A High Resolution,Hard X-ray Bio-imaging Facility at SSRL

The old saying that seeing is believing has particular resonance for studying biological cells and tissues. Since 1677, when Anton van Leeuwenhoek used a simple light microscope to discover single cell organisms, scientists have relied on structural information obtained from microscopes with improving capabilities to advance the understanding of how biological systems work. Optical and electron microscopes are essential for many of these important discoveries and have been widely employed in biomedical research laboratories. However, various limitations exist in these microscopy techniques. We describe below how the new X-ray imaging facility at the Stanford Synchrotron Radiation Laboratory (SSRL), based on an Xradia nano-XCT full-field transmission X-ray microscope (TXM), can provide complementary and unique capabilities to the current microscopy methods for studying complex biological systems.     

Photoinduced DNA cleavage and cellular damage in human dermal fibroblasts by 2,3-diaminophenazine

Aromatic amines, such as o-phenylenediamine (OPD), have been used extensively in commercial hair dyes and in the synthesis of agricultural pesticides. Air oxidation of OPD results in the formation of 2,3-diaminophenazine (DAP). Although the mutagenic toxicity of DAP has been shown in both prokaryotic and eukaryotic systems, its phototoxicity remains largely unexplored. This study focuses on the pH-dependent photophysical properties of DAP and demonstrates its ability to photoinduce DNA damage to pUC19 plasmid in vitro. The photocytotoxicity of DAP toward human skin fibroblasts was also measured. DAP exhibits weak intercalative binding to double-stranded DNA with a binding constant K(b) = 3.5 x 10(3) M(-1). Furthermore, upon irradiation with visible light, DAP is able to nick plasmid DNA in the presence of oxygen. The concentration of DAP that resulted in 50% cell death was 172 +/- 9 microM in the dark and 13 +/- 1 microM after irradiation of the DAP-treated cell cultures with visible light (400-700 nm, 30 min, 5 J/cm(2)). The 13-fold increase in toxicity upon exposure to visible light shows the need for further study of the photocytotoxicity of contaminants such as DAP.     

DNA binding and photocleavage in vitro by new dirhodium(II) dppz complexes: correlation to cytotoxicity and photocytotoxicity

Two new dirhodium(II) complexes possessing the intercalating dppz ligand (dppz = dipyrido[3,2-a:2',3'-c]phenazine), cis-[Rh(2)(mu-O(2)CCH(3))(2)(dppz)(eta(1)-O(2)CCH(3))(CH(3)OH)](+) (1) and cis-[Rh(2)(mu-O(2)CCH(3))(2)(dppz)(2)](2+) (2), were synthesized and characterized as potential agents for photochemotherapy. Various techniques show that 1 binds to DNA through intercalation, although some aggregation of the complex on the DNA surface is also present. In contrast, 2 does not intercalate between the DNA bases; however, strong hypochromic behavior is observed in the presence of DNA, which can be attributed to intermolecular pi-stacking of 2 enhanced by the polyanion. The apparent DNA binding constants determined using optical titrations are compared to those from dialysis experiments. Both complexes photocleave pUC18 plasmid in vitro under irradiation with visible light (lambda(irr) >or= 395 nm, 15 min), resulting in the nicked, circular form. Greater photocleavage is observed for 1 relative to 2, which may be due to the ability of 1 to intercalate between the DNA bases. The cytotoxicity toward human skin cells (Hs-27) measured as the concentration at which 50% cell death is recorded, LC(50), was found to be 135 +/- 8 microM for 2 in the dark (30 min), which is significantly lower than those of 1 (LC(50) = 27 +/- 2 microM) and Rh(2)(O(2)CCH(3))(4) (LC(50) = 15 +/- 2 microM). Irradiation of cell cultures containing 1 and Rh(2)(O(2)CCH(3))(4) with visible light (400-700 nm, 30 min) has little effect on their cytotoxicity, with LC(50) values of 21 +/- 3 and 13 +/- 2 microM, respectively. Interestingly, a 3.4-fold increase in the toxicity of 2 is observed when the cell cultures are irradiated (400-700 nm, 30 min), resulting in LC(50) = 39 +/- 1 microM. The greater toxicity of 1 compared to 2 in the dark may be related to the ability of the former compound to intercalate between the DNA bases. The lower cytotoxicity of 2, together with its significantly greater photocytotoxicity, makes this complex a potential agent for photodynamic therapy (PDT). These results suggest that intercalation or strong DNA binding may not be a desirable property of a potential PDT agent.     

