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Modeling the 3-D RNA distribution in the Balbiani ring granule.

Mature Balbiani Ring (BR) granules in situ were stained with the nucleic acid specific stain, osmium ammine-B, recorded by electron spectroscopic imaging and reconstructed by electron microscope tomography to examine the three-dimensional (3-D) distribution of BR heterogeneous nuclear RNA (hnRNA). The BR2 granules contain ca. 37 kb of mRNA. Reconstructed BR granules were selected to emphasize one of the prevalent conformations seen in the sectioned salivary glands, the en face or "pin-wheel" conformation. A variety of image processing and volume-rendering operations were applied to the set of reconstructed BR granules. Some of the conclusions of this study are the following: (1) RNA distribution is not uniform throughout the granule; (2) RNA is condensed into about ten particles per granule, which all appear to possess approximately the same RNA stain density; (3) heterogeneity exists in the positions and sizes of particles within the various BR granules. These data argue for the folding of a beaded ribbon, consisting of connected particulate condensations of BR mRNA, possessing considerable 3-D flexibility, even in the packaged state. A comparison of this beaded-ribbon model and a prior folded hnRNP fiber model is also presented.

Animals↗

The electron density of light and dark lysosomes in the proximal convoluted tubule of the rat kidney.

The present paper deals with the electron density of the lysosomal matrices in the renal proximal-convoluted-tubule cells of Wistar rats (20-60 days of age). Its purpose was to determine which physicochemical factors influence the electron density of lysosomes, and how their electron density is affected by various methods of fixation.--3 types of lysosomes can be distinguished in the proximal-convoluted-tubule cell; namely light, intermediate and dark lysosomes, which have an electron density lower, equal to or higher than the surrounding cytoplasm. All 3 types of lysosomes were invariably present after all methods of fixation tested. Light, intermediate and dark lysosomes differ in several respects. Staining intensity with uranyl acetate, lead citrate and potassium permanganate, osmiophilia, basophilia (in semithin sections) and --probably most importantly--the physical mass density of the lysosomal matrix are all low in light lysosomes, higher in intermediate lysosomes and highest in dark lysosomes. Light, intermediate and dark lysosomes of the proximal convoluted tubule do not form discrete classes, but one continuous spectrum of lysosomes of increasing electron density.

Animals↗

Ultrastructural investigation of DNA in megakaryoblastic leukemia by using osmium-ammine-B: comparison with several types of leukemia.

Using DNA staining with highly stable osmium-ammine-B, the blasts of AMKL were observed under an electron microscope, in comparison with AML, ALL, CML-MK crisis and TMD. The DNA within the nucleus of the megakaryoblasts was observed as a high electron-density substance and tended to be uniformly dispersed within the nucleus. DNA, associated with nucleoli, could be roughly divided in four types based on the presence or absence of peri-nucleolar clumps and intra-nucleolar clumps. In cases of AMKL, we often observed a type that did not have peri-nucleolar DNA clumps but did have DNA flecks within the nucleoli. By analytical evaluation there were no differences among cells from several subtypes of megakaryocytic leukemia, such as AMKL in children with Down's syndrome, AMKL in children without Down's syndrome, AMKL in adults, and CML-MK crisis. The DNA distribution of TMD blasts, which were self-limited and not malignant, resembled that of AMKL blasts.

Adolescent↗

Dinuclear versus mononuclear ruthenium(II) and osmium(II) complexes as potent mediators of glucose oxidase; crystal structure of [OsCl(4,4'-bpy)(bpy)2]BF4.

