Labeling of Salmonella Typhymurium with iodine-131 to study phagocytic function in rats

The present study describes a method for labeling Salmonella typhymurium with iodine-131 to evaluate both the morphological and the functional characteristics of the reticulo-endothelial system. A suspension containing 2 x 10(9) bacteria per ml was labeled with carrier-free Na131I without reductor, with a labeling yield of 46.5 ± 3% and 3.5 ± 1.3% of free Iodine-131. The biodistribution of the labeled bacteria in rats was studied with a large field-of-view scintillation camera equiped with a pinhole collimator. Whole body images were obtained 15 and 30 minutes after intravenous injection of the labeled microorganisms. Images showed accumulation of bacteria in the liver and both normal and transplanted spleens of the animals. Autoradiographs of liver and spleen demonstrated labeled bacteria within the cells of the reticulo-endothelial system. The method described is easy to perform, has a good labeling yield and allows the functional evaluation of the reticulo-monophagocytic system, including transplanted spleens.

A stratigraphic framework for the Miocene from the lower Tagus Basin (Lisbon, Setúbal Península, Portugal) Depositional sequences, biostratigraphy and isotopic ages

Rev. Soc. Geol. España, 12(1), ano 1999

Miocene sediments from Foz da Fonte and Penedo sections (Lower Tagus basin): clay minerals and isotopic data

Eight depositional sequences (DS) delimited by regional disconformities had been recognized in the Miocene of Lisbon and Setúbal Peninsula areas. In the case of the western coast of the Setúbal Peninsula, outcrops consisting of Lower Burdigalian to Lower Tortonian sediments were studied. The stratigraphic zonography and the environmental considerations are mainly supported on data concerning to foraminifera, ostracoda, vertebrates and palynomorphs. The first mineralogical and geochemical data determined for Foz da Fonte, Penedo Sul and Penedo Norte sedimentary sequences are presented. These analytical data mainly correspond to the sediments' fine fractions. Mineralogical data are based on X-ray diffraction (XRD), carried out on both the less than 38 nm and 2 nm fractions. Qualitative and semi-quantitative determinations of clay and non-clay minerals were obtained for both fractions. The clay minerals assemblages complete the lithostratigraphic and paleoenvironmental data obtained by stratigraphic and palaeontological studies. Some palaeomagnetic and isotopic data are discussed and correlated with the mineralogical data. Multivariate data analysis (Principal Components Analysis) of the mineralogical data was carried out using both R-mode and Q-mode factor analysis.

Functional brain perfusion evaluation with Arterial Spin Labeling at 3 Tesla

Dissertation submitted in Faculdade de Ciências e Tecnologia of Universidade Nova de Lisboa for the degree of Master of Biomedical Engineering

The effect of energy and traffic light labeling on parents purchasing decisions of cereals for their children

Objective: Nutritional labeling systems are considered a tool to fight obesity since they aim to contribute for more informed food choices as well as assist consumers to make healthier nutrition options and in this manner, contribute to a decrease in the obesity rate. This study intends to analyze the effect of different types of labeling systems on parents’ purchasing decisions for their children on a specific product: breakfast cereals. More precisely, how labels affect parents’ perception of healthiness regarding cereals and if the nutritional information has an effect on intended purchases for their children. Participants and methods: We conducted a study with 135 Portuguese parents of children aged 4 to12 years. Parents answered a questionnaire with one of three hypothetical cereals menus. Menus only differed in their nutritional labeling technique: no labels (control group), reference intake labels or traffic light labels. In addition, we conducted 20 face-to-face interviews to a different group of parents in order to perform a recall task. Findings: This paper provides no evidence to suggest that energy labeling or traffic light labeling systems alone were successful in helping parents making healthy purchases of cereals for their children. Therefore, there is the need to promote supplementary policies to encourage the consumption of healthier food and help fight obesity.

