Abstract: Human Arkadia is a nuclear protein consisted of 989 amino acid residues, with a characteristic RING domain in its C-terminus. The RING domain harbours the E3 ubiquitin ligase activity needed by Arkadia to ubiquitinate its substrates such as negative regulators of TGF-beta signaling. The RING finger domain of Arkadia is a RING-H2 type and its structure and stability is strongly dependent on the presence of two bound Zn(II) ions attached to the protein frame through a defined Cys3-His2-Cys3 motif. In the present paper we transform the RING-H2 type of Arkadia finger domain to nonnative RING sequence, substituting the zinc-binding residues Cys(955) or His(960) to Arginine, through site-directed mutagenesis. The recombinant expression, in Escherichia coli, of the mutants C955R and H960R reveal significant lower yield in respect with the native polypeptide of Arkadia RING-H2 finger domain. In particular, only the C955R mutant exhibits expression yield sufficient for recombinant protein isolation and preliminary studies. Atomic absorption measurements and preliminary NMR data analysis reveal that the C955R point mutation in the RING Finger domain of Arkadia diminishes dramatically the zinc binding affinity, leading to the breakdown of the global structural integrity of the RING construct.
Abstract: A simple, non-destructive, methodology based on FT-Raman spectroscopy was developed for the quantitative analysis of risperidone in commercially available film-coated tablets. A simple linear regression model was constructed based on standard tablets, prepared using the same manufacturing process as the commercially available. The tablets contained 0.27, 0.54, 1.08, 1.62, 2.16, 3.24 and 4.32 wt% risperidone. The most prominent Raman vibration of the active pharmaceutical ingredient at 1533 cm(-1), recorded using a home-made rotating system, was plotted against concentration. The model was tested on commercial film-coated tablets. The results were compared against those obtained by application of HPLC on the same samples.
Abstract: Two different nondestructive spectroscopy methods based on near-infrared (NIR) and Fourier transform (FT) Raman spectroscopy were developed for the determination of ticlopidine-hydrochloride (TCL) in pharmaceutical formulations and the results were compared to those obtained by high-performance liquid chromatography (HPLC). An NIR assay was performed by reflectance over the 850-1700 nm region using a partial least squares (PLS) prediction model, while the absolute FT-Raman intensity of TCL's most intense vibration was used for constructing the calibration curve. For both methodologies the spectra were obtained from the as-received film-coated tablets of TCL. The two quantitative techniques were built using five "manual compressed" tablets containing different concentrations and validated by evaluating the calibration model as well as the accuracy and precision. The models were applied to commercial preparations (Ticlid). The results were compared to those obtained from the application of HPLC using the methodology described by "Sanofi Research Department" and were found to be in excellent agreement, proving that NIR, using fiber-optic probes, and FT-Raman spectroscopy can be used for the fast and reliable determination of the major component in pharmaceutical analysis.
Abstract: Identification of the crystal phase of an active pharmaceutical ingredient (API) in a pharmaceutical tablet is of outmost importance since different polymorphs exhibit different physicochemical properties. Furthermore, some of the crystal phases are protected by patents. Identification of Risperidone polymorph A in film coated commercial tablets was attempted using IR spectroscopy, Raman spectroscopy and X-ray powder diffraction (XRPD). The stability of this polymorph through time and during the manufacturing process was also examined. The inability of IR and Raman techniques to identify the presence of polymorph A in the tablets, despite their lower detection limits for Risperidone, left the XRPD as the only technique that could be used for identifying the presence of Risperidone A against the other crystal phases in the presence of the excipients. Polymorph A was proved to be stable during the manufacturing process and after a storage period of 2 years.
Abstract: Raman spectroscopy was applied for the direct non-destructive analysis of amiodarone hydrochloride (ADH), the active ingredient of the liquid formulation Angoron((R)). The FT-Raman spectra were obtained through the un-broken as-received ampoules of Angoron((R)). Using the most intense vibration of the active pharmaceutical ingredient (API) at 1568cm(-1), a calibration model, based on solutions with known concentrations, was developed. The model was applied to the Raman spectra recorded from three as-purchased commercial formulations of Angoron((R)) having nominal strength of 50mgml(-1) ADH. The average value of the API in these samples was found to be 48.56+/-0.64mgml(-1) while the detection limit of the proposed technique was found to be 2.11mgml(-1). The results were compared to those obtained from the application of HPLC using the methodology described in the European Pharmacopoeia and found to be in excellent agreement. The proposed analytical methodology was also validated by evaluating the linearity of the calibration line as well as its accuracy and precision. The main advantage of Raman spectroscopy over HPLC method during routine analysis is that it is considerably faster and no solvent consuming. Furthermore, Raman spectroscopy is non-destructive for the sample. However, the detection limit for Raman spectroscopy is much higher than the corresponding for the HPLC methodology.