New and known homo- and heterodinuclear Ru(II) and Os(II) complexes with 4,4'-bipyridine (4,4'-bpy), pyrazine, and 4-pyCH=CHpy-4' as bridging ligands (LL) of the type [Cl(bpy)(2)M(LL)MCl(bpy)(2)]X(2) (bpy=2,2'-bipyridine; X=PF(6) or BF(4)) have been studied in their capacity to exchange electrons with a reduced active site of glucose oxidase (GO) from Aspergillus niger. Cyclic voltammograms (CVs) of the dimers in the aqueous buffered solution, when compared with CVs of the parent monomeric species [MCl(LL)(bpy)(2)]BF(4) and [MCl(2)(bpy)(2)] which could be generated at pH approximately 7, if the dimers undergo monomerization, indicate that the dimers are the dominating species under such conditions. All electrochemically oxidized dinuclear complexes studied show high rates of oxidation of GO reduced by D-glucose and the corresponding observed second-order rate constants are in the range (5-64)x10(5) M(-1) s(-1) at 25 degrees C. However, these values are lower than that for the mononuclear complex [OsCl(4,4'-bpy)(bpy)(2)]BF(4) (1.1x10(7) M(-1) s(-1)), suggesting that potentially two-electron dimeric mediators have no advantage compared with corresponding monomeric complexes of Ru(II) and Os(II). The structure of [OsCl(4,4'-bpy)(bpy)(2)]BF(4) was confirmed by X-ray crystallography. The monodentate 4,4'-bpy ligand is coordinated cis to the chloride. Its higher reactivity toward reduced GO is accounted for in terms of the antenna effect of the monodentate 4,4'-bpy ligand. The antenna length equals 9.2 A and matches the depth of the enzyme active site pocket of ca. 10 A. The mechanism of the antenna effect is discussed.

2,2'-Dipyridyl↗

Spectrophotometric kinetic study and analytical implications of the glucose oxidase-catalyzed reduction of [MIII(LL)2Cl2]+ complexes by D-glucose (M = Os and Ru, LL = 2,2'-bipyridine and 1,10-phenanthroline type ligands).

Glucose oxidase-catalyzed reduction of cis-[MIII(LL)2Cl2]+ (M = Os and Ru) complexes to cis-[MII(LL)2Cl2] (LL = 2,2'-bipyridine and 1,10-phenanthroline type ligands) by D-glucose is a first-order process in the complex and the enzyme in aqueous buffered solution. The reaction follows Michaelis-Menten kinetics in D-glucose and the rate is independent of D-glucose concentration above 0.03 M. The reactivity decreases in the series [Ru(bpy)2Cl2]+ > [Os(phen)2Cl2]+ > [Os(4,4'-Me2bpy)2Cl2]+ > [Os(4,7-Me2phen)2Cl2]+. The measured second-order rate constant for the oxidation of reduced glucose oxidase by [Os(phen)2Cl2]+ in air equals 1.2 x 10(5) M-1 s-1 at pH 6,7, [D-glucose] 0.05 M, and 25 degrees C, which is ca. 20% less than that when the reaction solutions are purged with argon. In the case of [Ru(bpy)2Cl2]+ the rate constant equals 1.8 x 10(5) M-1 s-1 under similar conditions in air, showing higher reactivity of Ru complexes compared with Os ones. The reduction is pH-dependent with a maximum around 7. Added for solubilization of poorly soluble metal complexes, surfactants decrease the rates of the enzymatic reaction. The retardation effect increases in the series: cetyltrimethylammonium bromide < Triton X-100 << sodium dodecyl sulfate, i.e. on going from positively charged to neutral and then to negatively charged surfactants. The behavior of the OsIII and RuIII complexes toward reduced glucose oxidase contrasts to that of recently studied ferricenium cations. As opposed to the latter, the former do not show kinetically meaningful binding with the enzyme, and the Michaelis kinetics typical of the ferricenium case is not realized for the OsIII, and RuIII species. The systems OsIII- or RuIII-glucose oxidase are convenient for routine "one pot" spectrophotometric monitoring of the D-glucose content in samples, since the metal reduction to MII is accompanied by a strong increase in absorbance in the visible spectral region.

Aspergillus niger↗

Water-soluble benzoheterocycle triosmium clusters as potential inhibitors of telomerase enzyme.