Use of nonradioactive labeling to detect large gene rearrangements in 21-hydroxylase deficiency

PURPOSE: To establish the Southern blotting technique using hybridization with a nonradioactive probe to detect large rearrangements of CYP21A2 in a Brazilian cohort with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH-21OH). METHOD: We studied 42 patients, 2 of them related, comprising 80 non-related alleles. DNA samples were obtained from peripheral blood, digested by restriction enzyme Taq I, submitted to Southern blotting and hybridized with biotin-labeled probes. RESULTS: This method was shown to be reliable with results similar to the radioactive-labeling method. We found CYP21A2 deletion (2.5%), large gene conversion (8.8%), CYP21AP deletion (3.8%), and CYP21A1P duplication (6.3%). These frequencies were similar to those found in our previous study in which a large number of cases were studied. Good hybridization patterns were achieved with a smaller amount of DNA (5 mug), and fragment signs were observed after 5 minutes to 1 hour of exposure. CONCLUSIONS: We established a non-radioactive (biotin) Southern blot/hybridization methodology for CYP21A2 large rearrangements with good results. Despite being more arduous, this technique is faster, requires a smaller amount of DNA, and most importantly, avoids problems with the use of radioactivity.

On the isotopic composition of precipitation in tropical stations (*).

Some of the trends and characteristics of the isotopic composition oh precipitation in tropical stations are discussed. Stations in small Pacific islands show a variation with latitude, with lower 8-values between 15°N and 1S°S and higher values at higher. Inland stations are depleted in heavy isotopes with respect to coastal stations but sometimes this continental effect is rather complex, as f,or instance in África. Mean monthly 8-values show a remarkable correlation with the amount of precipitation, but the slope variations do not show a clear dependence on the mean long term 8-value,as should be expected theoretically. In Southern American stations the seasonal variations of the meanmonthly 5-values are correlated and they are greater in inland stations due to con-tinentaly. The possible effects of recycling of water vapour by evapotranspiration are also discussed.

Development of metabolic labeling strategies to study changes in protein expression

