Associação entre polimorfismo inserção/delecção da enzima conversora da angiotensina na diabetes mellitus tipo 2: estudo da população portuguesa

A Diabetes mellitus tipo 2 (DM2) é uma patologia de etiologia múltipla à qual estão associados vários factores genéticos. A Enzima Conversora da Angiotensina (ECA) tem sido alvo de vários estudos pela sua relação com factores pró-inflamatórios, pró-oxidantes e pró-fibrose, sendo o polimorfismo de Inserção/Delecção o mais estudado. Neste contexto, o objectivo deste estudo é assim verificar a distribuição deste polimorfismo numa amostra de indivíduos de nacionalidade portuguesa e verificar a sua possível associação com a DM2. Para tal, foram analisadas 87 amostras (controlos n =24 e diabéticos n =63) de indivíduos de nacionalidade portuguesa. As amostras foram submetidas a um processo de extracção de ADN, sendo posteriormente amplificadas por Polymerase Chain Reaction e analisadas por eletroforese em gel de agarose a 1%. Observou-se uma prevalência de 8% (n=7) com genótipo I/I, 38% (n=33) com genótipo I/D e 54% (n=47) com genótipo D/D. A amostra em estudo demonstrou assim estar sob o equilíbrio Hardy-Weinberg. Observou-se também uma associação entre níveis mais elevados de glicemia e o genótipo I/I (p=0,019). Na análise da utilização de insulina no controlo dos níveis de glicemia na DM2, observou-se uma maior proporção de indivíduos com genótipo D/D. Este estudo demonstra a importância do investimento da caracterização genética em patologias metabólicas multifactoriais como a DM2.

Comparative analysis of two-dimensional electrophoresis maps (2-DE) of Helicobacter pylori from Brazilian patients with chronic gastritis and duodenal ulcer: a preliminary report

Helicobacter pylori is a bacterium recognized as the major cause of peptic ulcer and chronic gastritis. Recently, a proteome-based approach was developed to investigate pathogenic factors related to H. pylori. In this preliminary study, H. pylori strains were isolated from gastric biopsies of patients with chronic gastritis and duodenal ulcers. A partial proteomic analysis of H. pylori strains was performed by bacterial lyses and proteins were separated by two-dimensional gel electrophoresis (2-DE). A comparative analysis was performed to verify a differential protein expression between these two 2-DE maps. These data should be useful to clarify the role of different proteins related to bacterial pathogenesis. This study will be completed using a larger number of samples and protein identification of H. pylori by MALDI-TOF mass spectrometry.

Genetic variability among Anopheles species belonging to the Nyssorhynchus and Anopheles subgenera in the Amazon region

INTRODUCTION: Isoenzymatic analyses were performed involving species of the Nyssorhynchus and Anopheles subgenera in order to estimate the intra and interspecies genetic variability. METHODS: Mosquitoes were caught at different localities in the Amazon region. The collection and rearing of mosquitoes in the laboratory followed specific protocols. For the genetic variability analyses, the technique of horizontal electrophoresis on starch and starch-agarose gel with appropriate buffer systems was used. The alloenzyme variation was estimated using the Biosys-1 software. RESULTS: Out of the 13 loci, eight were polymorphic. Anopheles nuneztovari presented the largest number of alleles per locus, while the smallest number was detected in Anopheles marajoara from Macapá. The largest number of polymorphic loci was found for Anopheles marajoara from Maruanum and the smallest for Anopheles benarrochi (Guayará Mirim). Anopheles darlingi (Macapá) presented the greatest heterozygosity (Ho = 0.167 ± 0.071), while the lowest heterozygosity (Ho = 0.045 ± 0.019) was observed in Anopheles intermedius (Pacoval) of the subgenus Anopheles. Wright's F coefficient revealed considerable genetic structuring between the populations of Anopheles darlingi (Fst = 0.110) and between the populations of Anopheles marajoara (Fst = 0.082). CONCLUSIONS: Considering all the species studied, the genetic distance ranged from 0.008 to 1.114. The greatest distance was between Anopheles mattogrossensis and Anopheles oswaldoi, while the smallest was between the Anopheles benarrochi populations.

