Regioselective synthesis of 1I,1II,5I,5II,6I,6I,6II,6II-2H8-cellobiose

Partially deuteriated 1,5,6,6-(2)H(4)-d-glucose and 1(I),1(II),5(I),5(II),6(I),6(I),6(II),6(II)-(2)H(8)-d-cellobiose were synthesized in high yields and on a large scale from d-glucose. (2)H enrichment at C-5 and C-6 of each glucopyranosyl unit in excess of 85% and 90%, respectively, was realized by (1)H-(2)H exchange in (2)H(2)O containing deuteriated Raney Ni. Nucleophilic addition of LiAlD(4) to 5,6,6-(2)H(3)-2,3,4,6-tetra-O-benzyl-d-gluconolactone led to a 98% (2)H enrichment at C-1. Deuteriated cellobiose is of interest as building block for the synthesis of a model compound of cellulose I.     

Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a

We have synthesized several ganglio-oligosaccharide structures using glycosyltransferases from Campylobacter jejuni. The enzymes, alpha-(2-->3/8)-sialyltransferase (Cst-II), beta-(1-->4)-N-acetylgalactosaminyltransferase (CgtA), and beta-(1-->3)-galactosyltransferase (CgtB), were produced in large-scale fermentation from Escherichia coli and further characterized based on their acceptor specificities. 2-Azidoethyl-glycosides corresponding to the oligosaccharides of GD3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GM2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GD2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), and GM1 (beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) were synthesized in high yields (gram-scale). In addition, a mammalian alpha-(2-->3)-sialyltransferase (ST3Gal I) was used to sialylate GM1 and generate GD1a (alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) oligosaccharide. We also cloned and expressed a rat UDP-N-acetylglucosamine-4'epimerase (GalNAcE) in E. coli AD202 cells for cost saving in situ conversion of less expensive UDP-GlcNAc to UDP-GalNAc.     

Cytotoxicity, apoptosis, cellular uptake, cell cycle distribution, and DNA-binding investigation of ruthenium complexes

Three new ruthenium(II) polypyridyl complexes [Ru(bpy)(2)(BHIP)](2+) 1, [Ru(phen)(2)(BHIP)](2+) 2, and [Ru(dip)(2)(BHIP)](2+) 3 were synthesized and characterized. The cytotoxicity of the three complexes was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptosis induced by the complexes was studied by cell morphology and flow cytometry. The results showed that the percentage of apoptotic cells is 7.19%, 75.58%, and 3.51% in the presence of complexes 1, 2, and 3, respectively. The cellular uptakes were also performed and the results indicated that complexes 1, 2, and 3 can enter into the cytoplasm and also into the nucleus. The studies on antiproliferative mechanism showed the induction of S-phase arrest by complexes 1, 2, and 3. DNA-binding constants of these complexes with calf thymus DNA (CT-DNA) were determined to be 1.07 (± 0.47) × 10(5) M(-1) (s = 2.04), 1.21 (± 0.32) × 10(5) M(-1) (s = 1.88), and 2.75 (± 0.27) × 10(5) M(-1) (s = 2.17), respectively. Upon irradiation at 365 nm, complexes 1, 2, and 3 can induce cleavage of pBR322 DNA.     

Molecular orbital study of complexes of zinc(II) with sulphide, thiomethanolate, thiomethanol, dimethylthioether, thiophenolate, formiate, acetate, carbonate, hydrogen carbonate, iminomethane and imidazole. Relationships with structural and catalytic zinc in some metallo-enzymes.