Résumé : Les progrès techniques de la spectrométrie de masse (MS) ont contribué au récent développement de la protéomique. Cette technique peut actuellement détecter, identifier et quantifier des milliers de protéines. Toutefois, elle n'est pas encore assez puissante pour fournir une analyse complète des modifications du protéome corrélées à des phénomènes biologiques. Notre objectif était le développement d'une nouvelle stratégie pour la détection spécifique et la quantification des variations du protéome, basée sur la mesure de la synthèse des protéines plutôt que sur celle de la quantité de protéines totale. Pour cela, nous volions associer le marquage pulsé des protéines par des isotopes stables avec une méthode d'acquisition MS basée sur le balayage des ions précurseurs (precursor ion scan, ou PIS), afin de détecter spécifiquement les protéines ayant intégré les isotopes et d'estimer leur abondance par rapport aux protéines non marquées. Une telle approche peut identifier les protéines avec les plus hauts taux de synthèse dans une période de temps donnée, y compris les protéines dont l'expression augmente spécifiquement suite à un événement précis. Nous avons tout d'abord testé différents acides aminés marqués en combinaison avec des méthodes PIS spécifiques. Ces essais ont permis la détection spécifique des protéines marquées. Cependant, en raison des limitations instrumentales du spectromètre de masse utilisé pour les méthodes PIS, la sensibilité de cette approche s'est révélée être inférieure à une analyse non ciblée réalisée sur un instrument plus récent (Chapitre 2.1). Toutefois, pour l'analyse différentielle de deux milieux de culture conditionnés par des cellules cancéreuses humaines, nous avons utilisé le marquage métabolique pour distinguer les protéines d'origine cellulaire des protéines non marquées du sérum présentes dans les milieux de culture (Chapitre 2.2). Parallèlement, nous avons développé une nouvelle méthode de quantification nommée IBIS, qui utilise des paires d'isotopes stables d'acides aminés capables de produire des ions spécifiques qui peuvent être utilisés pour la quantification relative. La méthode IBIS a été appliquée à l'analyse de deux lignées cellulaires cancéreuses complètement marquées, mais de manière différenciée, par des paires d'acides aminés (Chapitre 2.3). Ensuite, conformément à l'objectif initial de cette thèse, nous avons utilisé une variante pulsée de l'IBIS pour détecter des modifications du protéome dans des cellules HeLa infectée par le virus humain Herpes Simplex-1 (Chapitre 2.4). Ce virus réprime la synthèse des protéines des cellules hôtes afin d'exploiter leur mécanisme de traduction pour la production massive de virions. Comme prévu, de hauts taux de synthèse ont été mesurés pour les protéines virales détectées, attestant de leur haut niveau d'expression. Nous avons de plus identifié un certain nombre de protéines humaines dont le rapport de synthèse et de dégradation (S/D) a été modifié par l'infection virale, ce qui peut donner des indications sur les stratégies utilisées par les virus pour détourner la machinerie cellulaire. En conclusion, nous avons montré dans ce travail que le marquage métabolique peut être employé de façon non conventionnelle pour étudier des dimensions peu explorées en protéomique. Summary : In recent years major technical advancements greatly supported the development of mass spectrometry (MS)-based proteomics. Currently, this technique can efficiently detect, identify and quantify thousands of proteins. However, it is not yet sufficiently powerful to provide a comprehensive analysis of the proteome changes correlated with biological phenomena. The aim of our project was the development of ~a new strategy for the specific detection and quantification of proteomé variations based on measurements of protein synthesis rather than total protein amounts. The rationale for this approach was that changes in protein synthesis more closely reflect dynamic cellular responses than changes in total protein concentrations. Our starting idea was to couple "pulsed" stable-isotope labeling of proteins with a specific MS acquisition method based on precursor ion scan (PIS), to specifically detect proteins that incorporated the label and to simultaneously estimate their abundance, relative to the unlabeled protein isoform. Such approach could highlight proteins with the highest synthesis rate in a given time frame, including proteins specifically up-regulated by a given biological stimulus. As a first step, we tested different isotope-labeled amino acids in combination with dedicated PIS methods and showed that this leads to specific detection of labeled proteins. Sensitivity, however, turned out to be lower than an untargeted analysis run on a more recent instrument, due to MS hardware limitations (Chapter 2.1). We next used metabolic labeling to distinguish the proteins of cellular origin from a high background of unlabeled (serum) proteins, for the differential analysis of two serum-containing culture media conditioned by labeled human cancer cells (Chapter 2.2). As a parallel project we developed a new quantification method (named ISIS), which uses pairs of stable-isotope labeled amino acids able to produce specific reporter ions, which can be used for relative quantification. The ISIS method was applied to the analysis of two fully, yet differentially labeled cancer cell lines, as described in Chapter 2.3. Next, in line with the original purpose of this thesis, we used a "pulsed" variant of ISIS to detect proteome changes in HeLa cells after the infection with human Herpes Simplex Virus-1 (Chapter 2.4). This virus is known to repress the synthesis of host cell proteins to exploit the translation machinery for the massive production of virions. As expected, high synthesis rates were measured for the detected viral proteins, confirming their up-regulation. Moreover, we identified a number of human proteins whose synthesis/degradation ratio (S/D) was affected by the viral infection and which could provide clues on the strategies used by the virus to hijack the cellular machinery. Overall, in this work, we showed that metabolic labeling can be employed in alternative ways to investigate poorly explored dimensions in proteomics.

