ABSTRACT
Personalized dosages of monoclonal antibodies are being used more regularly to treat various diseases, rendering their quantitation more essential than ever for the right dose administration to the patients. A promising alternative, which overcomes the obstacles of the well-established chromatographic techniques regarding the quantification of biopharmaceuticals, is Raman spectroscopy. This study aimed to develop and validate a novel analytical method for the quantitation of bevacizumab in solutions via Raman spectroscopy. For this purpose, a droplet of the solution was left to dry on a highly reflective carrier and a home-made apparatus was employed for rotation of the sample. Hence, each recorded Raman spectrum was the average of the signal acquired simultaneously from multiple points on a circular circumference. The method was validated, and the detection limit of the antibody was found to be 1.06 mg/mL. Bevacizumab was found to be highly distributed at the formed coffee ring of the dried droplet, though this was a function of solution concentration. Finally, Raman spectra at different distances on the coffee ring were obtained from the four quarters. The lowest bevacizumab detection limit was found at a distance of 75 µm from the external side of the coffee ring and it was determined to be equal to 0.53 mg/mL.
ABSTRACT
Raman microspectroscopy and X-ray microcomputed tomography (micro-CT) were used for assessment of the quality of the femur and tibia bones in apolipoprotein-deficient mice compared to control littermates. The cortical and trabecular bone was investigated separately. Raman spectra revealed no differences in the bioapatite-to-collagenous matrix ratio of the cortical bone. The quantities of calcium and collagen, which were measured using atomic absorption spectrometry and thermogravimetric analysis, respectively, were also found to be equal in the two groups. Density and morphometric parameters, which were measured using micro-CT, verified the cortical mineral stability. Bone quality indices were measured using Raman spectra. A decreased collagen crosslink (trivalent-to-divalent) ratio revealed delayed maturation of the collagen network. Such a decrease has been reported in the literature to be connected to decreased bone strength. For the trabecular bone, micro-CT revealed severe osteoporosis in the knock-out group, which was evident from a decreased mineral density, trabecular thickness and increased bone surface/volume ratio. The trabecular bone was not accessible for Raman spectroscopy. According to these results, the cortical and trabecular femur bone is expected to exhibit proneness to fracturing, each for a different reason. A combination of the two techniques was regarded as necessary for an overall assessment of bone quality.
Subject(s)
Bone Density , Collagen , Animals , Mice , X-Ray Microtomography , Collagen/chemistry , Models, Animal , MineralsABSTRACT
The severe effects of alcohols on humans trigger the continuous research on the alcohols level measurement in biological fluids. The officially established technique is Headspace Gas Chromatography (HS-GC), while breathalyzers are commonly used by police on the road. However, they all exhibit drawbacks; HS-GC is expensive and labor-intensive, while the precision of breathalyzers is controversial. In the present study, a novel method was developed, for ethanol and methanol detection and quantification in human urine, saliva and blood serum, based on Raman spectroscopy. Biological fluids from healthy adult volunteers were collected, standard solutions of the alcohols in a concentration range from 0.00 µL/mL to 5.00 µL/mL were prepared and analysed using an air-tight and small volume sample carrier. Calibration curves for each binary system (alcohol - biological fluid) were created. Ethanol calculated detectable concentrations were below permissible limits for all biological fluids. In the case of methanol, the limits were not as satisfactory, but lower than intoxication level, due to the difficult spectral discrimination. For both alcohols, the lowest detection limits were recorded for saliva. All detection limits were verified by visual inspection of the spectra. The proposed quantitative method was validated in all cases regarding their specificity, working range, accuracy, precision and sensitivity.
ABSTRACT
The measurement of ethanol and toxic alcohol (methanol and isopropanol) strengths in beverages and spirits is crucial for health reasons but also for the identification of adulterated products. Many methodologies have been reported in the literature, based mainly on chromatographic and on spectroscopic techniques. Chromatographic techniques are laborious and time-consuming, while spectroscopic techniques are rapid and need no special sample pretreatment. All techniques were only applied to off-line or at-line manner. In the present work, Raman spectroscopy was used for fast and non-destructive measurements. A "through the container" method was developed for a non-invasive analysis, i.e., analysis without unsealing the bottles. This method, coupled with a miniature portable Raman, can serve for in-line measurements in a production line. The optimum laser focus for maximum spirit signal and minimum glass-wall signal was investigated. Calibration curves for the alcohols of interest were constructed and validated. The limits of detections were calculated and proved to be lower than the legitimate values. The influences of the liquor color and the bottle color, shape, and thickness were checked. Twenty-eight alcoholic products were studied. The concentrations found were compared against the nominal values (from the bottle labels).