Geometry optimization and energy calculations have been performed at the density functional B3LYP/LANL2DZ level on hydrogen sulfide (HS-), dihydrogensulfide (H2S), thiomethanolate (CH3S-), thiomethanol (CH3SH), thiophenolate (C6H5S-), methoxyde (CH3O-), methanol (CH3OH), formiate (HCOO-), acetate (CH3COO-), carbonate (CO3(2-)), hydrogen carbonate (HCO3-), iminomethane (NH=CH2), [ZnS], [ZnS2]2-, [Zn(HS)]+, [Zn(H2S)]2+, [Zn(HS)4]2-, [Zn(CH3S)]+, [Zn(CH3S)2], [Zn(CH3S)3]-, [Zn(CH3S)4]2-, [Zn(CH3SH)]2+, [Zn(CH3SCH3)]2+, [Zn(C6H5S)]+, [Zn(C6H5S)2], [Zn(C6H5S)3]-, [Zn(HS)(NH=CH2)2]+, [Zn(HS)2(NH=CH2)2], [Zn(HS)(H2O)]+, [Zn(HS)(HCOO)], [Zn(HS)2(HCOO)]-, [Zn(CH3O)]+, [Zn(CH3O)2], [Zn(CH3O)3]-, [Zn(CH3O)4]2, [Zn(CH3OH)]2+, [Zn(HCOO)]+, [Zn(CH3COO)]+, [Zn(CH3COO)2], [Zn(CH3COO)3]-, [Zn(CO3)], [Zn(HCO3)]+, and [Zn(HCO3)(Imz)]+ (Imz, 1,3-imidazole). The computed Zn-S bond distances are 2.174A for [ZnS], 2.274 for [Zn(HS)]+, 2.283 for [Zn(CH3S)]+, and 2.271 for [Zn(C6H5S)]+, showing that sulfide anion forms stronger bonds than substituted sulfides. The nature of the substituents on sulfur influences only slightly the Zn-S distance. The optimized tetra-coordinate [Zn(HS)2(NH=CH2)2] molecules has computed Zn-S and Zn-N bond distances of 2.392 and 2.154A which compare well with the experimental values at the solid state obtained via X-ray diffraction for a number of complex molecules. The computed Zn-O bond distances for chelating carboxylate derivatives like [Zn(HOCOO)]+ (1.998A), [Zn(HCOO)]+ (2.021), and [Zn(CH3COO)]+ (2.001) shows that the strength of the bond is not much influenced by the substituent on carboxylic carbon atom and that CH3- and HO- groups have very similar effects. The DFT analysis shows also that the carboxylate Ligand has a preference for the bidentate mode instead of the monodentate one, at least when the coordination number is small.     

Cytotoxicity, apoptosis, cellular uptake, cell cycle arrest, photocleavage, and antioxidant activity of 1, 10-phenanthroline ruthenium(II) complexes

Two new ruthenium(II) complexes, [Ru(phen)2(DNPIP)](ClO4)2 (1) and [Ru(phen)2(DAPIP)](ClO4)2 (2), were synthesized and characterized. The DNA-binding properties of these complexes were investigated using UV/vis absorption titration, viscosity measurements, thermal denaturation, and photoactivated cleavage. The DNA binding constants for complexes 1 and 2 are 2.63?±?0.25×10(5) M(-1) (s=2.45) and 1.51±0.18×10(5) M(-1) (s=1.34). The results indicated that these complexes interacted with DNA through the intercalative mode. The cytotoxicity in vitro of complexes 1 and 2 were assessed against BEL-7402, HepG-2, and MCF-7 cell lines by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was studied with the acridine orange/ethidium bromide staining method. The antiproliferative mechanism was explored with flow cytometry. Cellular uptake studies showed that complexes 1 and 2 can enter into the cytoplasm and accumulate in the nuclei. Cell cycle arrest and antioxidant activity were also investigated.     

Raman spectroscopy of DNA-metal complexes. I. Interactions and conformational effects of the divalent cations: Mg, Ca, Sr, Ba, Mn, Co, Ni, Cu, Pd, and Cd.