Depleted arc volcanism in the Alboran Sea and shoshonitic volcanism in Morocco: geochemical and isotopic constraints on Neogene tectonic processes

Samples of volcanic rocks from Alboran Island, the Alboran Sea floor and from the Gourougou volcanic centre in northern Morocco have been analyzed for major and trace elements and Sr-Nd isotopes to test current theories on the tectonic geodynamic evolution of the Alboran Sea. The Alboran Island samples are low-K tholeiitic basaltic andesites whose depleted contents of HFS elements (similar to0.5xN-MORB), especially Nb (similar to0.2xN-MORB), show marked geochemical parallels with volcanics from immature intra-oceanic arcs and back-arc basins. Several of the submarine samples have similar compositions, one showing low-Ca boninite affinity. Nd-143/Nd-144 ratios fall in the same range as many island-arc and back-arc basin samples, whereas Sr-87/Sr-86 ratios (on leached samples) are somewhat more radiogenic. Our data point to active subduction taking place beneath the Alboran region in Miocene times, and imply the presence of an associated back-arc spreading centre. Our sea floor suite includes a few more evolved dacite and rhyolite samples with (Sr-87/Sr-86)(0) up to 0.717 that probably represent varying degrees of crustal melting. The shoshonite and high-K basaltic andesite lavas from Gourougou have comparable normalized incompatible-element enrichment diagrams and Ce/Y ratios to shoshonitic volcanics from oceanic island arcs, though they have less pronounced Nb deficits. They are much less LIL- and LREE-enriched than continental arc analogues and post-collisional shoshonites from Tibet. The magmas probably originated by melting in subcontinental lithospheric mantle that had experienced negligible subduction input. Sr-Nd isotope compositions point to significant crustal contamination which appears to account for the small Nb anomalies. The unmistakable supra-subduction zone (SSZ) signature shown by our Alboran basalts and basaltic andesite samples refutes geodynamic models that attribute all Neogene volcanism in the Alboran domain to decompression melting of upwelling asthenosphere arising from convective thinning of over-thickened lithosphere. Our data support recent models in which subsidence is caused by westward rollback of an eastward-dipping subduction zone beneath the westemmost Mediterranean. Moreover, severance of the lithosphere at the edges of the rolling-back slab provides opportunities for locally melting lithospheric mantle, providing a possible explanation for the shoshonitic volcanism seen in northern Morocco and more sporadically in SE Spain. (C) 2004 Elsevier B.V. All rights reserved.

Cell-specific localization of monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain revealed by double immunohistochemical labeling and confocal microscopy.

Recent evidence suggests that lactate could be a preferential energy substrate transferred from astrocytes to neurons. This would imply the presence of specific transporters for lactate on both cell types. We have investigated the immunohistochemical localization of two monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain. Using specific antibodies raised against MCT1 and MCT2, we found strong immunoreactivity for each transporter in glia limitans, ependymocytes and several microvessel-like elements. In addition, small processes distributed throughout the cerebral parenchyma were immunolabeled for monocarboxylate transporters. Double immunofluorescent labeling and confocal microscopy examination of these small processes revealed no co-localization between glial fibrillary acidic protein and monocarboxylate transporters, although many glial fibrillary acidic protein-positive processes were often in close apposition to elements labeled for monocarboxylate transporters. In contrast, several elements expressing the S100beta protein, another astrocytic marker found to be located in distinct parts of the same cell when compared with glial fibrillary acidic protein, were also strongly immunoreactive for MCT1, suggesting expression of this transporter by astrocytes. In contrast, MCT2 was expressed in a small subset of microtubule-associated protein-2-positive elements, indicating a neuronal localization. In conclusion, these observations are consistent with the possibility that lactate, produced and released by astrocytes (via MCT1), could be taken up (via MCT2) and used by neurons as an energy substrate.