We have studied the ability of several bioorganometallic clusters [(mu-H)Os(3)(CO)(9)(L)(mu(3)-eta(2)-(Q-H))], where L = [P(C(6)H(4)SO(3)Na)(3)] or [P(OCH(2)CH(2)NMe(3)I)(3)], and Q = quinoline, 3-aminoquinoline, quinoxaline or phenanthridine, of inhibiting telomerase, a crucial enzyme for cancer progression. In general, quinolines have shown interesting biological properties, especially in inhibiting enzymes. For example, the 2,3,7-trichloro-5-nitroquinoxaline (TNQX) exhibited strong anti-telomerase activity in vitro. Among the quinoline-clusters under study, only the negatively charged ones (by virtue of the sulfonated phosphines) exhibited good anti-telomerasic activity on semi-purified enzyme in a cell-free assay, while they were ineffective in vitro on Taq, a different DNA-polymerase. On the contrary, the treatment of breast cancer MCF-7 cell line did not evidence any activity of these clusters, suggesting a low aptitude for crossing cell membrane. Furthermore, all clusters exhibited non-specific, acute cytotoxicy, probably due to accumulation on cell membranes by virtue of their amphiphilic character. A detailed study of Os uptake and accumulation in MCF-7 cells supported this hypothesis.

Biological Transport, Active↗

Production and characterization of a polyclonal antibody for Os(II) and Ru(II) polypyridyl complexes.

The characterization of a polyclonal antibody produced via immunization with an [Os(bpy)(2)dcbpy] hapten is described. Bpy is 2,2'-bipyridine and dcbpy is 2,2'-bipyridine-4,4'-dicarboxylic acid. The cross-reactivity of the antibody for the Ru(II) analogue of the hapten was also investigated. Large increases in the emission and luminescent lifetime of a series of Os and Ru complexes were observed on binding of the antibody. Association equilibrium constants were derived from luminescence titration data and were found to be 5.6 x 10(8) and 5.0 x 10(8)M(-1) for [Os(bpy)(2)dcbpy] and [Ru(bpy)(2)dcbpy], respectively. Spectroscopic changes were likely due to the exclusion of H(2)O from the complex/antibody binding cleft and blocking of vibrational relaxation pathways of the Os/Ru excited state. D(2)O/H(2)O experiments confirmed that the antibody protected approx. 82% of [Os(bpy)(2)dcbpy] and 80% of [Ru(bpy)(2)dcbpy] from excited state deactivation by the aqueous solvent.

Animals↗

Theoretical studies on vibrational spectra of some mixed carbonyl-halide complexes of Osmium(II).

The vibrational spectra of Os(CO)(6)(2+) and some of its mixed carbonyl-halide complexes, cis-Os(CO)(2)X(4)(2-), fac-Os(CO)(3)X(3)(-) and Os(CO)(5)X(+) (X=F, Cl, Br and I), have been systematically investigated by ab initio RHF and density functional B3LYP methods with LanL2DZ and SDD basis sets. The calculated vibrational frequencies of complexes Os(CO)(6)(2+), cis-Os(CO)(2)X(4)(2-) and fac-Os(CO)(3)X(3)(-) are evaluated via comparison with the experimental values. In infrared frequency region, the C-O stretching vibrational frequencies calculated at B3LYP level with two basis sets are in good agreement with the observed values with deviations less than 5%. In the far-infrared region, the B3LYP/SDD method achieved the best results with deviations less than 9% for Os-X stretching and less than 8% for Os-C stretching vibrational frequencies. The vibrational frequencies for Os(CO)(5)X(+) that have not been experimentally reported were predicted.

Carbon↗

Electron transfer between cytochrome c and metal hexacyanide complexes. Effect of thermodynamic driving force on the electron transfer rate.

The electron transfer reactions between ferrocytochrome c and three isomorphic hexacyanide complexes, [Fe(CN)6]3-, [Os(CN)6]3- and [Ru(CN)6]3-, have been studied using the method of photoexcitation. The transfer rates for [Os(CN)6]3- and [Ru(CN)6]3- are, respectively, about 45- and 200-times higher than that of [Fe(CN)6]3-. A reorganization energy of approx. 0.8 eV was found for the cytochrome c-hexacyanide system when the data were analyzed according to the theory of Marcus.

Cyanides↗

Electron microscopic demonstration of hyaluronidase sensible proteoglycans at the presynaptic area in mouse cerebellar cortex.

