Molecular Analysis of NOTCH2 in Patients with Primary Open-Angle Glaucoma.

BACKGROUND AND PURPOSE: Transgenic mice overexpressing Notch2 in the uvea exhibit a hyperplastic ciliary body leading to increased IOP and glaucoma. The aim of this study was to investigate the possible presence of NOTCH2 variants in patients with primary open-angle glaucoma (POAG). METHODS: We screened DNA samples from 130 patients with POAG for NOTCH2 variants by denaturing high-performance liquid chromatography after PCR amplification and validated our data by direct Sanger sequencing. RESULTS: No mutations were observed in the coding regions of NOTCH2 or in the splice sites. 19 known SNPs (single nucleotide polymorphisms) were detected. An SNP located in intron 24, c.[4005+45A>G], was seen in 28.5% of the patients (37/130 patients). As this SNP is reported to have a minor allele frequency of 7% in the 1000 genomes database, it could be associated with POAG. However, we evaluated its frequency in an ethnic-matched control group of 96 subjects unaffected by POAG and observed a frequency of 29%, indicating that it was not related to POAG. CONCLUSION: NOTCH2 seemed to be a good candidate for POAG as it is expressed in the anterior segment in the human eye. However, mutational analysis did not show any causative mutation. This study also shows that proper ethnic-matched control groups are essential in association studies and that values given in databases are sometimes misleading.

Brown algal phlorotannins: Improving and applying chemical methods

Phlorotannins are the least studied group of tannins and are found only in brown algae. Hitherto the roles of phlorotannins, e.g. in plant-herbivore interactions, have been studied by quantifying the total contents of the soluble phlorotannins with a variety of methods. Little attention has been given to either quantitative variation in cell-wall-bound and exuded phlorotannins or to qualitative variation in individual compounds. A quantification procedure was developed to measure the amount of cell-wall-bound phlorotannins. The quantification of soluble phlorotannins was adjusted for both large- and small-scale samples and used to estimate the amounts of exuded phlorotannins using bladder wrack (Fucus vesiculosus) as a model species. In addition, separation of individual soluble phlorotannins to produce a phlorotannin profile from the phenolic crude extract was achieved by high-performance liquid chromatography (HPLC). Along with these methodological studies, attention was focused on the factors in the procedure which generated variation in the yield of phlorotannins. The objective was to enhance the efficiency of the sample preparation procedure. To resolve the problem of rapid oxidation of phlorotannins in HPLC analyses, ascorbic acid was added to the extractant. The widely used colourimetric method was found to produce a variation in the yield that was dependent upon the pH and concentration of the sample. Using these developed, adjusted and modified methods, the phenotypic plasticity of phlorotannins was studied with respect to nutrient availability and herbivory. An increase in nutrients decreased the total amount of soluble phlorotannins but did not affect the cell-wall-bound phlorotannins, the exudation of phlorotannins or the phlorotannin profile achieved with HPLC. The presence of the snail Thedoxus fluviatilis on the thallus induced production of soluble phlorotannins, and grazing by the herbivorous isopod Idotea baltica increased the exudation of phlorotannins. To study whether the among-population variations in phlorotannin contents arise from the genetic divergence or from the plastic response of algae, or both, algae from separate populations were reared in a common garden. Genetic variation among local populations was found in both the phlorotannin profile and the content of total phlorotannins. Phlorotannins were also genetically variable within populations. This suggests that local algal populations have diverged in their contents of phlorotannins, and that they may respond to natural selection and evolve both quantitatively and qualitatively.

N-acetylcysteine treatment ameliorates the skeletal phenotype of a mouse model of diastrophic dysplasia.

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by mutations in SLC26A2, a cell membrane sulfate-chloride antiporter. Sulfate uptake impairment results in low cytosolic sulfate, leading to cartilage proteoglycan (PG) undersulfation. In this work, we used the dtd mouse model to study the role of N-acetyl-l-cysteine (NAC), a well-known drug with antioxidant properties, as an intracellular sulfate source for macromolecular sulfation. Because of the important pre-natal phase of skeletal development and growth, we administered 30 g/l NAC in the drinking water to pregnant mice to explore a possible transplacental effect on the fetuses. When cartilage PG sulfation was evaluated by high-performance liquid chromatography disaccharide analysis in dtd newborn mice, a marked increase in PG sulfation was observed in newborns from NAC-treated pregnancies when compared with the placebo group. Morphometric studies of the femur, tibia and ilium after skeletal staining with alcian blue and alizarin red indicated a partial rescue of abnormal bone morphology in dtd newborns from treated females, compared with pups from untreated females. The beneficial effect of increased macromolecular sulfation was confirmed by chondrocyte proliferation studies in cryosections of the tibial epiphysis by proliferating cell nuclear antigen immunohistochemistry: the percentage of proliferating cells, significantly reduced in the placebo group, reached normal values in dtd newborns from NAC-treated females. In conclusion, NAC is a useful source of sulfate for macromolecular sulfation in vivo when extracellular sulfate supply is reduced, confirming the potential of therapeutic approaches with thiol compounds to improve skeletal deformity and short stature in human DTD and related disorders.