Chlamydia pneumoniae and atherosclerosis. Identification of bacterial DNA in the arterial wall

OBJECTIVE: The intracellular Gram-negative bacterium Chlamydia pneumoniae has been associated with atherosclerosis. The presence of Chlamydia pneumoniae has been investigated in fragments of the arterial wall with a technique for DNA identification. METHODS: Arterial fragments obtained from vascular surgical procedures in 58 patients were analyzed. From these patients, 39 were males and the mean age was 65±6 years. The polymerase chain reaction was used to identify the bacterial DNA with a pair of primers that codify the major outer membrane protein (MOMP) of Chlamydia pneumoniae. The amplified product was visualized by electrophoresis in the 2% agarose gel stained with ethidium bromide, and it was considered positive when migrating in the band of molecular weight of the positive controls. RESULTS: Seven (12%) out of the 58 patients showed positive results for Chlamydia pneumoniae. CONCLUSION: DNA from Chlamydia pneumoniae was identified in the arterial wall of a substantial number of patients with atherosclerosis. This association, which has already been described in other countries, corroborates the evidence favoring a role played by Chlamydia pneumoniae in atherogenesis.

Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins

Electrophoretic characteristics of monoclonal immunoglobulin G of different subclasses.

Monoclonal IgG are commonly observed in various B cell disorders, of which multiple myeloma is the most clinically relevant. In a series of serum samples, we identified by immunofixation 73 monoclonal IgG, including 63 IgG(1), 4 IgG(2), 5 IgG(3), and 1 IgG(4). The light chains were of kappa type in 45 cases, and of lambda type in 28 cases. These monoclonal IgG were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) in various isoelectric focusing conditions, as well as by 3-DE (2-DE of the proteins extracted from agarose after serum protein agarose electrophoresis). After 2-DE, 38 out of 73 monoclonal gamma chains (52%) were visualized using immobilized pH 3-10 gradients for isoelectric focusing. In 6 cases (8%), gamma chains were only detected using alkaline immobilized pH 6-11 gradients. In 3 cases (4%), 3-DE revealed monoclonal gamma chains hidden by polyclonal gamma chains. Finally, in 26 cases (36%), no monoclonal gamma chains were clearly visualized. Sixty-one monoclonal light chains (84%) were detected using immobilized pH 3-10 gradients, whereas 12 (16%) were not. Monoclonal gamma chains and light chains were highly heterogeneous in terms of pI and M(r). However, a statistically significant correlation (P<0.05) was observed between the position of the monoclonal IgG in agarose gel and the pI of their heavy and light chains (R=0.733, multiple linear regression). Because of the extreme diversity of their heavy and light chains, it appears that a classification of monoclonal IgG based only on their electrophoretic properties is not possible.

Simulations of electrophoretic collisions of DNA knots with gel obstacles

Gel electrophoresis can be used to separate nicked circular DNA molecules of equal length but forming different knot types. At low electric fields, complex knots drift faster than simpler knots. However, at high electric field the opposite is the case and simpler knots migrate faster than more complex knots. Using Monte Carlo simulations we investigate the reasons of this reversal of relative order of electrophoretic mobility of DNA molecules forming different knot types. We observe that at high electric fields the simulated knotted molecules tend to hang over the gel fibres and require passing over a substantial energy barrier to slip over the impeding gel fibre. At low electric field the interactions of drifting molecules with the gel fibres are weak and there are no significant energy barriers that oppose the detachment of knotted molecules from transverse gel fibres.

Sedimentation of macroscopic rigid knots and its relation to gel electrophoretic mobility of DNA knots.

We address the general question of the extent to which the hydrodynamic behaviour of microscopic freely fluctuating objects can be reproduced by macrosopic rigid objects. In particular, we compare the sedimentation speeds of knotted DNA molecules undergoing gel electrophoresis to the sedimentation speeds of rigid stereolithographic models of ideal knots in both water and silicon oil. We find that the sedimentation speeds grow roughly linearly with the average crossing number of the ideal knot configurations, and that the correlation is stronger within classes of knots. This is consistent with previous observations with DNA knots in gel electrophoresis.

Coeficientes de difusão de metais em materiais não convencionais (agarose e acetato de celulose) usados na técnica de difusão em filmes finos por gradientes de concentração

The DGT technique allows one to measure quantitatively free and labile metal species in aquatic systems. Nevertheless, for this approach, knowledge is required of the diffusion coefficients of the analytes in a diffusive layer. In this study, the diffusion coefficients of Hg(II), As(III), Mn(II), Mg(II), Cu(II), Cd(II) were determined in agarose gel and those of Ba(II), Cd(II), Cu(II), Mg(II), Mn(II) e Zn(II) in cellulose acetate membranes. These materials presented good performance and the reported results can be used as a data base for further DGT studies.