Deprotonation-substitution reactions of cyclic methylphenylphosphazenes: synthesis and structures of nongeminal P-ethyl,P-phenyl cyclotriphosphazenes

The deprotonation-substitution reactions of both the cis and trans isomers of nongeminally substituted [(Me)(Ph)P=N](3) were investigated. Treatment of the trans isomer, 1, with 3 equiv of n-BuLi followed by 3 equiv of MeI gave only nongeminal trans-[(Et)(Ph)P=N](3), 3, while the same reaction sequence on cis-[(Me)(Ph)P=N](3), 2, gave a mixture of nongeminal di- and trisubstitution products, cis-Et(2)MePh(3)P(3)N(3), 4, and cis-Et(3)Ph(3)P(3)N(3), 5. These trimers were separated by column chromatography. No changes in the stereochemistry of the rings occurred during these reactions. Compound 4 was also prepared using 2 equiv of the reactants and was then converted to 5 by treatment with a single equivalent of BuLi and MeI. Thermal analysis of the new cyclic trimers indicates that ring-opening polymerization does not occur and that sublimation occurs at ca. 300 degrees C. The structures of 4 and 5, obtained by X-ray diffraction, illustrate the basketlike shape of these molecules with an aromatic bowl formed by the phenyl rings on the top rim, while the structure of 3 clearly shows the trans orientation of the substituents. Crystal data for trans-Et(3)Ph(3)P(3)N(3), 3, at 20 degrees C are as follows: C(24)H(30)N(3)P(3) monoclinic, a = 14.273(2) A, b = 9.370(2) A, c = 19.600(3) A, beta = 107.16(1) degrees, P2(1/n), Z = 4. Crystal data for cis-Et(2)MePh(3)P(3)N(3), 4, at 20 degrees C are as follows: C(23)H(28)N(3)P(3), triclinic, a = 10.276(2) A, b = 10.699(2) A, c = 11.925(2) A, alpha = 72.07(2) degrees, beta = 73.79(1) degrees, gamma = 85.87(1) degrees, P1, Z = 2. Crystal data for cis-Et(3)Ph(3)P(3)N(3), 5, at 20 degrees C are as follows: C(24)H(30)N(3)P(3) monoclinic, a = 29.488(2) A, b = 9.8391(1) A, c = 21.172(2) A, beta = 126.30(1) degrees, C2/c, Z = 8.     

Oligomers of a polyester network

Oligomers of glycerol and succinic acid have been prepared by three different methods and characterised by solution ¹³C-NMR spectroscopy at 125.8 MHz. The first series of five oligomers was prepared by reacting glycerol with succinic anhydride; only one acid function of each succinic acid residue formed an ester by this means. They were readily distinguished as the shifts of their glycerol residues were dispersed over 16 ppm, and their shifts provided a guide to the assignment of shifts in the more elaborate oligomers. The second set of oligomers was prepared by treating glycerol with a small quantity of succinic acid, ester links being promoted by means of the reagent dicyclohexylcarbodiimide (DCCDI). When we used 2 mol of DCCDI/mol of acid in the presence of an excess of glycerol, no free acid functions remained, and a new set of oligomers was obtained. Furthermore, within this set the proportion of ring molecules was enhanced by repeating the reaction under much more dilute conditions. In this way the shifts of two ring oligomers were recognized in the spectrum. A different set of oligomers again was obtained when the esterification was performed with 1 mol of DCCDI/mol of succininic acid. After the first generation of oligomers had been identified in the mixtures produced by these three experiments, the reactions continued to produce larger oligomers with new fine structure features in the spectra. Glycerol trisuccinate was prepared in a pure form and heated in a vacuum to eliminate succinic acid groups and allow the formation of oligomers with two and three branch points. The structures successively produced by this reaction were readily recognized. In all we were able to recognize the formation of component structures in at least 17 different oligomers. The shifts of the carbons of the glycerol residues are sensitive to the substitution pattern at that residue, to whether the succinic acid residue to which they are linked has reacted a second time, and in some cases the methine shift is sensitive to how the succinic acid residue attached to a neighboring methylene carbon has reacted. © 1994 John Wiley & Sons, Inc.     