By application of electron cytochemical techniques to cerebellar tissue, the presence of proteoglycans was demonstrated at the axoplasmic matrix of mossy fiber endings. Blocks of glutaraldehyde (G) fixed mouse cerebellum were processed according to the following procedures: a) Some pieces of tissue were post-fixed in osmium tetroxide, dehydrated by ethanol and embedded in araldite. b) Other pieces were sectioned to 30 mum thick and then immersed in Alcian blue solution pH = 2.7 followed by osmium tetroxide fixation, dehydrated and embedded in araldite (GABOUL procedure). c) Parallel slices of (b) previous to Alcian blue immersion were washed and incubated in either methanol-HCl, neuraminidase, ribonuclease or testicular hyaluronidase with their respective controls. d) Other blocks of G fixed tissue without any other treatment and fixation were dehydrated and embedded in araldite. Ultrathin sections of a, b and c were doubly stained with uranyl acetate and lead citrate while ultrathin sections of (d) were stained with the osmium coordination compound Os-DMEDA. The electron microscopic study revealed at the presynaptic axoplasm of mossy fiber rosettes, the presence of a GABOUL and Os-DMEDA positive electron dense material surrounding synaptic vesicles and continuous with presynaptic dense projections. This material which coincides with cytonet distribution was resistant to neuraminidase and ribonuclease and sensible to hyaluronidase and carboxymethylation. These findings permit us to conclude that the axoplasmic material of mossy fiber endings is constituted by proteoglycans in which hyaluronic acid and chondroitin 4-and/or 6-sulphate are present. The probable importance of these proteoglycans in synaptic mechanisms is also discussed.

Alcian Blue↗

Morphological and cytochemical analysis of human cytomegalovirus inoculum: correlation of free particles in inoculum with counterparts in infected cells.

Electron microscopic examination of human cytomegalovirus (HCMV) inoculum, as used in the laboratory and generated by infection of human fibroblasts at low multiplicity, led to the distinction of 7 different structures. Complete virions constituted 38% of the inoculum. Non-infectious enveloped particles (NIEP) were also quite numerous (4.7%). Inoculum also contained other enveloped and non-enveloped particles. Dense bodies were the most numerous (50.2%). The Feulgen-like osmium ammine/SO2 reaction applied to ultrathin sections of inoculum suggests that NIEP, considered to be lacking DNA, may contain small and varying amounts of DNA. This DNA was lightly stained and appeared as a filamentous ring in the core structure, extending to the limits of the capsid. A correlation was established between particles identifiable in HCMV-infected cells and their free counterparts in the inoculum, which revealed that all intracytoplasmic particles are present in the inoculum. All of these elements could potentially contribute to virus-induced phenomena associated with HCMV infection of cells in vitro.

Cell Nucleus↗

DNA sensor with a dipyridophenazine complex of osmium(lI) as an electrochemical probe.

Application of a dipyrido[3,2-a:2',3'-c]phenazine (DPPZ)-type metal complex as an DNA electrochemical probe was studied. The introduction of electron-donating groups (NH2) was effective for controlling the redox potential and binding affinities of the DPPZ-type osmium complex. The [Os(DA-bpy)2DPPZ]2+ complex (DA-bpy; 4,4'-diamino-2,2'-bipyridine) had a lower half-wave potential (E 1/2) of 147 mV (vs Ag/AgCl) and higher binding affinity with DNA (binding constant, K = 3.1 x 10(7) M(-1)) than those of other complexes. With a single-stranded DNA immobilized gold electrode, the hybridization signal (deltaI) of the [Os(DA-bpy)2DPPZ]2+ complex was linear in the concentration range of 1.0 pg mL(-1) - 0.12 microg mL(-1) for the targeted DNA with a regression coefficient of 0.999. The detection limit was 0. 1 pg mL(-1). The 400-bp yAL3 gene was also detected with good sensitivity and selectivity using the [Os(DA-bpy)2DPPZ]2+ complex.

Biosensing Techniques↗

Study of layer-by-layer self-assembled viscoelastic films on thickness-shear mode resonator surfaces.