Subject(s)
Alcoholic Beverages , Ethanol , Ethanol/analysis , Alcoholic Beverages/analysis , Spectrum Analysis, Raman/methods , Beverages/analysis , Methanol/analysisABSTRACT
Vibrational spectroscopic techniques and especially Raman spectroscopy are gaining ground in substituting the officially established chromatographic methods in the identification of ethanol and other volatile substances in body fluids, such as blood, urine, saliva, semen, and vaginal fluids. Although a couple of different carriers and substrates have been employed for the biochemical analysis of these samples, most of them are suffering from important weaknesses as far as the analysis of volatile compounds is concerned. For this reason, in this study three carriers are proposed, and the respective sample preparation methods are described for the determination of ethanol in human urine samples. More specifically, a droplet of the sample on a highly reflective carrier of gold layer, a commercially available cuvette with a mirror to enhance backscattered radiation sealed with a lid, and a home designed microscope slide with a cavity coated with gold layer and covered with transparent cling film have been evaluated. Among the three proposed carriers, the last one achieved a quick, simple, and inexpensive identification of ethanol, which was used as a case study for the volatile compound, in the biological samples. The limit of detection (LoD) was found to be 1.00 µL/mL, while at the same time evaporation of ethanol was prevented.
Subject(s)
Body Fluids , Spectrum Analysis, Raman , Body Fluids/chemistry , Ethanol/analysis , Female , Gold/analysis , Humans , Saliva/chemistry , Spectrum Analysis, Raman/methodsABSTRACT
The assessment of active pharmaceutical ingredient (API) particle size and morphology is of great importance for the pharmaceutical industry since it is expected to significantly affect physicochemical properties. However, very few methods are published for the determination of API morphology and particle size of film-coated (FC) tablets. In the current study we provide a methodology for the measurement of API particle size and morphology which could be applied in several final products. Bismuth Oxide 120 mg FC Tabs were used for our method development, which contain bismuth oxide (as tripotassium dicitratobismuthate (bismuth subcitrate)) as the active substance. The sample preparation consists of partial excipient dissolution in different solvents. Following this procedure, the API particles were successfully extracted from the granules. Particle size and morphology identification in Bismuth Oxide 120 mg FC Tabs was conducted using micro-Raman mapping spectroscopy and ImageJ software. The proposed methodology was repeated for the raw API material and against a reference listed drug (RLD) for comparative purposes. The API particle size was found to have decreased compared to the raw API, while the API morphology was also affected from the formulation manufacturing process. Comparison with the RLD product also revealed differences, mainly in the API particle size and secondarily in the crystal morphology.
Subject(s)
Bismuth , Excipients , Excipients/chemistry , Particle Size , Tablets/chemistryABSTRACT
Posaconazole is an API added as Form I for the production of oral suspensions, but it is found as Form-S in the final formulation. In this study, it was found that this polymorphic conversion, which may affect the bioavailability, is due to an interaction with water. However, the relatively poor wettability of posaconazole Form I renders the complete wetting of its particles and production of pure Form-S challenging. Consequently, for its isolation, Form I should be dispersed in water followed by application of sonication for at least 10 min. Pure posaconazole Form-S was characterised using X-ray powder diffraction (XRPD), Raman spectroscopy, attenuated total reflection (ATR) spectroscopy, thermogravimetric analysis (TGA) and optical microscopy. From these techniques, posaconazole Form-S was characterised as a hydrate form, which includes three molecules of water per API molecule.
ABSTRACT
Warfarin sodium is a low-dose pharmaceutical blood thinner that exists in two forms: the clathrate form and the amorphous form. In commercially available warfarin sodium oral suspension, the active pharmaceutical ingredient (API) is added in the amorphous state. This study investigates the apparent instability of the commercially available warfarin liquid oral formulation using Raman and IR spectroscopy, X-ray diffraction, differential scanning calorimetry, UV spectroscopy, and optical microscopy. Warfarin, not its sodium salt, was identified as the undissolved solid existing in the suspension. This was found to be due to the dissociation of sodium salt and the protonation of the warfarin ion in the liquid phase, which triggered the crystallization of the sparingly soluble unsalted form. The coexistence of protonated and unprotonated warfarin ions in the supernatant, as detected by Raman and UV spectroscopy, confirmed this assumption. Study of the dissolution of warfarin sodium amorphous salt and crystalline sodium clathrate in the placebo and pure water verified the results. The effect of pH and temperature on warfarin precipitation was also explored.