Interactions of divalent metal cations (Mg2+, Ca2+, Ba2+, Sr2+, Mn2+, Co2+, Ni2+, Cu2+, Pd2+, and Cd2+) with DNA have been investigated by laser Raman spectroscopy. Both genomic calf-thymus DNA (> 23 kilobase pairs) and mononucleosomal fragments (160 base pairs) were employed as targets of metal interaction in solutions containing 5 weight-% DNA and metal:phosphate molar ratios of 0.6:1. Raman difference spectra reveal that transition metal cations (Mn2+, Co2+, Ni2+, Cu2+, Pd2+, and Cd2+) induce the greatest structural changes in B-DNA. The Raman (vibrational) band differences are extensive and indicate partial disordering of the B-form backbone, reduction in base stacking, reduction in base pairing, and specific metal interaction with acceptor sites on the purine (N7) and pyrimidine (N3) rings. Many of the observed spectral changes parallel those accompanying thermal denaturation of B-DNA and suggest that the metals link the bases of denatured DNA. While exocyclic carbonyls of dT, dG, and dC may stabilize metal ligation, correlation plots show that perturbations of the carbonyls are mainly a consequence of metal-induced denaturation of the double helix. Transition metal interactions with the DNA phosphates are weak in comparison to interactions with the bases, except in the case of Cu2+, which strongly perturbs both base and phosphate group vibrations. On the other hand, the Raman signature of B-DNA is largely unperturbed by Mg2+, Ca2+, Sr2+, and Ba2+, suggesting much weaker interactions of the alkaline earth metals with both base and phosphate sites. A notable exception is a moderate perturbation by alkaline earths of purine N7 sites in 160-base pair DNA, with Ca2+ causing the greatest effect. Correlation plots demonstrate a strong interrelationship between perturbations of Raman bands assigned to ring vibrations of the bases and those of bands assigned to exocyclic carbonyls and backbone phosphodiester groups. However, strong correlations do not occur between the Raman phosphodioxy band (centered near 1092 cm-1) and other Raman bands, suggesting that the former is not highly sensitive to the structural changes induced by divalent metal cations. The structural perturbations induced by divalent cations are much greater for > 23-kilobase pair DNA than for 160-base pair DNA, as evidenced by both the Raman difference spectra and the tendency toward the formation of insoluble aggregates. In the presence of transition metals, aggregation of high-molecular-weight DNA is evident at temperatures as low as 11 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis, antimalarial, antileishmanial, antimicrobial, cytotoxicity, and methemoglobin (MetHB) formation activities of new 8-quinolinamines

We report the synthesis, in vitro antiprotozoal (against Plasmodium and Leishmania), antimicrobial, cytotoxicity (Vero and MetHb-producing properties), and in vivo antimalarial activities of two series of 8-quinolinamines. N1-{4-[2-(tert-Butyl)-6-methoxy-8-quinolylamino]pentyl}-(2S/2R)-2-aminosubstitutedamides (21-33) and N1-[4-(4-ethyl-6-methoxy-5-pentyloxy-8-quinolylamino)pentyl]-(2S/2R)-2-aminosubstitutedamides (51-63) were synthesized in six steps from 6-methoxy-8-nitroquinoline and 4-methoxy-2-nitro-5-pentyloxyaniline, respectively. Several analogs displayed promising antimalarial activity in vitro against Plasmodium falciparum D6 (chloroquine-sensitive) and W2 (chloroquine-resistant) clones with high selectivity indices versus mammalian cells. The most promising analogs (21-24) also displayed potent antimalarial activity in vivo in a Plasmodium berghei-infected mouse model. Most interestingly, many analogs exhibited promising in vitro antileishmanial activity against Leishmania donovani promastigotes, and antimicrobial activities against a panel of pathogenic bacteria and fungi. Several analogs, notably 21-24, 26-32, and 60, showed less MetHb formation compared to primaquine indicating the potential of these compounds in 8-quinolinamine-based antimalarial drug development.     

Synthesis,Characterization, Cellular Uptake,Apoptosis, Cytotoxicity,Dna-Binding,and Antioxidant Activity Studies of Ruthenium(II) Complexes

Two new ruthenium(II) polypyridyl complexes [Ru(dmb)₂(HECIP)](ClO₄)₂ (1) (HECIP = N-ethyl-4-[(1,10)-phenanthroline(5,6-f)imidazol-2-yl]carbazole, dmb = 4,4’-dimethyl-2,2’-bipyridine) and [Ru(dmp)₂(HECIP)](ClO₄)₂ (2) (dmp = 2,9-dimethyl-1,10-phenanthroline) have been synthesized and characterized. The DNA-binding behaviors of the two complexes were investigated by absorption spectra, viscosity measurements, and photoactivated cleavage. The DNA-binding constants for complexes 1 and 2 were determined to be 8.03 (± 0.12) × 10⁴ M^?1 (s = 1.62) and 2.97 (± 0.15) × 10⁴ M^?1 (s = 1.82), respectively. The results suggest that these complexes interact with DNA through intercalative mode. The photocleavage of pBR322 DNA by Ru(II) complexes was investigated. The cytotoxicity of complexes 1 and 2 has been evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)] method. Complex 1 shows higher anticancer potency than 2 against the four tumor cell lines. Apoptosis and cellular uptake were investigated. The antioxidant activities of the ligand and these complexes were also performed.     

