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Electron probe X-ray microanalysis of K, Rb, Cs, and T1 in cryosections of striated muscle.

Muscles containing the normal amount of K+ or loaded with Rb+, Cs+, or T1+ were cryofixed, cryosectioned and analysed by electron probe X-ray microanalysis. Previously reported results obtained with independent methods were confirmed: The alkali-metals and T1 are mainly localized in the A bands and at Z lines of the striated muscle. The results are in accordance with the association-induction hypothesis and the concept that K+ is physically adsorbed onto beta- and gamma-carboxyl side chains of myosin and other proteins.

Animals↗

Aluminium in rat cerebellar neural cultures.

A systematic microchemical analysis of unstained and uncoated neurone cultures was performed with synchrotron radiation photoemission spectromicroscopy after exposure to an aluminium solution. Clear evidence was found for localized aluminium uptake in a few cells. Their possible identification based on morphology is discussed.

Aluminum↗

Leaching and corrosion of polyvinyl chloride (PVC) tubes in a dialysis water distribution system.

PVC tubes, from the dialysis water distribution system of a 14 and a 7 year old plant, were analysed by scanner electron microscopy (SEM) and X-ray microanalysis (EDAX). Results showed that the leaching of material occurs depending on time of use. The phenomenon does not modify the chemical composition of the material. Results suggest that PVC alterations, induced by time, could support bacterial proliferation thus reducing the quality of dialysate.

Corrosion↗

[Flow cytometry and laser scanning confocal microscopy analysis of the receptor distribution].

To study the physiological regulation of the receptor protein, a fluorescent probe and detection system for alpha 1B-adrenergic receptors have been developed. By using the anti-peptide antibody developed against the alpha 1B-adrenergic receptor NH2-terminus, we have examined the agonist-regulated alpha 1B-adrenergic receptor redistribution in desensitized cells. Flow cytometry analysis showed that anti-peptide antibody against alpha 1B-adrenergic receptor specifically identifies the receptor in CHO cells, COS-7 cells that were transfected with alpha 1B-adrenergic receptor cDNA and rat hepatocytes. Using a fluoro-labeled receptor ligand, BODIPY FL-prazosin, as a probe, cell surface alpha 1-adrenergic receptor subtypes can be detected by flow cytometry. Laser scanning confocal microscopy visualized the agonist-regulated redistribution process of alpha 1B-adrenergic receptor in living cells; thus, following phenylephrine (10(-6) M) stimulation, receptor antigen at the cell surface rapidly internalized and clustered together in a cell within 30 min. The results showed that the antibody and fluoro-labeled ligand are valuable tools for studying the localization and functional role of the alpha 1-adrenergic receptor subtype.

Animals↗

[Effect of morphological properties on drug release from biodegradable microspheres].

The morphological properties of poly(beta-hydroxybutyric acid) (PHB) or poly(L-lactic acid) microspheres loading flomoxef sodium (FMOX) were investigated with regard to FMOX release. The release profiles of FMOX from the microspheres could be divided into two types, a sustained release type and a burst one. Two representative PHB microspheres, the release profiles of which were quite different from those of FMOX, were compared in detail from a morphological point of view. The shapes of their surfaces and sections were observed by using scanning electron microscopy (SEM), and FMOX distribution was analyzed by using electron probe microanalysis. The crystallinity of polymers was further measured by powder X-ray diffratometry. There was little difference in the FMOX distribution and their microscopic properties such as sphere size, specific surface area, shape of surface and section. In contrast, water penetration into the inside of the microspheres was found to be clearly different by use of cryogenic SEM. A significant difference was also observed in the crystallinity of polymers forming the microspheres. The release of FMOX from the microspheres was affected by the crystallinity of polymers forming the microspheres, and burst phenomena occurred in case the polymer was highly crystallized. It was speculated that the crystallization of polymer induced micro voids in the microspheres which functioned as channels for water penetration.

Biodegradation, Environmental↗

Liquid phase SPR imaging experiments for biosensors applications.

Surface plasmon resonance (SPR) has recently gained attention as a label-free method for the detection of biological molecules binding onto functionalised surfaces. It is one of the most sensitive detection method for monitor variations in the thickness and refractive index in ultra-thin films. Here, the adsorption processes of oligonucleotides onto gold substrates have been investigated in aqueous buffer solution using SPR imaging measurements. The hybridization of a thiol-modified, single stranded oligonucleotide anchored to a gold surface via thiol group, with its complementary sequence has been observed and characterised monitoring the hybridization process by SPR equipment. In situ investigation of smallest changes in SPR imaging measurements dynamically performed in liquid phase in the presence of DNA complementary probes was performed. Infrared spectroscopy and scanning electron microscopy characterisation of the functionalised gold surfaces of the biosensor were compared with the images obtained by SPR experimental apparatus.

Biosensing Techniques↗

Quantitative fluorescence of 5-FU-treated fetal rat limbs using confocal laser scanning microscopy and Lysotracker Red.