Acoustical analysis and model-based sound synthesis of the kantele

The five-string Finnish kantele is a traditional folk music instrument that has unique structural features, resulting in a sound of bright and reverberant timbre. This article presents an analysis of the sound generation principles in the kantele, based on measurements and analytical formulation. The most characteristic features of the unique timbre are caused by the bridgeless string termination around a tuning pin at one end and the knotted termination around a supporting bar at the other end. These result in prominent second-order nonlinearity and strong beating of harmonics, respectively. A computational model of the instrument is also formulated and the algorithm is made efficient for real-time synthesis to simulate these features of the instrument timbre.     

Synthesis and structure of nongeminally substituted cyclic phosphazenes with haloalkyl and thioester functional groups

Nongeminally substituted cyclic phosphazenes with various haloalkyl substituents were prepared using deprotonation-substitution reactions at the methyl groups of the cis isomers of nongeminally substituted cis-[Me(Ph)P=N]3, 2. Treatment of 2 with n-BuLi followed by reaction with organic halogenated reagents (RX=C2Cl6, BrC(O)CMe2Br, and ICH2COOEt) at low temperature afforded the various cyclic derivatives cis-[(XCH2)(Ph)PN]3 (3, X=Cl, 4, Br, and 5, I). The mono- and dibromoalkyl derivatives, cis-[Ph3(BrCH2)Me2P3N3], 6, and [Ph3(BrCH2)2MeP3N3], 7, were also isolated along with 4 when the electrophile was dibromoethane. Reaction of cis-[Ph(BrCH2)PN]3, 4, with KSC(O)Me gave cis-[Ph(MeC(O)SCH2)PN]3, 8. The structures of all the cis cyclic phosphazenes were determined by NMR spectroscopy and X-ray diffraction. All retained the basketlike shape with the hydrophobic phenyl groups opposite the haloalkyl groups on the P3N3 ring. Thermal analysis of the new cyclic trimers indicates that ring-opening polymerization does not occur. The melting points and the thermal stabilities of haloalkyl cyclophosphazenes were higher than those of the parent compound 2.     

NEW NONGEMINAL CYCLOPHOSPHAZENES

A series of new nongeminally-substituted cyclic phosphazenes with various substituents has been prepared via deprotonation-substitution reactions at the Me groups of both the cis and trans isomers of [(Me)(Ph)PN] ₃ . Treatment of [(Me)(Ph)PN] ₃ with n-BuLi followed by reaction with organic electrophilic reagents affords a variety of cyclic derivatives, [(RCH ₂ )(Ph)PN] ₃ , [R = Me, Cl, Br, I, (CH ₂ ) ₂ Br, CH ₂ CH═CH ₂ , SR, C(═O)OLi, C(═O)OMe, C(═O)OEt]. The structures of theses cis cyclic phosphazenes, which were obtained by x-ray diffraction, illustrate the basket-like shape of the molecules. Heating the cis and trans isomers of the parent [(Me)(Ph)PN] ₃ produced mixtures of cyclic trimers and tetramers. The latter were isolated and characterized by x-ray crystallography. Nanoparticles of gold and silver were prepared by reduction of metal salts with a reducing agent in the presence of selected trimers.