Subject(s)
Warfarin/chemistry , Administration, Oral , Drug Compounding , Drug Stability , Molecular Structure , Particle Size , Warfarin/administration & dosageABSTRACT
Medical errors associated with IV preparation and administration procedures in a hospital workflow can even cost human lives due to the direct effect they have on patients. A large number of such incidents, which have been reported in bibliography up to date, indicate the urgent need for their prevention. This study aims at proposing an analytical methodology for identifying and quantifying IV drugs before their administration, which has the potential to be fully harmonized with clinical practices. More specifically, it reports on the analysis of a piperacillin (PIP) and tazobactam (TAZ) IV formulation, using Raman spectroscopy. The simultaneous analysis of the two APIs in the same formulation was performed in three stages: before reconstitution in the form of powder without removing the substance out of the commercial glass bottle (non-invasively), directly after reconstitution in the same way, and just before administration, either the liquid drug is placed in the infusion set (on-line analysis) or a minimal amount of it is transferred from the IV bag to a Raman optic cell (at-line analysis). Except for the successful identification of the APIs in all cases, their quantification was also achieved through calibration curves with correlation coefficients ranging from 0.953 to 0.999 for PIP and from 0.965 to 0.997 for TAZ. In any case, the whole procedure does not need more than 10 min to be completed. The current methodology, based on Raman spectroscopy, outweighs other spectroscopic (UV/Vis, FT-IR/ATR) or chromatographic (HPLC, UHPLC) protocols, already applied, which are invasive, costly, time-consuming, not environmentally friendly, and require specialized staff and more complex sample preparation procedures, thus exposing the staff to hazardous materials, especially in cases of cytotoxic drugs. Such an approach has the potential to bridge the gap between experimental setup and clinical implementation through exploitation of already developed handheld devices, along with the presence of digital spectral libraries.
Subject(s)
Anti-Bacterial Agents/administration & dosage , Hospitals/standards , Piperacillin/administration & dosage , Spectrum Analysis, Raman/methods , Tazobactam/administration & dosage , Workflow , Anti-Bacterial Agents/analysis , Humans , Piperacillin/analysis , Tazobactam/analysisABSTRACT
Determination of the polymorphic form of an active pharmaceutical ingredient (API) in a suspension could be really challenging because of the water phase and the low concentration of the API in this formulation. Posaconazole is an antifungal drug available also as an oral suspension. The aim of this study was to develop a sample-preparation method for polymorphic identification of the dispersed API by increasing the concentration of the API but with no compromise of polymorph stability. For this purpose, filtration, drying and centrifugation were tested for separating the API from the suspending medium. Centrifugation was selected because it succeeded in separating Posaconazole API with no polymorph transformation during the process. During this study, it was found that Posaconazole in oral suspensions is Form-S. However, when slower scanning rates were used for acquiring an XRPD pattern with better signal/noise ratio, Posaconazole was converted to Form I due to water loss. In order to protect the sample from conversion, different approaches were tested to secure an airtight sample including a commercially available XRPD sample holder with a dome-like transparent cap, standard polymethylmethacrylate (PMMA) sample holders covered with Mylar film, transparent pressure-sensitive tape and a transparent food membrane. Only usage of the transparent food membrane was found to protect the API from conversion for a period of at least two weeks and resulted in a Posaconazole Form-S XRPD pattern with no artificial peaks.
Subject(s)
Suspensions/chemistry , Triazoles/chemistry , Administration, Oral , Chemistry, Pharmaceutical/methods , Polymethyl Methacrylate/chemistryABSTRACT
Silver nanoparticles (AgNPs) were synthesized using hydroalcoholic extracts of dittany (Origanum dictamnus), sage (Salvia officinalis), sea buckthorn (Elaeagnus rhamnoides, syn. Hippophae rhamnoides), and calendula (Calendula officinalis) as reducing agents. AgNPs synthesized using NaBH4 and citric acid were used as control. The impact of the origin of the extract and preparation conditions (light, temperature, reaction time) on the properties of the synthesized AgNPs was investigated. The structure, morphology, composition, physicochemical characteristics, and colloidal stability were characterized using dynamic laser scattering (DLS), ultraviolet-visible spectrophotometry (UV-/Vis), XRD, X-ray fluorescence (XRF), TEM, and FTΙR. The reduction of total phenolic and flavonoid content of the extracts after the reaction of AgNPs synthesis was also determined. Low IC50 values for all types of AgNPs revealed good antioxidant activity, attributable to the phenolic and flavonoid content of their surface. The results suggest that plant extract selection is important to the green synthesis of AgNPs because it affects the kinetics of their synthesis as well as their morphology, physicochemical characteristics, and colloidal stability. In vitro permeation studies on porcine skin revealed that AgNPs remained at the upper layers of stratum corneum and did not penetrate the skin barrier after 4 h of cutaneous application suggesting the safety of their application on intact skin for a relatively short time.