Validació del mètode analític per la determinació d'aminoàcids i carbohidrats d'un producte farmacèutic per HPLC

Per poder desenvolupar un producte farmacèutic és necessari establir un mètode d’anàlisis que permeti determinar i quantificar totes aquelles substàncies que conté, ja sigui referent als principis actius; a les impureses i productes de degradació, conservants, antioxidants,... Grans entitats com la ICH remarquen la importància de validar els mètodes analítics ja que és la via per demostrar que aquell producte compleix les garanties de qualitat prèviament establertes. Així doncs, l’objectiu d’aquest Treball Final de Grau és poder desenvolupar i validar dos mètodes analítics per a la determinació d’aminoàcids i carbohidrats respectivament, d’un producte farmacèutic per cromatografia líquida (HPLC). Per tal de poder concloure que aquell mètode és adequat per la determinació per la qual ha estat desenvolupat, és necessari obtenir resultats que compleixin els criteris d’acceptació corresponents als paràmetres que han de ser avaluats en una validació analítica. Aquests paràmetres són: la precisió, la selectivitat, l’exactitud i la linealitat i el rang. Els resultats d’aquest projecte han demostrat que els dos mètodes desenvolupats són adequats per a la determinació de tres dels principis actius (aminoàcid 1, aminoàcid 2 i carbohidrat 1) que conté el producte farmacèutic d’ús veterinari analitzat; i poden ser validats ja que compleixen els criteris d’acceptació dels paràmetres avaluats que proposa la ICH. El mètode per la determinació de carbohidrats no és vàlid per el carbohidrat 2, ja que durant el desenvolupament es va detectar que una bona part d’aquest passava a carbohidrat 1 (desplaçament de l’equilibri ceto-enòlic que hi ha entre el carbohidrat 1 i el carbohidrat 2 a pHs alts). És per aquest motiu, que es pot concloure que aquest mètode no és vàlid i es recomana seguir investigant per a poder desenvolupar un mètode analític adient.

Influência do material do alambique na composição química das aguardentes de cana-de-açúcar

This paper describes variations in the profile of the main volatile organic compounds present in Brazilian sugar cane spirits distilled in copper and stainless steel distillers. The main organic compounds: aldehydes, ketones, carboxylic acids, alcohols and esters, were determined through High Performance Liquid Chromatography (HPLC) and High Resolution Gas Cromatography (HRGC). The spirits produced in copper distillers exhibit higher contents of aldehydes with respect to the ones produced in stainless steel. The inverse is true with respect to the higher alcohol and ester contents. No significant variation has been observed for the carboxylic acids.

Drosophila melanogaster Meigen: 3. sensibilidade ao carbofuran e biomonitoramento de seus resíduos em repolho

The susceptibility of Drosophila melanogaster to carbofuran and the use of this organism in biomonitoring residues of the insecticide in cabbage was evaluated. Under the conditions of the bioassay, residues-film bioassay in Petri dish, carbofuran degraded depending on the temperature and time of exposure. Bioassays conducted with D. melanogaster showed that its toxicity increases with temperature (20 to 35 °C). LC50 values, calculated as a function of temperature, ranged from 3.6 to 10.5 mg/g body weight (bw) for males and from 2.9 to 8.7 mg/g bw for females. The formulated product Furadan® G was applied on cabbage (Brassica oleracea, var. capitata) and the residues of carbofuran were determined by bioassay. The determination limit of the bioassay was 0.1 mg/kg and the method presented reproducibility with coefficient variation of 17 %. The validation of the bioassay by high performance liquid chromatography confirms the viability of the bioassay with D. melanogaster in monitoring the residues of carbofuran in cabbage.

Perfil cromatográfico de duas espécies de Plumbaginaceae: Plumbago scandens L. E Plumbago auriculata Lam

The genus Plumbago belongs to the family Plumbaginaceae, order Plumbaginales. Comparative chemical profile of P. scandens (native) and P. auriculata (cultivated) was obtained by normal and reversed-phase high performance liquid chromatography with photodiode array detector. Comparison of the ultraviolet espectra and the retention times for the compounds allowed to find similar metabolic patterns in roots, stems and leaves. Four flavonoids, one phenolic acid or derivative and the naphtoquinone plumbagin were comparatively identified to standards.

Determinação de alfa-Tocoferol em alho irradiado utilizando cromatografia líquida de alta eficiência (CLAE)

The effects of 60Co ionizing radiations in doses of 0, 75, 100, 150, 200 and 250Gy on garlic, upon the alpha-tocopherol concentration were studied. The alpha-tocopherol contents were established by high performance liquid chromatography (HPLC), after direct hexane extraction from the garlic samples. The alpha-tocopherol was determined through normal-phase column, and mobile phase was composed by hexane: iso-propyl alcohol (99:01 v/v), with 2mL/min flow rate and fluorescence detector. It is statistically shown that an irradiation dose of up to 150 Gy does not affect the garlic alpha-tocopherol content.