Nitrotyrosine, dityrosine, and nitrotryptophan formation from metmyoglobin, hydrogen peroxide, and nitrite

The biological relevance of tyrosine nitration is a subject of much interest, because extensive evidence supports formation of 3-nitrotyrosine in vivo under a variety of different pathological conditions. Several reagents are likely to be responsible for nitration in vivo, among others peroxynitrite and nitrite in the presence of H(2)O(2)/peroxidases. In this work we show that also metmyoglobin and methemoglobin can nitrate free tyrosine in the presence of nitrite and H(2)O(2). The results of these studies are simulated rather well by using a scheme that comprehends all the possible reactions that can take place in the system. Thus, a good understanding of the factors that determine the yields is achieved. Finally, we demonstrate that the system metMb/H(2)O(2)/NO(2)(-) can also lead to the nitration of tryptophan and produces, in particular, 6-, 4-, and 5-nitrotryptophan.     

Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav.

Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.     

Design,Drug-Likeness,Synthesis, Characterization,Antimicrobial Activity,Molecular Docking,and MTT Assessment of 1,3-Thiazolidin-4-one Bearing Piperonal and Pyrimidine Moieties

Russian Journal of Bioorganic Chemistry - The recent study reported the designing of substituted 3-[4-(1,3-benzodioxol-5-yl)-6-(pyridin-2-yl)pyrimidin-2-yl]-2-(pyridin-2-yl)-1,3-thiazolidin-4-one...     

Metabolism of the endocannabinoids, 2-arachidonylglycerol and anandamide,into prostaglandin,thromboxane, and prostacyclin glycerol esters and ethanolamides

Cyclooxygenase-2 (COX-2) action on the endocannabinoids, 2-arachidonylglycerol (2-AG) and anandamide (AEA), generates prostaglandin glycerol esters (PG-G) and ethanolamides (PG-EA), respectively. The diversity of PG-Gs and PG-EAs that can be formed enzymatically following COX-2 oxygenation of endocannabinoids was examined in cellular and subcellular systems. In cellular systems, glycerol esters and ethanolamides of PGE(2), PGD(2), and PGF(2alpha) were major products of the endocannabinoid-derived COX-2 products, PGH(2)-G and PGH(2)-EA. The sequential action of purified COX-2 and thromboxane synthase on AEA and 2-AG provided thromboxane A(2) ethanolamide and glycerol ester, respectively. Similarly, bovine prostacyclin synthase catalyzed the isomerization of the intermediate endoperoxides, PGH(2)-G and PGH(2)-EA, to the corresponding prostacyclin derivatives. Quantification of the efficiency of prostaglandin and thromboxane synthase-directed endoperoxide isomerization demonstrated that PGE, PGD, and PGI synthases catalyze the isomerization of PGH(2)-G at rates approaching those observed with PGH(2). In contrast, thromboxane synthase was far more efficient at catalyzing PGH(2) isomerization than at catalyzing the isomerization of PGH(2)-G. These results define the in vitro diversity of endocannabinoid-derived prostanoids and will permit focused investigations into their production and potential biological actions in vivo.     

The synthesis and characterisation of some MnLnX2 complexes (n = 1, L = pyridine, 4-pyrrolidinopyridine,X = Cl,Br, I,NCS; n = 1, L = EtOH,X = Cl,Br; n = 2, L = EtOH,X = I,NCS)

Mono-ligand complexes MnLX₂ (L = pyridine, 4-pyrrolidinopyridine, X = Cl, Br, I, NCS) have been prepared. The pyridine complexes contain only bridging halide and pseudohalide groups, whereas the 4-py complexes contain both bridging and terminal bound anions. Ethanol is coordinated in Mn(EtOH)X₂ (X=C1, Br) and Mn(EtOH)₂X₂ (X=I, NCS). Although the MnLX₂ complexes have the same stoichiometry as the Mn(PR₃)X₂complexes none show any activity towaxds binding dioxygen either in the solid state or in solution.     

Rat liver ATP-sulfurylase: purification, kinetic characterization, and interaction with arsenate, selenate, phosphate, and other inorganic oxyanions