Frequency of CCR5delta32 in Brazilian populations

A sample of 103 randomly chosen healthy individuals from Alegrete, RS, Brazil, was tested for the CCR5delta32 allele, which is known to influence susceptibility to HIV-1 infection. The CCR5delta32 allele was identified by PCR amplification using specific primers flanking the region of deletion, followed by electrophoresis on a 3% agarose gel. The data obtained were compared to those reported for other populations and interpreted in terms of Brazilian history. The individuals studied came from a highly admixed population. Most of them were identified as white (N = 59), while blacks and browns (mulattoes) were N = 13 and N = 31, respectively. The observed frequencies, considering the white, black and brown samples (6.8, 3.8, and 6.4%, respectively), suggest an important European parental contribution, even in populations identified as black and brown. However, in Brazil as a whole, this allele shows gradients indicating a relatively good correlation with the classification based on skin color and other physical traits, used here to define major Brazilian population groups.

DNA damage by ochratoxin A in rat kidney assessed by the alkaline comet assay

There are few studies of ochratoxin A (OTA) genotoxicity in experimental animals and the results obtained with cell cultures are inconsistent, although the carcinogenic potential of OTA for the kidney of experimental animals has been well established. We studied the genotoxic potential of OTA in the kidney of adult female Wistar rats (5 in each group) treated intraperitoneally with OTA (0.5 mg kg body weight-1 day-1 for 7, 14, and 21 days) measuring DNA mobility on agarose gel stained with ethidium-bromide using standard alkaline single-cell gel electrophoresis (comet assay). Negative control animals were treated with solvent (Tris buffer, 1.0 mg/kg) and positive control animals were treated with methyl methanesulfonate (40 mg/kg) according to the same schedule. OTA concentrations in plasma and kidney homogenates in 7-, 14-, and 21-day treated animals were 4.86 ± 0.53, 7.52 ± 3.32, 7.85 ± 2.24 µg/mL, and 0.87 ± 0.09, 0.99 ± 0.06, 1.09 ± 0.15 µg/g, respectively. In all OTA-treated groups, the tail length, tail intensity, and tail moment in kidney tissue were significantly higher than in controls (P < 0.05). The tail length and tail moment were higher after 14 days than after 7 days of treatment (P < 0.05), and still higher after 21 days (P < 0.05). The highest tail intensity was observed in animals treated for 21 days, and it differed significantly from animals treated for 7 and 14 days (P < 0.05). OTA concentrations in plasma and kidney tissue increased steadily and OTA concentration in kidney tissue strongly correlated with tail intensity and tail moment values. These results confirm the genotoxic potential of OTA, and show that the severity of DNA lesions in kidney correlates with OTA concentration.

Effect of castration on renal glycosaminoglycans and their urinary excretion in male and female rats with chronic renal failure

Glycosaminoglycans (GAGs) participate in a variety of processes in the kidney, and evidence suggests that gender-related hormones participate in renal function. The aim of this study was to analyze the relationship of GAGs, gender, and proteinuria in male and female rats with chronic renal failure (CRF). GAGs were analyzed in total kidney tissue and 24-h urine of castrated (c), male (M), and female (F) Wistar control (C) rats (CM, CMc, CF, CFc) and after 30 days of CRF induced by 5/6 nephrectomy (CRFM, CRFMc, CRFF, CRFFc). Total GAG quantification and composition were determined using agarose and polyacrylamide gel electrophoresis, respectively. Renal GAGs were higher in CF compared to CM. CRFM presented an increase in renal GAGs, heparan sulfate (HS), and proteinuria, while castration reduced these parameters. However, CRFF and CRFFc groups showed a decrease in renal GAGs concomitant with an increase in proteinuria. Our results suggest that, in CRFM, sex hormones quantitatively alter GAGs, mainly HS, and possibly the glomerular filtration barrier, leading to proteinuria. The lack of this response in CRFMc, where HS did not increase, corroborates this theory. This pattern was not observed in females. Further studies of CRF are needed to clarify gender-dependent differences in HS synthesis.

Marquage fluorescent des protéines pour étudier les enzymes protéolytiques solubles et immobilisées par la cartographie peptidique électrophorétique

