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PubMed1997

The conformation of DNA packaged in bacteriophage G.

When packaged in a bacteriophage capsid, double-stranded DNA occupies a cavity whose volume is roughly twice the volume of the DNA double helix. The data thus far have not revealed whether the compactness of packaged bacteriophage DNA is achieved by folding of the DNA, undirectional winding of the DNA, or a combination of both folding and winding. To assist in discriminating among these possibilities, the present study uses electron microscopy, together with ultraviolet light-induced DNA-DNA cross-linking, to obtain the following information about the conformation of DNA packaged in the comparatively large bacteriophage, G: 1) At the periphery of some negatively stained particles of bacteriophage G, electron microscopy reveals standards of DNA that are both parallel to each other and parallel to the polyhedral bacteriophage G capsid. However, these strands are not visible toward the center of the zone of packaged DNA. 2) Within some positively stained particles, electron microscopy reveals DNA-associated stain in relatively high concentration at corners of the polyhedral bacteriophage G capsid. 3) When cross-linked DNA is expelled from its capsid during preparation for electron microscopy, some DNA molecules consist primarily of a compacted central region, surrounded by DNA strands that appear to be unravelling at multiple positions uniformly distributed around the compacted DNA region. The above results are explained by a previously presented model in which DNA is compacted by folding to form 12 icosahedrally arranged pear-shaped rings.

Bacteriophages↗
PubMed2005

Comparative bioelectrochemical study of core-shell nanocluster films with ordinary layer-by-layer films containing heme proteins and CaCO3 nanoparticles.

Negatively charged heme protein hemoglobin (Hb) or myoglobin (Mb) at pH 9.0 and positively charged poly(diallyldimethylammonium) (PDDA) were alternately adsorbed on the surface of CaCO(3) nanoparticles, forming core-shell CaCO(3)-[PDDA/(protein/PDDA)(m)] ([protein-m]) nanoclusters. Oppositely charged [protein-m] and poly(styrenesulfonate) (PSS) were then assembled layer by layer on various solid substrates, forming {[protein-m]/PSS}(n) films. In the meantime, ordinary layer-by-layer films of heme proteins with CaCO(3) nanoparticles ({protein/CaCO(3)}(n)) were also grown on solid surfaces. Transmission electron microscopy (TEM), ultraviolet-visible (UV-vis) spectroscopy, quartz crystal microbalance (QCM), and cyclic voltammetry (CV) were used to characterize the nanoclusters and monitor the growth of the two types of films. Both kinds of protein films assembled on pyrolytic graphite (PG) electrodes exhibited well-defined, nearly reversible CV reduction-oxidation peaks, characteristic of heme Fe(III)/Fe(II) redox couples, and were used to catalyze the electrochemical reduction of hydrogen peroxide. The {[protein-m]/PSS}(n) films demonstrate distinct advantages over the {protein/CaCO(3)}(n) films due to their larger fraction of electroactive proteins, higher catalytic efficiency, and better thermostability. The penetration experiments of the electroactive probe into these films indicate that the {[protein-m]/PSS}(n) nanocluster films possess more pores or channels than the simple {protein/CaCO(3)}(n) films, which may be beneficial to counterion transport in the charge-hopping mechanism and helpful for the diffusion of catalysis substrates into the films. In addition, the electrochemical and biocatalytic activity of protein nanocluster films can be tailored by controlling the number of bilayers assembled on the nanoparticle cores (m) as well as the film thickness or the number of nanocluster layers on the electrodes (n).

Biochemistry↗
PubMed1973

Contrast in the photoelectric effect of organic and biochemical surfaces. A first step towards selective labeling in photoelectron microscopy.

The photoelectric effect can provide the physical basis for a new method of mapping organic and biological surfaces. The technique, photoelectron microscopy, is similar to fluorescence microscopy using incident ultraviolet light except that photoejected electrons form the image of the specimen surface. In this work the minimum wavelengths of incident light required to produce an image were determined for the molecules 3,6-bis(dimethylamino)acridine (acridine orange) (I), benzo[a]pyrene (II), N,N,N',N'-tetraphenylbenzidine (III), and copper phthalocyanine (IV). The photoelectron image thresholds for these compounds are 220 (I), 215 (II), 220 (III), and 240 nm (IV), all +/-5 nm. Contrast of I-IV with respect to typical protein, lipid, nucleic acid, and polysaccharide surfaces was examined over the wavelength range 240-180 nm. The low magnification micrographs exhibited bright areas corresponding to I-IV but dark regions for the biochemical surfaces. The high contrast suggests the feasibility of performing extrinsic photoelectron microscopy experiments through selective labeling of sites on biological surfaces.