Immunogold labeling and cerium cytochemistry of the enzyme ecto-5'-nucleotidase in promastigote forms of Leishmania species

We have applied both enzyme cytochemistry and immunological labeling techniques to characterize the enzyme 5'-nucleotidase (5'-Nase), at the ultrastructural level, in promastigote forms of four Leishmania species: Leishmania amazonensis, Leishmania mexicana, Leishmania donovani and Leishmania chagasi. The cerium phosphate staining was localized at the surface of the cell body, the flagellum and the flagellar pocket membranes of all the parasites studied. The immunogold labelling technique confirmed these results. In this report we localized 5'-Nase in L. chagasi and L. amazonensis which have been implicated respectively in visceral and cutaneous forms of leishmaniasis. In addition, we confirmed the localization of this phosphomonoesterase in the other two species studied. The superior quality of the images, obtained with both methodologies, confirms that these parasites possess mechanisms capable of hydrolyzing nucleotide monophosphates, and that the expression of 5'-Nase is associated with the outer surface of the plasma membrane.

Alteration of brain glycogen turnover in the conscious rat after 5h of prolonged wakefulness.

Although glycogen (Glyc) is the main carbohydrate storage component, the role of Glyc in the brain during prolonged wakefulness is not clear. The aim of this study was to determine brain Glyc concentration ([]) and turnover time (tau) in euglycemic conscious and undisturbed rats, compared to rats maintained awake for 5h. To measure the metabolism of [1-(13)C]-labeled Glc into Glyc, 23 rats received a [1-(13)C]-labeled Glc solution as drink (10% weight per volume in tap water) ad libitum as their sole source of exogenous carbon for a "labeling period" of either 5h (n=13), 24h (n=5) or 48 h (n=5). Six of the rats labeled for 5h were continuously maintained awake by acoustic, tactile and olfactory stimuli during the labeling period, which resulted in slightly elevated corticosterone levels. Brain [Glyc] measured biochemically after focused microwave fixation in the rats maintained awake (3.9+/-0.2 micromol/g, n=6) was not significantly different from that of the control group (4.0+/-0.1 micromol/g, n=7; t-test, P>0.5). To account for potential variations in plasma Glc isotopic enrichment (IE), Glyc IE was normalized by N-acetyl-aspartate (NAA) IE. A simple mathematical model was developed to derive brain Glyc turnover time as 5.3h with a fit error of 3.2h and NAA turnover time as 15.6h with a fit error of 6.5h, in the control rats. A faster tau(Glyc) (2.9h with a fit error of 1.2h) was estimated in the rats maintained awake for 5h. In conclusion, 5h of prolonged wakefulness mainly activates glycogen metabolism, but has minimal effect on brain [Glyc].

Relative protein quantification by isobaric SILAC with immonium ion splitting (ISIS)

Metabolic labeling techniques have recently become popular tools for the quantitative profiling of proteomes. Classical stable isotope labeling with amino acids in cell cultures (SILAC) uses pairs of heavy/light isotopic forms of amino acids to introduce predictable mass differences in protein samples to be compared. After proteolysis, pairs of cognate precursor peptides can be correlated, and their intensities can be used for mass spectrometry-based relative protein quantification. We present an alternative SILAC approach by which two cell cultures are grown in media containing isobaric forms of amino acids, labeled either with 13C on the carbonyl (C-1) carbon or 15N on backbone nitrogen. Labeled peptides from both samples have the same nominal mass and nearly identical MS/MS spectra but generate upon fragmentation distinct immonium ions separated by 1 amu. When labeled protein samples are mixed, the intensities of these immonium ions can be used for the relative quantification of the parent proteins. We validated the labeling of cellular proteins with valine, isoleucine, and leucine with coverage of 97% of all tryptic peptides. We improved the sensitivity for the detection of the quantification ions on a pulsing instrument by using a specific fast scan event. The analysis of a protein mixture with a known heavy/light ratio showed reliable quantification. Finally the application of the technique to the analysis of two melanoma cell lines yielded quantitative data consistent with those obtained by a classical two-dimensional DIGE analysis of the same samples. Our method combines the features of the SILAC technique with the advantages of isobaric labeling schemes like iTRAQ. We discuss advantages and disadvantages of isobaric SILAC with immonium ion splitting as well as possible ways to improve it