3 resultados para FLUORESCENCE SPECTROMETRY
em Greenwich Academic Literature Archive - UK
Resumo:
Macromolecular therapeutics and nano-sized drug delivery systems often require localisation to specific intracellular compartments. In particular, efficient endosomal escape, retrograde trafficking, or late endocytic/lysosomal activation are often prerequisites for pharmacological activity. The aim of this study was to define a fluorescence microscopy technique able to confirm the localisation of water-soluble polymeric carriers to late endocytic intracellular compartments. Three polymeric carriers of different molecular weight and character were studied: dextrin (Mw~50,000 g/mol), a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Mw approximately 35,000 g/mol) and polyethylene glycol (PEG) (Mw 5000 g/mol). They were labelled with Oregon Green (OG) (0.3-3 wt.%; <3% free OG in respect of total). A panel of relevant target cells were used: THP-1, ARPE-19, and MCF-7 cells, and primary bovine chondrocytes (currently being used to evaluate novel polymer therapeutics) as well as NRK and Vero cells as reference controls. Specific intracellular compartments were marked using either endocytosed physiological standards, Marine Blue (MB) or Texas-red (TxR)-Wheat germ agglutinin (WGA), TxR-Bovine Serum Albumin (BSA), TxR-dextran, ricin holotoxin, C6-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled ceramide and TxR-shiga toxin B chain, or post-fixation immuno-staining for early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins (LAMP-1, Lgp-120 or CD63) or the Golgi marker GM130. Co-localisation with polymer-OG conjugates confirmed transfer to discreet, late endocytic (including lysosomal) compartments in all cells types. The technique described here is a particularly powerful tool as it circumvents fixation artefacts ensuring the retention of water-soluble polymers within the vesicles they occupy.
Resumo:
Major and trace elemental composition provides a powerful basis for forensic comparison of soils, sediments and rocks. However, it is important that the potential 'errors' associated with the procedures are fully understood and quantified, and that standard protocols are applied for sample preparation and analysis. This paper describes such a standard procedure and reports results both for instrumental measurement precision (repeatability) and overall 'method' precision (reproducibility). Results obtained both for certified reference materials and example soils show that the instrumental measurement precision (defined by the coefficient of variation, CV) for most elements is better than 2-3%. When different solutions were prepared from the same sample powder, and from different sub-sample powders prepared from the same parent sample, the CV increased to c. 5-6% for many elements. The largest variation was found in results for certified reference materials generated from 23 instrument runs over an 18 month period (mean CV=c. 11%). Some elements were more variable than others. W was found to be the most variable and the elements V, Cr, Co, Cu, Ni and Pb also showed higher than average variability. SiO2, CaO, Al2O3 and Fe2O3, Rb, Sr, La, Ce, Nd and Sm generally showed lower than average variability, and therefore provided the most reliable basis for inter-sample comparison. It is recommended that, whenever possible, samples relating to the same investigation should be analysed in the same sample run, or at least sequential runs.
Resumo:
The secondary structure of the trimeric protein 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1, the second of three enzymes involved in the dechlorination of 4-chlorobenzoate to form 4-hydroxybenzoate, has been examined. E(mM) for the enzyme was 12.59. Analysis by circular dichroism spectrometry in the far uv indicated that 4-chlorobenzoyl coenzyme A dehalogenase was composed mostly of alpha-helix (56%) with lesser amounts of random coil (21%), beta-turn (13%) and beta-sheet (9%). These data are in close agreement with a computational prediction of secondary structure from the primary amino acid sequence, which indicated 55.8% alpha-helix, 33.7% random coil and 10.5% beta-sheet; the enzyme is, therefore, similar to the 4-chlorobenzoyl coenzyme A dehalogenase from Pseudomonas sp. CBS-3. The three-dimensional structure, including that of the presumed active site, predicted by computational analysis, is also closely similar to that of the Pseudomonas dehalogenase. Study of the stability and physicochemical properties revealed that at room temperature, the enzyme was stable for 24 h but was completely inactivated by heating to 60 degrees C for 5 min; thereafter by cooling at 1 degrees C min(-1) to 45 degrees C, 20.6% of the activity could be recovered. Mildly acidic (pH 5.2) or alkaline (pH 10.1) conditions caused complete inactivation, but activity was fully recovered on returning the enzyme to pH 7.4. Circular dichroism studies also indicated that secondary structure was little altered by heating to 60 degrees C, or by changing the pH from 7.4 to 6.0 or 9.2. Complete, irreversible destruction of, and maximal decrease in the fluorescence yield of the protein at 330-350 nm were brought about by 4.5 M urea or 1.1 M guanidinium chloride. Evidence was obtained to support the hypothetical three-dimensional model, that residues W140 and W167 are buried in a non-polar environment, whereas W182 appears at or close to the surface of the protein. At least one of the enzymes of the dehalogenase system (the combined 4-chlorobenzoate:CoA ligase, the dehalogenase and 4-hydroxybenzoyl coenzyme A thioesterase) appears to be capable of association with the cell membrane.