Acridines↗
PubMed2005

Sustained release of ganciclovir from poly(lactide-co-glycolide) microspheres.

Biodegradable poly(lactide-co-glycolide) microspheres loaded with ganciclovir were produced using the emulsification/solvent evaporation technique. The effects of drug-to-polymer ratio and dispersion time on the drug content in the microspheres were investigated. The release rate of the drug was studied for 20 weeks in a phosphate buffered solution of pH 7 at 37 degrees C. Data revealed that lower drug content was obtained with increasing drug-to-polymer ratio and decreasing dispersion time. The release of the drug followed a triphasic release pattern, i.e. an initial burst, a diffusive phase and a second burst. The initial burst occurred within the first 2 days of immersion. After the burst, the release was by diffusion for up to 13 weeks, followed by another burst release, which signals the onset of bulk degradation of the polymer. Gel permeation chromatography (GPC), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and ultraviolet spectroscopy (UV) were used to follow the hydrolytic degradation and drug release rate of the microspheres.

Antiviral Agents↗
PubMed2004

Different deswelling behavior of temperature-sensitive microgels of poly(N-isopropylacrylamide) crosslinked by polyethyleneglycol dimethacrylates.

Polymerization of N-isopropylacrylamide (NIPAM) with polyethyleneglycol dimethacrylates (n G, n representing the number of --CH2CH2O-- units in polyethyleneglycol dimethacrylates) through surfactant-free radical polymerization was used to prepare the temperature-sensitive microgels. The morphology, dispersity, and deswelling behavior of the microgels were investigated by means of transmission electron microscopy (TEM), ultraviolet-visible spectroscopy, differential scanning calorimetry (DSC), and dynamic light scattering (DLS) techniques. TEM micrographs revealed that it was feasible to obtain regular spherical microgels for crosslinking agents with short chain. Turbidity, DSC, and DLS analysis showed that in marked contrast to 1G and 3G crosslinked microgels, the collapse of microgels crosslinked by 9G, 14G, and 23G proceeded in a two-step mechanism. The amide groups dehydrated at the lower temperature leading to the first-step transition. In the transition, the hydrophilic long --(--CH2CH2O--)n-- segments could be enriched on the surface of the microgels, which was further verified by variable temperature 1H NMR spectroscopy. The hydrophilic long --(--CH2CH2O--)n-- segments can be dehydrated at the higher temperature.

Acrylamides↗
PubMed2006

One-step synthesis of monodisperse silver nanoparticles beneath vitamin E Langmuir monolayers.

The monodisperse silver nanoparticles were synthesized by one-step reduction of silver ions in the alkaline subphase beneath vitamin E (VE) Langmuir monolayers. The monolayers and silver nanocomposite LB films were characterized by surface pressure-area (pi-A) isotherms, transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-vis), selected area electron diffraction (SAED), Fourier transform infrared transmission spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS), respectively. The results showed that the limiting area/VE molecule on different subphases varied. The phenolic groups in the VE molecules were converted to a quinone structure, and the silver ions were mainly reduced to ellipsoidal and spherical nanoparticles. The arrangement of the nanoparticles changed from sparseness to compactness with reaction time. The electron diffraction pattern indicated that the silver nanoparticles were face-centered cubic (fcc) polycrystalline. Silver nanocomposite LB films with excellent quality could be formed on different substrates, indicating that the transfer ratio of monolayer containing silver nanoparticles is close to unity. The dynamic process of reduction of silver ions by VE LB films was also studied through monitoring the conductivity of an Ag2SO4 alkaline solution.

Cations↗
PubMed2006

Resonance light scattering spectroscopy study of interaction between norfloxacin and calf thymus DNA and its analytical application.