Determinação de corantes artificiais por cromatografia líquida de alta eficiência (CLAE) em pó para gelatina

Synthetic dyes were determined by high performance liquid chromatography in 76 samples of regular jelly and diet jelly powders, of several flavors produced by seven different manufacturers. Three sample of each product, from different batches, were analyzed. The same qualitative composition was observed for products of the same flavor from different manufacturers, but the quantitative composition varied markedly from one manufacturer to the other. There were no or few significant differences between batches from a given manufacturer, demonstrating good quality control in the use of these additives by the food industry. None of the samples exceeded the limit stipulated by Brazilian legislation.

Adsorção/dessorção do explosivo tetril em turfa e em argissolo vermelho amarelo

This paper presents the study of adsorption/desorption of the explosive tetryl (2,4,6-trinitrophenylmethyl-nitramine) in different matrices, such as in natura soil, roasted soil, humic acid of soil, in natura peat, roasted peat and humic acid of peat. The aim of the study is to evaluate the interaction capacity of those matrices with the explosive. The analytic technique used was HPLC (high performance liquid chromatography), with UV-detection at 230 nm. The Freundlich isotherms were utilized for the mathematical treatment of the data. The results indicated that in natura soil and in natura peat (with organic substances) are excellent matrices for the retention of tetryl, adsorbing it and keeping it immovable, preventing it from contaminating the groundwater. The largest adsorption of the explosive ocurred in in natura soil, while the smallest desorption was observed in in natura peat. After the calcination of the matrices, the smallest adsorption was observed, indicating that the retention occurs in the organic substance.

Bisfosfonatos: síntese, análises químicas e aplicações farmacológicas

Bisphosphonates are drugs that have been widely used in different bone diseases, and have recently been used successfully against many parasites. Various synthetic routes to prepare different types of bisphosphonates have been described, with distinct potency and pharmacological activity. A number of analytical techniques are currently being used to analyze these drugs; among these, the high performance liquid chromatography (HPLC), with different systems of detection, is worth highlighting. However, the development of more sensitive methods is still necessary, once they are essential for bioavailability and bioequivalence studies. This paper reports the major synthesis routes, chemical analysis methodologies and pharmacological applications of bisphosphonates.

Método para a determinação de hexazinone e tebutiuron em água

This work presents an alternative method for determination of the herbicides tebuthiuron and hexazinone in ground water. The extraction was made with dichloromethane and the analyses by high performance liquid chromatography (HPLC), using reversed-phase column, C-18, mobile phase methanol/water 50:50, v/v, detection and quantification at 247 nm. The following validation parameters were obtained: limit of detection of method 0.02 and 0.03 µg L-1, limit of quantification of method 0.07 and 0.09 µg L-1; linear range limit of quantification of instrument - 300 µg L-1 (r² > 0.998); recoveries from 90.3 to 108.2% and 90.3 to 101.6%; intermediary precision (%RSD) < 8 and < 6%, for hexazinone and tebuthiuron, respectively. The method showed to be efficient and reliable for determination of the herbicides in ground water.

Cassava and corn starch in maltodextrin production

Maltodextrin was produced from cassava and corn starch by enzymatic hydrolysis with alpha-amylase. The cassava starch hydrolysis rate was higher than that of corn starches in maltodextrin production with shorter dextrose equivalent (DE). DE values do not show directly the nature of the obtained oligosaccharides. Maltodextrin produced from cassava and corn starch was analysed by high performance liquid chromatography (HPLC), and the analysis showed that maltodextrin production differs according to the source of the starch. This is important in defining the application of the maltodextrin, according to its desired function.

Análise enantiosseletiva de fármacos: contribuições da cromatografia líquida de alta eficiência e eletroforese capilar

The demand for analytical methods suitable for accurate and reproducible determination of drug enantiomers has increased significantly in the last years. High-performance liquid chromatography (HPLC) using chiral stationary phases and capillary electrophoresis (CE) are the most important techniques used for this purpose. In this paper, the fundamental aspects of chiral separations using both techniques are presented. Some important aspects for the development of enantioselective methods, particularly for the analysis of drugs and metabolites in biological samples, are also discussed.

Análise de fármacos em material biológico: acoplamento microextração em fase sólida "no tubo" e cromatografia líquida de alta eficiência

A new solid phase microextraction (SPME) system, known as in-tube SPME, was recently developed using an open tubular fused-silica capilary column, instead of an SPME fiber, as the SPME device. On-line in-tube SPME is usually used in combination with high performance liquid chromatography. Drugs in biological samples are directly extracted and concentrated in the stationary phase of capillary columns by repeated draw/eject cycles of sample solution, and then directly transferred to the liquid chromatographic column. In-tube SPME is suitable for automation. Automated sample handling procedures not only shorten the total analysis time, but also usually provide better accuracy and precision relative to manual techniques. In-tube SPME has been demonstrated to be a very effective and highly sensitive technique to determine drugs in biological samples for various purposes such as therapeutic drug monitoring, clinical toxicology, bioavailability and pharmacokinetics.