ABSTRACT
FT-IR/ATR analytical technique is one of the most applicable techniques worldwide. It is closely associated with easy-to-use equipment, rapid analysis, and reliable results. This study reports the simultaneous qualitative and quantitative analysis of two active pharmaceutical ingredients (APIs), of a piperacillin and tazobactam formulation using a film formation method. This method requires film formation on the ATR crystal, resulting from solvent evaporation of a small amount of liquid sample. Good contact between the film and the crystal led to the identification of both APIs, although tazobactam was of low content in the formulation mixture. The quantification of the APIs in the commercial mixture was also achieved, using a single calibration line with a correlation coefficient equal to 0.999, not only after film formation but also in the initial dry formulation before reconstitution. The present spectroscopic technique combined with the proposed relatively simple sample treatment outweighs chromatographic protocols already applied, which require specialized staff and are costly, time-consuming, and not environmentally friendly. Taking all the above into consideration, it turns out that such an approach has the potential to be used for off-line quality control procedures in manufacture or, in terms of portable equipment and automated software, anywhere for on-site analysis, even in a hospital workflow.
Subject(s)
Piperacillin/chemistry , Tazobactam/chemistry , Calibration , Chemistry, Pharmaceutical/methods , Evaluation Studies as Topic , Pharmaceutical Preparations/chemistry , Spectroscopy, Fourier Transform Infrared/methodsABSTRACT
The functional role(s) of plant calcium oxalate (CaOx) crystals are still poorly understood. Recently, it was shown that crystals function as dynamic carbon pools whose decomposition could provide CO2 to photosynthesis when stomata are closed (e.g. under drought conditions) and CO2 starvation conditions may be created within the mesophyll. This biochemical process, named as 'alarm photosynthesis', can become crucial for plant survival under adverse conditions. Here, we study crystal decomposition under controlled CO2 starvation conditions (either in the shoot or in the root) to obtain a better insight into the process of crystal formation and function. Hydroponically grown pigweed plants were kept in CO2 -free air and/or CO2 -free nutrient medium for 9 days. Crystal volume was monitored daily, and carbon stable isotope composition (δ13 C) and Fourier transformation Raman spectra were obtained at the end of the experiment. A considerable reduction in the leaf crystal volume was observed in shoot-CO2 -starved plants at the end of the experiment. The smallest crystals were isolated from the plants in which carbon was excluded from both the shoot and the root and contained potassium nitrate. Crystal δ13 C of CO2 -starved plants was altered in a predicted way. Specifically, it depended on the average calculated isotope fractionation of all carbon fixation processes considered to be contributing in each experimental treatment. The results of the present study confirmed the correlation between CO2 starvation conditions and the CaOx crystal decomposition. Inorganic carbon fixed in the root may represent a major carbon source for CaOx formation.
Subject(s)
Amaranthus/metabolism , Calcium Oxalate/metabolism , Carbon Dioxide/metabolism , Carbon Isotopes/analysis , Photosynthesis/physiology , Plant Leaves/metabolism , Spectrum Analysis, RamanABSTRACT
Electronic cigarettes are considered healthier alternatives to conventional cigarettes containing tobacco. They produce vapor through heating of the refill liquids (e-liquids) which consist of propylene glycol, vegetable glycerin, nicotine (in various concentrations), water and flavoring agents. Heavy metals may enter the refill liquid during the production, posing a risk for consumer's health due to their toxicity. The objective of the present study was the development of a methodology for the detection and quantitative analysis of cadmium (Cd), lead (Pb), nickel (Ni), copper (Cu), arsenic (As) and chromium (Cr), employing Total Reflection X-Ray Fluorescence Spectroscopy (TXRF) as an alternative technique to ICP-MS or ICP-OES commonly used for this type of analysis. TXRF was chosen due to its advantages, which include short analysis time, promptness, simultaneous multi-element analysis capability and minimum sample preparation, low purchase and operational cost. The proposed methodology was applied to a large number of electronic cigarette liquids commercially available, as well as their constituents, in order to evaluate their safety. TXRF may be a valuable tool for probing heavy metals in electronic cigarette refill liquids to serve for the protection of human health.