Abstract: The capability of FT-Raman spectroscopy for the fast and non-destructive quantitative analysis of liquid formulations was tested and the results were compared to those obtained by HPLC. Diphenhydramine hydrochloride (DPH), the active ingredient of Benadryl, was determined in the presence of the numerous excipients of the elixir. A Raman calibration model was developed by measuring the peak intensities of different standard solutions of DPH vibration at 1003 cm(-1). Application of the calibration model on the peak intensity recorded from the as-received commercially available sample with 2.5 mg ml(-1) DPH nominal value yielded a value of 2.49+/-0.05 mg ml(-1) DPH. The reliability of this method was verified by testing it against the conventionally used HPLC. The results from both methods were in excellent agreement. The main advantage of Raman over HPLC method during routine analysis is that is considerably faster and less solvent consuming. Furthermore, Raman spectroscopy is non-destructive for the sample. On the other hand, the detection limit for Raman spectroscopy is much higher than the corresponding for the HPLC methodology.
Abstract: Methodologies based on FT-Raman spectroscopy and HPLC were developed for monitoring the stability of lovastatin in the solid state in the presence of gallic acid, a natural antioxidant. A Raman calibration curve was constructed using the area of the strong but overlapping vibration mode of lovastatin at 1645 cm(-1) and of the gallic acid at 1595 cm(-1). Mixtures of the active ingredient with the antioxidant were heated in the presence of atmospheric air up to 120 degrees C. The molar ratios of lovastatin and gallic acid in the artificially oxidized mixtures were determined from their Raman spectra using the calibration curve. The results were compared to those obtained from the application of the HPLC methodology and found to match satisfactorily. The HPLC analysis was based on a reserved-phase Zorbax C(g), 10 microm (4.6mm x 25 cm i.d.) column, using a gradient elution program by varying the proportion of solvent A acetonitrile 100% to solvent B 0.1% v/v phosphoric acid, and a programmable diode array detection at 225 nm. The Raman methodology was simpler and non-destructive for the sample but yielded only molar ratios as opposed to the HPLC technique where the moles of the both ingredients were determined.
Abstract: In the present study, the effect of mesopore and particle size distributions on the kinetics of dissolution of powdered Pentelic marble was investigated. Powders obtained by grinding marble slabs in an agate mortar with a pestle (1-3.5 min) were found to be nonporous by nitrogen absorption measurements. These powders, upon dissolution under conditions of constant undersaturation, 25 degreesC, pH 8.25, showed that the kinetics in the absence of mesopores depended on the number of active sites on the exposed surface. Thus, powders consisting of smaller particles, having higher specific surface areas, yielded higher rates of dissolution. Powders, however, which were prepared by grinding marble slabs in a cylinder mill for time periods between 2.0 and 30.0 min, and which exhibited considerable mesoporosity, showed the opposite trend. The rates of dissolution measured for these powders in undersaturated solutions increased with increasing mean particle size and decreased with increasing specific surface area. This finding suggested that the presence of mesopores resulted in lower dissolution rates even though the exposed total surface area was larger. Furthermore, the larger the number of mesopores in a powder sample the slower the corresponding dissolution rates in undersaturated solutions. (C) 2003 Elsevier Science (USA). All rights reserved.
Abstract: Dissolution processes play an important role in marble weathering. Investigation of the dissolution kinetics of powdered Pentelic marble, a calcitic natural stone, showed that the rates measured at constant undersaturation decreased with time. It was found that the rate of dissolution decreased sharply to about 50% of the initial value before 5% of the initially suspended powder was dissolved. Past a dissolution extent corresponding to 25% with respect to the suspended solid, the rates of dissolution at the same constant undersaturation reached a stable value corresponding to 20-25% of the initial rate measured. The reduction in the rates of dissolution up to 25% of mass loss was in agreement with the reduction in the total surface area exposed to the solution, suggesting a dependence of the rate on the number of active sites on the surface of the dissolving material. The decrease in the dissolution rates did not depend on the undersaturation of the solution or on the physical characteristics of the particles such as specific surface area, porosity, and particle size distribution. Equal mass losses, with respect to the initially suspended solid, of different samples exhibited the same relative decrease in rate regardless of the conditions of the aqueous solutions. (C) 2002 Elsevier Science (USA).