The interaction between norfloxacin and calf thymus double-stranded DNA (dsDNA) has been studied by a resonance light scattering (RLS) technique with a common spectrofluorometer. The characteristics of RLS spectra, the effective factors and optimum conditions of the reaction have been investigated. In Britton-Robinson (BR) buffer (pH 5.87), norfloxacin has a maximum peak 405.5 nm and the RLS intensity is remarkably enhanced by trace amount of calf thymus dsDNA due to the interaction between norfloxacin and dsDNA. The binding of norfloxacin to DNA forms large particles, which were characterized by RLS spectrum, scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectrum, and fluorescence spectrum. Based on the enhanced RLS intensity, a novel method for sensitive determination of calf thymus dsDNA concentration ranging from 0.02 to 2.3 microg ml(-1) was developed. The determination limit (3 sigma) was 1.2 ng ml(-1). The method is simple, rapid, practical and relatively free from interference generated by coexisting substance, as well as much more sensitive than most of the reported methods. Three synthetic samples of ctDNA were determined with satisfactory results.

Animals↗
PubMed1998

Effect of anti-neutrophil cytoplasmic antibodies on proteinase 3-induced apoptosis of human endothelial cells.

Wegener's granulomatosis is characterized by crescentic necrotizing glomerulonephritis and systemic vasculitis. Both proteinase 3 (PR3) and anti-neutrophil cytoplasmic antibodies (ANCA), directed against this enzyme, are thought to play a pathogenic role. PR3 has been shown to cause detachment and cytolysis of human umbilical vein endothelial cells (HUVEC) in vitro and to induce apoptosis of bovine pulmonary artery endothelial cells. In the present study we investigated the effect of PR3 and ANCA on the induction of apoptosis of human endothelial cells in vitro. HUVEC were cultured in the absence or presence of varying concentrations of PR3 for different time periods and apoptosis was assessed by three different methods. Staining of the cells with Hoechst 33258 and assessment of nuclear morphology by ultraviolet (UV) light microscopy revealed a dose-dependent induction of apoptosis, as determined by cell counts. A concentration of 8 microg/ml PR3 was found to induce 16% apoptosis after 16 h incubation. Analysis of apoptosis by flow cytometry using the terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling (TUNEL) method also demonstrated a dose-dependent induction of apoptosis by PR3. DNA fragmentation was confirmed by agarose gel electrophoresis. To investigate the effect of ANCA on PR3-mediated apoptosis, HUVEC were exposed to immunoglobulin G (IgG) from patients with Wegener's granulomatosis or systemic vasculitis, and from normal controls, in the presence or absence of PR3. Enhancement of PR3-mediated apoptosis was found in two of 10 IgG samples with anti-PR3 activity, whereas a reduction in apoptosis was observed in two others. Anti-MPO (myeloperoxidase)-positive IgG, six additional anti-PR3 positive IgG samples and control IgG samples did not have any detectable effect on apoptosis. These studies suggest that ANCA may modulate the relative degree of injury in some cases of Wegener's granulomatosis.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1999

Extracellular matrix regulates apoptosis in human neutrophils.

BACKGROUND: During inflammation, polymorphonuclear neutrophils (PMNs) migrate into the affected tissue interacting with extracellular matrix (ECM) proteins. We tested the hypothesis that PMN-matrix interaction affects PMN apoptosis. METHODS: Apoptosis of human PMNs was detected by DNA-fragmentation assay and was quantitated by flow cytometry, ultraviolet and light microscopy. Cell adhesion was assessed by a toluidine blue assay, and cell spreading was detected by phase contrast microscopy. Protein tyrosine phosphorylation was studied using Western blotting and confocal microscopy. RESULTS: PMN apoptosis was not different in unstimulated cultures on either surface-adherent fibronectin or on PolyHema, a surface that prevents cell adherence. However, tumor necrosis factor-alpha (TNF alpha) treatment significantly increased apoptosis on fibronectin (37 +/- 4%) compared with PolyHema (20 +/- 3%). Tests on other matrix substances revealed that the percentage of apoptotic PMNs in the presence of TNF alpha was 8 +/- 1% on PolyHema, 26 +/- 4% on fibronectin, 17 +/- 2% on collagen I, 16 +/- 2% on collagen IV, and 16 +/- 3% on laminin (P < 0.05 for all matrices compared with PolyHema). Preincubation with genistein (50 microM) significantly inhibited TNF alpha-mediated apoptosis on fibronectin (39 +/- 4% to 21 +/- 4%) but not on PolyHema (21 +/- 4% to 16 +/- 4%). Genistein also reduced PMN spreading on fibronectin. In contrast, inhibitors of mitogen-activated protein kinase and protein kinase C showed no effect on PMN apoptosis. Fibronectin strongly increased tyrosine phosphorylation of three 102, 63, and 54 kDa proteins. Five newly tyrosine-phosphorylated 185, 85, 66, 56, and 42 kDa bands were also visible. Using confocal microscopy, highest tyrosine phosphorylation was localized to sites of cell-matrix interaction. CONCLUSIONS: ECM influences apoptosis in TNF alpha-activated, adherent, spreading PMNs. The process is regulated by tyrosine phosphorylation. Acceleration of apoptosis may shorten the PMN lifespan and thereby locally regulate inflammation.