Subject(s)
Electronic Nicotine Delivery Systems , Metals, Heavy/analysis , Spectrometry, X-Ray Emission/methods , Flavoring Agents/analysis , Limit of Detection , Nicotine/analysisABSTRACT
Land plants face the perpetual dilemma of using atmospheric carbon dioxide for photosynthesis and losing water vapors, or saving water and reducing photosynthesis and thus growth. The reason behind this dilemma is that this simultaneous exchange of gases is accomplished through the same minute pores on leaf surfaces, called stomata. In a recent study we provided evidence that pigweed, an aggressive weed, attenuates this problem exploiting large crystals of calcium oxalate as dynamic carbon pools. This plant is able to photosynthesize even under drought conditions, when stomata are closed and water losses are limited, using carbon dioxide from crystal decomposition instead from the atmosphere. Abscisic acid, an alarm signal that causes stomatal closure seems to be implicated in this function and for this reason we named this path "alarm photosynthesis." The so-far "enigmatic," but highly conserved and widespread among plant species calcium oxalate crystals seem to play a crucial role in the survival of plants.
Subject(s)
Calcium Oxalate/metabolism , Abscisic Acid/metabolism , Amaranthus/metabolism , Amaranthus/physiology , Droughts , Photosynthesis/physiology , Plant Leaves/metabolism , Plant Leaves/physiology , Plant Stomata/metabolism , Plant Stomata/physiologyABSTRACT
Calcium oxalate crystals are widespread among animals and plants. In land plants, crystals often reach high amounts, up to 80% of dry biomass. They are formed within specific cells, and their accumulation constitutes a normal activity rather than a pathological symptom, as occurs in animals. Despite their ubiquity, our knowledge on the formation and the possible role(s) of these crystals remains limited. We show that the mesophyll crystals of pigweed (Amaranthus hybridus) exhibit diurnal volume changes with a gradual decrease during daytime and a total recovery during the night. Moreover, stable carbon isotope composition indicated that crystals are of nonatmospheric origin. Stomatal closure (under drought conditions or exogenous application of abscisic acid) was accompanied by crystal decomposition and by increased activity of oxalate oxidase that converts oxalate into CO2 Similar results were also observed under drought stress in Dianthus chinensis, Pelargonium peltatum, and Portulacaria afra Moreover, in A. hybridus, despite closed stomata, the leaf metabolic profiles combined with chlorophyll fluorescence measurements indicated active photosynthetic metabolism. In combination, calcium oxalate crystals in leaves can act as a biochemical reservoir that collects nonatmospheric carbon, mainly during the night. During the day, crystal degradation provides subsidiary carbon for photosynthetic assimilation, especially under drought conditions. This new photosynthetic path, with the suggested name "alarm photosynthesis," seems to provide a number of adaptive advantages, such as water economy, limitation of carbon losses to the atmosphere, and a lower risk of photoinhibition, roles that justify its vast presence in plants.
