient paresis; but 9 of 23 control cows developed parturient paresis. Prior to calving, none of the injected cows developed hypercalcemia and there was no local or systemic clinically detectable signs of toxiosis. When given at the right time prepartum, 1alpha(OH)CC is considered to be an improvement over previous methods of preventing bovine parturient paresis.

The release of cytotoxic drugs from acrylic bone cement.

The ability of cis-platinum, 5-fluorouracil, and methotrexate to leach out from various types of bone cements was examined in three different experimental systems: (1) in vitro release in physiological solution; (2) in vitro release in the presence of mouse fibrosarcoma cells; and (3) in vivo release in rabbits. The amount of drugs released was measured either spectrophotometrically by atomic absorption or by using tritium-labeled drugs. In vitro cement pellets were found to release these drugs slowly for up to six months; release was greater in the first few days, rapidly declining with time. Up to 6% of the methotrexate implanted leached out during the entire experiment. The figures for cis-platinum and 5-fluorouracil were much less: 3.3% and 3.4%, respectively. Palacos bone cement had the best leaching properties. The drugs leached out were also effective in reducing the numbers of viable mouse fibrosarcoma cells in tissue culture. In vivo, high levels of the drugs were recovered from blood drained from the operative wound of the rabbits, while very low levels of these drugs were found in the serum. Anticancer drug-loaded cement may be used effectively in the treatment of pathologic fractures and tumor surgery. This delivery method may reduce the side effects that result from systemic administration of such drugs.

Flavanone Glycoside Biosynthesis in Citrus: Chalcone Synthase, UDP-Glucose:Flavanone-7-O-Glucosyl-Transferase and -Rhamnosyl-Transferase Activities in Cell-Free Extracts.

Previous indirect evidence suggested that the biosynthesis of flavonoids in Citrus may not proceed via the usual chalcone synthase reaction and that glycosylation occurs during chalcone formation and not afterward, as has been reported in other species. We detected chalcone-synthase and UDP-glucose:flavanone-7-O-glucosyl-transferase activities in cell-free extracts of Citrus. The glucosylated flavanone was further rhamnosylated when exogenous UDP-glucose and NADPH were added to the extract. Chalcone-synthase activity was detected in cell-free extracts derived from young leaves and fruits. Young fruits (2 millimeter diameter) had the highest chalcone synthase activity. UDP-glucose:flavanone-7-O-glucosyl-transferase activity was measured in cell-free extracts derived from young leaves and fruits of Citrus mitis and Citrus maxima. The highest UDP-glucose:flavanone-7-O-glucosyl-transferase activity was found in young C. maxima leaves. These data indicate that Citrus contains a flavonoid pathway similar to that studied in other species.

Targeted cross-linking of a molten globule form of acetylcholinesterase by the virucidal agent hypericin.

The natural product hypericin is a photosensitive polycyclic aromatic dione compound, which has been widely investigated because of its virucidal and antitumor properties. Although it has been suggested that singlet oxygen or a radical species might be responsible for its biological action, its mechanism of action remains unknown. Due to its amphiphilic characteristics, we considered the possibility that it might interact preferentially with partially unfolded proteins which exhibit exposed hydrophobic surfaces. We here demonstrate that hypericin binds to a molten globule species generated from Torpedo acetylcholinesterase, but not to the corresponding native enzyme. Irradiation with visible light, under aerobic conditions, causes chemical cross-linking of the catalytic subunits, to dimers and heavier species, under conditions where no cross-linking is observed for the native enzyme. Both anaerobiosis and sodium azide greatly reduce the extent of cross-linking, suggesting that singlet oxygen is responsible for the phenomenon. This agrees with our observation, using spin traps, that mainly singlet oxygen is produced by the complex of hypericin with the molten globule of acetylcholinesterase. Cross-linking is enhanced in the presence of liposomes to which the molten globule of acetylcholinesterase is quantitatively adsorbed. This may be due to high local concentrations of both hypericin and the protein resulting in close proximity, and hence in a high yield of cross-linking. Molten globule species are believed to be intermediates in both protein folding and translocation through biological membranes. Thus, hypericin may serve as a valuable tool for trapping such intermediates. This might also explain its therapeutic effectiveness toward virus-infected or tumor cells.