Apoptosis↗
PubMed2006

A Rudimentary labellar speculum in Cymbidium lowianum (Rchb.f.) Rchb.f. and Cymbidium devonianum Paxton (Orchidaceae).

BACKGROUND AND AIMS: The labellar 'hairs' of some Cymbidium spp. are said to be thin-walled and to contain 'plasma', oil and sugars and it has long been speculated that they may function as food-hairs. However, the present authors' preliminary studies showed that certain atypical papillae may have a different role and, by reflecting light, function as a speculum. The purpose of the paper is to test this hypothesis. METHODS: Light microscopy, scanning electron microscopy, transmission electron microscopy, histochemistry and ultraviolet photography were used to investigate the structure, food content and light-reflecting properties of these papillae. KEY RESULTS AND CONCLUSIONS: The labellum of Cymbidium lowianum (Rchb.f.) Rchb.f. is densely clothed with obconical to conical papillae with wide bases and pointed tips. However, on either side of the median axis of the lip occur silvery patches comprising papillae with truncated tips and it is thought that these reflect light and thereby attract insect pollinators. Similar patches are also found in Cymbidium devonianum Paxton, and in both species, they are set against a reddish background, which, since bees cannot perceive this colour, probably appears dark to the insect thus enhancing the visual impact of the light-reflecting patches. In Cymbidium tigrinum Parish ex Hook. and Cymbidium mastersii Griff. ex Lindl., however, the labellum is mainly white and no light-reflecting patches were observed. Instead, unlike C. lowianum and C. devonianum, these species are highly fragrant and the attraction of insects probably depends to a greater extent on olfactory cues. In C. lowianum both types of papillae contain protein, starch and lipid bodies but only protein is seemingly present at elevated concentrations. However, lipoidal material also occurs upon the surface of the labellum and it is possible that this may be gathered by insects as reported for C. iridifolium A. Cunn (syn. C. madidum Lindl.). The labellar papillae of C. lowianum, thus, have the potential to function as food-hairs, although direct evidence for this is lacking.

Animals↗
PubMed1995

Parthenogenetic activation of human oocytes following cryopreservation using 1,2-propanediol.

Fresh and aged human oocytes were cryopreserved using 1,2-propanediol (PROH). After thawing, the oocytes were cultured for 20 h and examined for parthenogenetic activation using light microscopy and an ultraviolet DNA stain. Control fresh or aged oocytes and oocytes exposed to PROH without cryoperservation were also examined for activation. No control oocytes were observed to activate spontaneously (n = 43) and parthenogenetic activation was not induced by exposure to PROH alone (n = 26). In both fresh and aged cryopreserved oocytes, 27 and 29% of the oocytes respectively were activated, and these proportions were significantly elevated compared with the controls (P < 0.01). Although a similar rate of activation was observed for the cryopreserved fresh and aged oocytes, the form of parthenogenetic activation varied between these two types of oocyte. A single pronucleus was observed in 18% of the fresh and 5% of the aged cryopreserved oocytes. In contrast, the presence of two or more pronuclei was observed in 0% of the fresh and 19% of the aged cryopreserved oocytes.

Cell Nucleus↗