Abstract: Dissolution of natural stone such as marble is not limited to its surface. The porous structure, known to play an important role in stone decay, is also affected by the conditions of dissolution. In the present work, the changes in pore size distribution of Pentelic marble particles accompanying chemical dissolution in undersaturated solutions and at alkaline pH 8.25 were investigated. The specific surface area and the mesopore distribution of the Pentelic marble tested showed a pronounced decrease to very low values. On the other hand, the sizes of macropores exhibited a tendency to increase with the extent of dissolution due either to dissolution in the interior of the pores or to fusion of small pores into larger. Furthermore, the number of small particles decreased significantly, reaching complete disappearance, depending on the extent of dissolution. At the same time, the relative number of particles of intermediate size increased. (C) 2001 Academic Press.
Abstract: The kinetics of dissolution of powdered Pentelic marble from two different sites located on Mount Pentelicon of Attica, Dionysos and Cave, were investigated. The results were compared with those of synthetically precipitated calcite, the main inorganic constituent (>98%) of the tested marbles. The dissolution process at solution undersaturation near equilibrium in all cases showed a linear dependence. of the rates of dissolution on the relative solution undersaturation. The dissolution was found to be polynuclear, surface diffusion controlled. Morphological examination of the specimens showed that dissolution proceeds through formation of steps, cracks, pores, and layerwise disintegration, corroborating the conclusion from the kinetics results. Powdered marble is suggested to be an excellent model system for the investigation of marble dissolution as it exhibited remarkable similarities with the dissolution of whole marble blocks.
Abstract: The wettability of calcium carbonate, an important parameter in the evaluation of the interaction characteristics of materials used in conservation, was examined by measurements with the Wilhelmy plate technique. Heterogeneous surfaces consisting of glass and calcium carbonate were examined. Stable calcium carbonate layers were deposited on microscope glass slides using supersaturated solutions in which the equimolar concentrations of total calcium and total carbonate used ranged between 0.1 and 0.4 M, over a pH range between 9 and 12 and temperature between 25 and 80 degrees C. In all cases amorphous calcium carbonate was deposited, the extent of surface coverage increasing with increasing supersaturation and temperature. Higher surface coverage of the glass slides with calcium carbonate yielded lower contact angles suggesting increased wettability of the heterogeneous surface. (C) 1999 Elsevier Science B.V. All rights reserved.
Abstract: A non-destructive method based on the use of Raman spectroscopy (RS) for the determination of the percentage of gypsum in sulfated marble is presented, The Raman spectra of well mixed powder samples of calcite-aragonite, calcite-gypsum and gypsum-aragonite pairs of mixtures were recorded and the characteristic bands at 280 cm(-1) for calcite, 205 cm(-1) for aragonite and 412 cm(-1) for gypsum were used as the basis for the quantitative analysis of specimens in which the most stable calcium carbonate phases, calcite and aragonite, were present, The detection limits mere found to be 0.3 mol% for calcite, 0.5 mol% for aragonite and 0.6 mol% for gypsum, For samples containing only one calcium carbonate phase the use of the strong and sharp Raman band at 1085 cm(-1), common for aragonite and calcite, together with the intensity of the Raman peak at 1006 cm(-1) for gypsum, yielded lower detection limits: calcite 0.1, aragonite 0.1 and gypsum 0.05 mol%, The analysis by RS was compared with X-ray powder diffraction (XRD), In this analysis, the calibration curves mere constructed using the relative intensities corresponding to the 113, the 111 and the 12 (1) over bar reflections of the calcite, aragonite and gypsum, respectively, The detection limits for calcite, aragonite and gypsum mere 4, 5 and 1-2 mol%, respectively, The potential of using RS for a point-by-point analysis ('mapping') of a surface by focusing the laser beam on the selected spots was also demonstrated on a marble sample removed from Athens National Garden, exposed in the open air.
Abstract: Laser Raman spectroscopy was used for the quantitative determination of aspirin in aspirin-maize starch tablets. A calibration curve was constructed from spectra obtained from tablets with known quantities of aspirin and starch. The calibration curve is given from the relationship: I(552)/I(478) = (W(aspirin)/W(starch)) x 4.21, where I(552) and I(478) are the relative Raman intensities for the 552 and 478 cm(-1) Raman shift, respectively. W(aspirin) and W(starch) represent the weight of aspirin and starch in a pellet.
Abstract: Vibrational Raman spectroscopy was employed successfully for the simultaneous quantitative determination of the cubic, tetragonal and monoclinic phases of partially stabilized zirconia. Pressed pellets with known composition were prepared and used as standard mixtures. From the standard pellets, a calibration curve was obtained which was found to be a straight line if (/(640) - 0.47 /(260))//(177) is plotted against the inverse monoclinic molar fraction. /(260), /(177) are the Raman intensities for the tetragonal and monoclinic peaks at 260 and 177 cm(-1) respectively, and were calculated by subtracting the ''background''. In contrast, /(640) represents the intensity for the 640 cm(-1) band, which is attributed to all three phases, and was measured including the ''background''.