ATP-sulfurylase (ATP:sulfate adenylyltransferase; EC 2.7.7.4), the first enzyme of the two-step sulfate activation sequence, was purified extensively from rat liver cytosol. The enzyme has a native molecular mass of 122 +/- 12 kDa and appears to be composed of identical 62 +/- 6-kDa subunits. At 30 degrees C and pH 8.0 (50 mM Tris-Cl buffer containing 5 mM excess Mg2+), the best preparations have "forward reaction" specific activities of about 20 and 2 units X mg protein-1 with MoO4(2-) and SO4(2-), respectively. The reverse (ATP synthesis) specific activity is about the same as the forward molybdolysis activity. The kinetic constants under the above conditions are as follows: KmA = 0.21 mM, Kia = 0.87 mM, KmB = 0.18 mM, KmQ = 0.65 microM, Kiq = 0.11 microM, and KmP = 5.0 microM where A = MgATP, B = SO4(2-), Q = APS, and P = total PPi at 5 mM Mg2+. PPi is a mixed-type inhibitor with respect to MgATP and SO4(2-). SeO4(2-) is an alternative inorganic substrate with a Vmax about 20% that of SO4(2-). The product, APSe, is unstable. But in the presence of a sufficient excess of APS kinase, APSe is completely converted to PAPSe. The rate constant for nonenzymatic PAPSe hydrolysis was determined from measurements of the final steady-state reaction rate in the presence of limiting initial SeO4(2-) and a large excess of MgATP, ATP sulfurylase, APS kinase, and the other coupling enzymes and their cosubstrates. The results yielded a k of 2.4 +/- 0.5 X 10(-3) sec-1 (t1/2 ca. 5 min). Phosphate is an effective buffer for enzyme purification and storage but inhibits catalytic activity, particularly at low substrate concentrations. In the presence of buffer levels of Pi, the MgATP reciprocal plot of the SO4(2-)-dependent reaction is concave-up. Inorganic monovalent oxyanions are dead end inhibitors competitive with SO4(2-) and apparently uncompetitive with respect to MgATP. The relative potencies are in the order ClO3- greater than ClO4- greater than FSO3- greater than NO3-. Thiosulfate is also competitive with SO4(2-) but noncompetitive with respect to MgATP. Several divalent oxyanions (MoO4(2-), WO4(2-), CrO4(2-), and HAsO4(2-] promote the enzyme-catalyzed cleavage of MgATP to AMP and MgPPi. The ratio Vmaxf/KmA ranged from 0.7 to 200 for various reactive inorganic substrates. The cumulative results suggest the random binding of MgATP and the inorganic substrate but the ordered release of MgPPi before APS.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic structure of the Mongols as derived from the ABO, MN, Rh, EsD, GLO1, PGM1, AcP, 6-PGD, Hp, Gc, Tf, C'3 and ChE2 loci

According to integral characterization of gene frequencies of the investigated loci AB0, MN, Rh, GLO1, PGM1, EsD, AcP, 6-PGD, Hp, Tf, Gc, C'3 and ChE2, Mongolian population has high level of polymorphism, with the exception of haplotypes R" (cdE) and Ry(CdE) at the Rh locus and TfB0-1 at the Tf locus. The data on biochemical and immunological polymorphic gene markers analysed in the population of Mongolia show that the Mongolians have some distinctive features, in comparison with the mean-in-the-world characteristics: high frequencies of the B genes at the AB0 locus; D, E, R1 and R2 at the Rh locus; GLO11, PGDc, TfDChi, E2(C5+), PGM1(1+); low frequencies of the genes A(AB0), R0(Rh), AcPc, Hp1, Gc2, C'3F, PGM 1(2-); the rest of the genes at the above-mentioned loci and the genes of the locus MN have the mean-in-the-world frequencies.     

Superoxide radical detection in cells, tissues, organisms (animals, plants, insects, microorganisms) and soils

A simple protocol is presented for the assessment of superoxide radical in organisms (animal/plant tissues, microorganisms, cell cultures, biological/culture fluids) and soils, through the quantification of 2-hydroxyethidium (2-OH-E+), its specific reaction product with hydroethidine (HE). It is an alternative to the quantification of 2-OH-E+ by HPLC (restricted to cell cultures), offering the advantage of the in vivo assessment of superoxide radical in a wide range of experimental systems. The protocol includes alkaline-acetone extraction of the sample, purification by microcolumn cation exchange and hydrophobic chromatographies, and fluorescence detection of the isolated 2-OH-E+/HE-oxidation products mixture before and after consumption of 2-OH-E+ by a horseradish peroxidase/hydrogen peroxide system. The protocol is sensitive at <1 pmol 2-OH-E+ per mg protein (extended to the femto level when using large samples) in biological systems, and in soils at 9 pmol superoxide radical per gram of soil. The protocol includes a cytochrome c-based subprotocol for superoxide radical detection in soils at 770 pmol g(-1) soil. For processing ten samples and depending on the experimental material used (soil or biological), the approximate procedure time would be 2-7 h.     