BACKGROUND: LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and tissues. After cell death, a high level of lysosomal activity (acidic enzyme) is expressed in tissues resulting from phagocytosis of apoptotic bodies by neighboring cells. LT was shown previously to be an indicator of cell death in a manner similar to other standard assays (Annexin, terminal dUTP nick end labeling, Nile blue sulfate, neutral red, and acridine orange). METHODS: LT fluorescence in fetal rat hindlimbs at gestational day 14 was measured 8 h after administration of the teratogen, 5-fluorouracil (5-FU), with the use of confocal laser scanning microscopy (CLSM). Four dose levels of 5-FU (0, 20, 30, and 40 mg/kg) were studied. The preparation technique involved staining with LT, paraformaldehyde fixation, methanol dehydration, and clearance with benzyl alcohol and benzyl benzoate. After this treatment, the limb was nearly transparent and ready for CLSM analysis. RESULTS: LT staining was observed in specific regions undergoing apoptosis in normal (control) hindlimbs. After 5-FU treatment, highly fluorescent regions appeared in the progress zone (PZ) of the limb. A dose-dependent response to 5-FU treatment was observed. Compared with controls, hindlimbs treated with 20, 30, and 40 mg/kg of 5-FU exhibited more fluorescence within the highly proliferative PZ. These results showed a dose-response relation between 5-FU exposure and LT uptake. CONCLUSIONS: We found that three-dimensional volumetric regions indicating a high level of fluorescence in the embryonic limb bud can be quantified with three different computer analysis programs. The combination of a sample preparation procedure that clears tissue, a CLSM technique that addresses the equipment variables, and an application of statistical population analysis procedures enabled the visualization and quantification of fluorescence in entire fetal rat hindlimbs that were approximately 500 microm in thickness.

Animals↗

SEM and elemental analysis of composite resins.

Twenty-four chemically cured, 21 light-cured anterior, three light-cured anterior/posterior, and 18 light-cured posterior composite resins were examined using scanning electron microscopy, and the elemental composition of their filler particles was analyzed with an energy dispersive electron probe microanalyzer. According to the results obtained, the composite resins were divided into five groups (traditional, microfilled type, submicrofilled type, hybrid type, and semihybrid), with two additional hypothetical categories (microfilled and hybrid). Characteristics of each type were described with clinical indications for selective guidance of respective composite resins for clinical use.

Aluminum↗

Gold-ATP.

The synthesis, purification, and chemical analysis of two covalent conjugates between ATP and undecagold are described, one in which gold is attached to the ribose moiety of ATP and the other in which it is attached to the N-6 position of the adenine base. The former probe was then used to bind to two ATP binding proteins, the helicase DnaB and the chaperone DnaK. After purification from unbound gold by column chromatography, binding was measured by UV-Vis spectroscopy, then the protein and gold were visualized by scanning transmission electron microscopy. Binding was observed with the conjugates, and virtually no binding occurred in the control of undecagold without the ATP attached. This new probe may be useful for studying nucleotide binding sites on proteins or for labeling nucleic acids or oligonucleotides directly.

Adenosine Triphosphate↗

In situ detection of rhodococci associated with activated sludge foams.

Genus-specific 16S rRNA targeted oligonucleotide probes, Rco1 and Rco2, were designed and used to detect rhodococci in activated sludge foam samples by confocal laser scanning microscopy. Pure cultures were used to find the optimal hybridisation conditions which were determined by comparing the mean fluorescent intensities of target and non-target cells from images captured using a confocal laser scanning microscope (CLSM). The combination of fluorescent in situ hybridisation with rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy provides an effective way of detecting rhodococci in environmental samples.

Bacteriological Techniques↗

Direct effects of dopamine on colonic mucosal pH: implications for tonometry.

Tonometric measurements of colonic and gastric mucosa pH are used as indirect determinants of splanchnic perfusion in shocked patients or those undergoing aortic cross-clamp. Mucosal acidification in response to splanchnic vasodilators such as dopamine has been assumed to signify ischemia. However, cellular acidification may occur independent of oxygenation and the direct effects of dopamine on mucosal acid-base are unknown. We examined the effects of dopamine on cellular pH (independent of oxygenation) of intestinal mucosa in vitro. Crypts isolated from the distal colon of Sprague-Dawley rats were loaded with a pH-sensitive fluorescent probe, perfused with a Hepes-buffered Ringers solution, and imaged with confocal laser scanning microscopy. In separate experiments, crypts were loaded with a calcium-sensitive probe (Fura-2) and concentrations of free cytosolic calcium were measured with fluorescence imaging. Dopamine perfusion produced a reversible cytosolic acidification of crypts which was not significantly affected by (i) the nominal absence of bicarbonate, (ii) alpha- and beta-adrenergic receptor blockade, or (iii) protein kinase C inhibition. Dopamine did not significantly affect intracellular calcium concentrations. However, dopamine-induced acidification was inhibited by (a) blocking sodium-hydrogen exchange with amiloride, (b) prior exposure to adenosine 3', 5'-cyclic monophosphate (cAMP), or (c) protein kinase A blockade (all P < 0.01). Dopamine directly acidifies mucosal crypt cells in a mechanism that involves a cAMP-mediated inhibition of sodium-hydrogen exchange. This finding accounts for the acidification of intestinal mucosa during low-dose dopamine infusion despite a demonstrable improvement in splanchnic perfusion. Direct mucosal effects of pharmacological agents must be considered in the evaluation of perfusion parameters based on tonometric data.

Animals↗

New polyurethane compositions able to bond high amounts of both albumin and heparin. II: Copolymers and polymer blends.

Haemocompatible new urethane copolymers and polymer blends containing, in the chain extender, a long chain alkyl group (able to bond albumin) or a tertiary ammonium group able, after suitable quaternization reaction, to bind ionically significant amounts of heparin, were prepared. The copolymers were characterized by differential scanning calorimetry, intrinsic viscosity determinations, infrared spectroscopy and nuclear magnetic resonance (1H and 13C). Biological in vitro evaluation has shown that the adsorption sequence for albumin and heparin, respectively, onto films of the various copolymers and blends, exerts a great influence. From scanning electron microscopy measurements it was seen that the bonding type of albumin to the polymer films plays a determining role on the platelet activation. A phase segregation occurring on the polymer blends surface was demonstrated by X-ray photoelectron spectroscopy measurements.

Biocompatible Materials↗