We describe quartz crystal electroacoustic admittance studies in thickness shear mode resonators loaded with self-assembled multilayers composed of alternate layers of glucose oxidase (GOx) and poly(allylamine) covalently attached to [Os(bpy)2ClPyCOH]- , (PAH-Os), deposited on a 3-mercaptopropanesulfonic acid (MPS)-modified gold on the quartz crystal. The complex acoustic impedance parameters, R(S) and X(LS) of a lumped-element Butterworth-Van Dyke (BVD) resonator have been determined for organized thin films of different thickness obtained by varying the number of enzyme layers, n, in (PAH-Os)n(GOx)n structures. The ellipsometric film thickness and mass for dry enzyme multilayer films and films in contact with water were evaluated, and the average film density was estimated. By combination of the estimated film thickness and density, the expression for the surface mechanical impedance of the lumped-element modified resonator (Granstaff and Martin model), and the liquid density and viscosity, we simulate the layer-by-layer film growth on the basis of the measured electroacoustic impedance. The complex impedance X(LS) and R(S) increase with film thickness and the enzyme films can be regarded as acoustically thin in the reduced state for films thinner than 600 nm. We have also measured electroacoustic parameters for PAH-Os/GOx self-assembled multilayers under electrochemical perturbation in a buffer electrolytic solution. The electrostatically self-assembled multilayers behaved as lossy viscoelastic films at 10 MHz with G'f and G''f on the order of 10(6) Pa. The films became viscoelastic upon oxidation to Os(III), resulting in an increase of R(S) and X(LS) in the oxidized state with the number of (PAH-Os)(GOx) bilayers due to film swelling and an increase in the shear moduli during oxidation.

Allylamine↗

Digital simulation of catalytic cyclic voltammograms for oxidation of DNA by a heterobimetallic dimer: effects of DNA binding and mass transport.

The electrocatalytic oxidation of DNA by a heterodimer, [(bpy)2Ru(tpphz)Os(bpy)2]4+ (tpphz: tetrapyrido[3,2-alpha: 2',3'-c:3'',2''-h:2''',3'''-j]phenazine) (1), was studied using cyclic voltammetry with digital simulation. This dimer was chosen because the Ru(III/II) couple (E1/2 = 1.09 V vs Ag/AgCl) is capable of catalyzing guanine oxidation while the Os(III/II) couple (E1/2 = 0.63 V) provides a convenient reporter on the binding and mass transport of the complex, which can then be determined in the same voltammetric sweep as the electrocatalysis. Proper description of the electrochemical response required careful measurement of the binding constant of 1 to herring testes (HT) DNA, which was (2.0 +/- 0.1) x 10(4) M(-1) by both absorption titration and normal pulse voltammetry. Thermal denaturation experiments were consistent with a nonintercalative binding mode and gave a deltaTm of only (2.4 +/- 0.5) degrees C. The minor groove binder distamycin did not displace 1 from HT DNA, suggesting that the complex binds in the major groove. As expected, acquisition of the cyclic voltammogram of 1 in the presence of DNA produced catalytic current for the Ru(III/II) couple, while a suppression of current was observed for the Os(III/II) couple. Although the catalytic current for the Ru(III/II) couple initially appeared as a current enhancement, higher concentrations suppressed the catalytic wave as a result of the slower mass transport of the DNA-bound complex. The binding studies were used to create a model for digital simulation that reproduced the behavior of 1 with DNA and gave rate constants that were independent of DNA concentration. The apparent second-order rate constant at 25 mV/s for oxidation of guanine in HT DNA (av 1000 bp, 25% guanine) by 1 was 3 +/- 1 x 10(4) M(-1) s(-1); similar values were obtained for a 200-bp fragment (7 +/- 3 x 10(4) M(-1) s(-1)) and a 435-bp fragment (8 +/- 2 x 10(4) M(-1) s(-1)). As observed in previous studies of these reactions, biphasic kinetics in the catalytic reaction led to a dependence of the rate constant determined by simulation on the sweep rate. Increasing the sweep rate led to a systematic increase in the simulated rate constant, consistent with a fast phase of the homogeneous catalytic reaction.

Animals↗