Subject(s)
Calcium Oxalate/metabolism , Carbon Dioxide/metabolism , Photosynthesis , Plants/metabolism , Abscisic Acid/pharmacology , Circadian Rhythm/drug effects , Crystallization , Metabolome/drug effects , Metabolomics , Photosystem II Protein Complex/metabolism , Plant Leaves/drug effects , Plant Leaves/metabolism , Plant Stomata/drug effects , Plant Stomata/physiology , Plants/drug effects , Spectrum Analysis, Raman , WaterABSTRACT
Imbalances in lipid metabolism affect bone homeostasis, altering bone mass and quality. A link between bone mass and high-density lipoprotein (HDL) has been proposed. Indeed, it has been recently shown that absence of the HDL receptor scavenger receptor class B type I (SR-B1) causes dense bone mediated by increased adrenocorticotropic hormone (ACTH). In the present study we aimed at further expanding the current knowledge as regards the fascinating bone-HDL connection studying bone turnover in apoA-1-deficient mice. Interestingly, we found that bone mass was greatly reduced in the apoA-1-deficient mice compared with their wild-type counterparts. More specifically, static and dynamic histomorphometry showed that the reduced bone mass in apoA-1(-/-) mice reflect decreased bone formation. Biochemical composition and biomechanical properties of ApoA-1(-/-) femora were significantly impaired. Mesenchymal stem cell (MSC) differentiation from the apoA-1(-/-) mice showed reduced osteoblasts, and increased adipocytes, relative to wild type, in identical differentiation conditions. This suggests a shift in MSC subtypes toward adipocyte precursors, a result that is in line with our finding of increased bone marrow adiposity in apoA-1(-/-) mouse femora. Notably, osteoclast differentiation in vitro and osteoclast surface in vivo were unaffected in the knock-out mice. In whole bone marrow, PPARγ was greatly increased, consistent with increased adipocytes and committed precursors. Further, in the apoA-1(-/-) mice marrow, CXCL12 and ANXA2 levels were significantly decreased, whereas CXCR4 were increased, consistent with reduced signaling in a pathway that supports MSC homing and osteoblast generation. In keeping, in the apoA-1(-/-) animals the osteoblast-related factors Runx2, osterix, and Col1a1 were also decreased. The apoA-1(-/-) phenotype also included augmented CEPBa levels, suggesting complex changes in growth and differentiation that deserve further investigation. We conclude that the apoA-1 deficiency generates changes in the bone cell precursor population that increase adipoblast, and decrease osteoblast production resulting in reduced bone mass and impaired bone quality in mice.
Subject(s)
Adipocytes/metabolism , Apolipoprotein A-I/metabolism , Mesenchymal Stem Cells/metabolism , Osteoblasts/metabolism , Adipocytes/cytology , Adipogenesis , Adrenocorticotropic Hormone/metabolism , Animals , Apolipoprotein A-I/deficiency , Apolipoprotein A-I/genetics , Bone Density , Cell Differentiation , Chemokine CXCL12/genetics , Hydrocortisone/biosynthesis , Lipoproteins, HDL/metabolism , Male , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteoblasts/cytology , Osteogenesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, CXCR4/genetics , Receptors, Lipoprotein/metabolism , Scavenger Receptors, Class B/geneticsABSTRACT
In the present study, the possible reversal effects of saffron against established aluminum (Al)-toxicity in adult mice, were investigated. Control, Al-treated (50 mg AlCl(3)/kg/day diluted in the drinking water for 5 weeks) and Al+saffron (Al-treatment as previously plus 60 mg saffron extract/kg/day intraperitoneally for the last 6 days), groups of male Balb-c mice were used. We assessed learning/memory, the activity of acetylcholinesterase [AChE, salt-(SS)/detergent-soluble(DS) isoforms], butyrylcholinesterase (BuChE, SS/DS isoforms), monoamine oxidase (MAO-A, MAO-B), the levels of lipid peroxidation (MDA) and reduced glutathione (GSH), in whole brain and cerebellum. Brain Al was determined by atomic absorption spectrometry, while, for the first time, crocetin, the main active metabolite of saffron, was determined in brain after intraperitoneal saffron administration by HPLC. Al intake caused memory impairment, significant decrease of AChE and BuChE activity, activation of brain MAO isoforms but inhibition of cerebellar MAO-B, significant elevation of brain MDA and significant reduction of GSH content. Although saffron extract co-administration had no effect on cognitive performance of mice, it reversed significantly the Al-induced changes in MAO activity and the levels of MDA and GSH. AChE activity was further significantly decreased in cerebral tissues of Al+saffron group. The biochemical changes support the neuroprotective potential of saffron under toxicity.
Subject(s)
Aluminum/toxicity , Brain/drug effects , Crocus/chemistry , Memory/drug effects , Neuroprotective Agents/pharmacology , Acetylcholinesterase/metabolism , Aluminum/analysis , Aluminum/pharmacokinetics , Animals , Behavior, Animal/drug effects , Brain/metabolism , Carotenoids/analysis , Carotenoids/metabolism , Cognition/drug effects , Glutathione/metabolism , Injections, Intraperitoneal , Learning/drug effects , Lipid Peroxidation/drug effects , Liver/drug effects , Liver/enzymology , Malondialdehyde/metabolism , Memory Disorders/chemically induced , Memory Disorders/drug therapy , Mice , Mice, Inbred BALB C , Monoamine Oxidase/metabolism , Oxidative Stress , Plant Extracts/pharmacology , Vitamin A/analogs & derivativesABSTRACT
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
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 non-porous by nitrogen absorption measurements. These powders, upon dissolution under conditions of constant under-saturation, 25 degrees C, 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 under-saturated 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 under-saturated solutions.