Effects of prostaglandins, dibutyryl cAMP, LHRH, estrogens, progesterone, and potassium on output of prostaglandin F2 alpha, 13, 14-dihydro-15-keto-prostaglandin F2 alpha, hCG, estradiol, and progesterone by placental minces

In order to compare the endocrine response of placental minces to luteinizing hormone releasing hormone (LHRH) and dibutyryl cAMP (dbcAMP) and to screen for effects of potential stimulatory and inhibitory substances, the simultaneous outputs of PGF2 alpha, 13, 14-dihydro-15-keto-prostaglandin F2 alpha (PGFM), progesterone, 17 beta-estradiol, and hCG were evaluated during a 4 hour incubation in 5 placentas. The output of hCG was highest for 12-week placentas, intermediate for a 16 week placenta, and lowest for term placentas. The output of 17 beta-estradiol by 12 and 16 week placentas in the presence of 30 microM dehydroepian-drosterone sulfate (DHEAS) was greater than that by term placentas. Progesterone output was apparently independent of gestational age although some variation between 12-week placentas was demonstrated. Output of PGF2 alpha was lower in 12 and 16-week placentas than in term placentas and that of PGFM was lower in 12-week placentas than in term placentas. LHRH (100 nM) produced stimulation of PGF2 alpha output (P less than .005) and a trend toward inhibition of progesterone output (which failed to achieve statistical significance) but no stimulation of hCG under these conditions. Stimulation of the outputs of hCG (P less than .005) and PGF2 alpha (P less than .001) and inhibition of that of progesterone (P less than .005) was produced by 20 mM dbcAMP. DHEAS inhibited output of progesterone (P less than .01) and PGF2 alpha (P less than .01). There were no effects of potassium, estrogens, progesterone, or prostaglandins on output of any measured substance.     

Ruthenium(II) polypyridyl complexes: synthesis and studies of DNA binding, photocleavage, cytotoxicity, apoptosis, cellular uptake, and antioxidant activity

Two ruthenium (II) complexes [Ru(dmb)2(APIP)](ClO4)2 (APIP=2-(2-aminophenyl)imidazo[4,5-f?][1,10]phenanthroline, dmb=4,4'-dimethyl-2,2'-bipyridine; 1) and [Ru(dmb)2(HAPIP)](ClO4)2 (HAPIP=2-(2-hydroxyl-4-aminophenyl)imidazo[4,5-f?][1,10]phenanthroline; 2) were synthesized and characterized. DNA binding was investigated by electronic absorption titration, luminescence spectra, thermal denaturation, viscosity measurements, and photocleavage. The DNA binding constants for complexes 1 and 2 were 4.20 (±0.14)×10(4) and 5.45 (±0.15)×10(4) M(-1). The results suggest that these complexes partially intercalate between the base pairs. The cytotoxicity of complexes 1 and 2 was evaluated by MTT assay. Cellular uptake was observed under fluorescence microscopy; complexes 1 and 2 can enter into the cytoplasm and accumulate in the nuclei. Apoptosis and the antioxidant activity against hydroxyl radicals (?OH) were also explored.     

Effect of fibroblast growth factors 1, 2, 4, 5, 6, 7, 8, 9, and 10 on avian chondrocyte proliferation.

It has been demonstrated that fibroblast growth factor receptors are key regulators of endochondral bone growth. However, it has not been determined what fibroblast growth factor ligand(s) (FGFs) are important in this process. This study sought to determine whether FGFs 1, 2, 4, 5, 6, 7, 8, 9, and 10 were capable of stimulating avian chondrocyte proliferation in vitro. We have found that FGFs 2, 4, and 9 strongly stimulate avian chondrocyte proliferation while FGFs 6 and 8 stimulate proliferation to a lesser extent. RT-PCR indicates that FGF-2 and FGF-4 are expressed in the postnatal avian epiphyseal growth plate (EGP) while FGF-8 and FGF-9 are not. Thus, FGF-2 and FGF-4 stimulate chondrocyte proliferation and are both present in the EGP. This suggests that FGF-2 and FGF-4 may be important ligands, in vivo, for the regulation of endochondral bone growth. These observations coupled with our observation that multiple avian FGF receptors (Cek1, Cek2, Cek3, and FREK) are expressed in proliferative chondrocytes highlights the complexity of FGF signaling pathways in postnatal endochondral bone growth.