La cartographie peptidique est une méthode qui permet entre autre d’identifier les modifications post-traductionnelles des protéines. Elle comprend trois étapes : 1) la protéolyse enzymatique, 2) la séparation par électrophorèse capillaire (CE) ou chromatographie en phase liquide à haute performance (HPLC) des fragments peptidiques et 3) l’identification de ces derniers. Cette dernière étape peut se faire par des méthodes photométriques ou par spectrométrie de masse (MS). Au cours de la dernière décennie, les enzymes protéolytiques immobilisées ont acquis une grande popularité parce qu’elles peuvent être réutilisées et permettent une digestion rapide des protéines due à un rapport élevé d’enzyme/substrat. Pour étudier les nouvelles techniques d’immobilisation qui ont été développées dans le laboratoire du Professeur Waldron, la cartographie peptidique par CE est souvent utilisée pour déterminer le nombre total de peptides détectés et leurs abondances. La CE nous permet d’avoir des séparations très efficaces et lorsque couplée à la fluorescence induite par laser (LIF), elle donne des limites de détection qui sont 1000 fois plus basses que celles obtenues avec l’absorbance UV-Vis. Dans la méthode typique, les peptides venant de l’étape 1) sont marqués avec un fluorophore avant l’analyse par CE-LIF. Bien que la sensibilité de détection LIF puisse approcher 10-12 M pour un fluorophore, la réaction de marquage nécessite un analyte dont la concentration est d’au moins 10-7 M, ce qui représente son principal désavantage. Donc, il n’est pas facile d’étudier les enzymes des peptides dérivés après la protéolyse en utilisant la technique CE-LIF si la concentration du substrat protéique initial est inférieure à 10-7 M. Ceci est attribué à la dilution supplémentaire lors de la protéolyse. Alors, afin d’utiliser le CE-LIF pour évaluer l’efficacité de la digestion par enzyme immobilisée à faible concentration de substrat,nous proposons d’utiliser des substrats protéiques marqués de fluorophores pouvant être purifiés et dilués. Trois méthodes de marquage fluorescent de protéine sont décrites dans ce mémoire pour étudier les enzymes solubles et immobilisées. Les fluorophores étudiés pour le marquage de protéine standard incluent le naphtalène-2,3-dicarboxaldéhyde (NDA), la fluorescéine-5-isothiocyanate (FITC) et l’ester de 6-carboxyfluorescéine N-succinimidyl (FAMSE). Le FAMSE est un excellent réactif puisqu’il se conjugue rapidement avec les amines primaires des peptides. Aussi, le substrat marqué est stable dans le temps. Les protéines étudiées étaient l’-lactalbumine (LACT), l’anhydrase carbonique (CA) et l’insuline chaîne B (INB). Les protéines sont digérées à l’aide de la trypsine (T), la chymotrypsine (CT) ou la pepsine (PEP) dans leurs formes solubles ou insolubles. La forme soluble est plus active que celle immobilisée. Cela nous a permis de vérifier que les protéines marquées sont encore reconnues par chaque enzyme. Nous avons comparé les digestions des protéines par différentes enzymes telles la chymotrypsine libre (i.e., soluble), la chymotrypsine immobilisée (i.e., insoluble) par réticulation avec le glutaraldéhyde (GACT) et la chymotrypsine immobilisée sur billes d’agarose en gel (GELCT). Cette dernière était disponible sur le marché. Selon la chymotrypsine utilisée, nos études ont démontré que les cartes peptidiques avaient des différences significatives selon le nombre de pics et leurs intensités correspondantes. De plus, ces études nous ont permis de constater que les digestions effectuées avec l’enzyme immobilisée avaient une bonne reproductibilité. Plusieurs paramètres quantitatifs ont été étudiés afin d’évaluer l’efficacité des méthodes développées. La limite de détection par CE-LIF obtenue était de 3,010-10 M (S/N = 2,7) pour la CA-FAM digérée par GACT et de 2,010-10 M (S/N = 4,3) pour la CA-FAM digérée par la chymotrypsine libre. Nos études ont aussi démontrées que la courbe d’étalonnage était linéaire dans la région de travail (1,0×10-9-1,0×10-6 M) avec un coefficient de corrélation (R2) de 0,9991.

Quantification of major camel milk proteins by capillary electrophoresis

Proteins from dromedary camel milk (CM) produced in Europe were separated and quantified by capillary electrophoresis (CE). CE analysis showed that camel milk lacks b-lactoglobulin and consists of high concentration of a-lactalbumin (2.01 ± 0.02 mg mL-1), lactoferrin (1.74 ± 0.06 mg mL-1) and serum albumin (0.46 ± 0.01 mg mL-1 ). Among caseins, the concentration of b-casein (12.78 ± 0.92 mg mL-1) was found the highest followed by a-casein (2.89 ± 0.29 mg mL-1) while k-casein represented only minor amount (1.67 ± 0.01 mg mL-1). These results were in agreement with sodium dodecyl sulphatepolyacrylamide gel electrophoresis patterns. Overall, CE offers a quick and reliable method for the determination of major CM proteins, which may be responsible for the many nutritional and health properties of CM.

MRI study of radiation effect on Fricke gel solutions

The quality control optimization of medical processes that use ionizing radiation in the treatment of diseases like cancer is a key element for patient safety and success of treatment. The major medical application of radiation is radiotherapy, i.e. the delivery of dose levels to well-defined target tissues of a patient with the purpose of eliminating a disease. The need of an accurate tumour-edge definition with the purpose of preserving healthy surrounding tissue demands rigorous radiation treatment planning. Dosimetric methods are used for dose distribution mapping region of interest to assure that the prescribed dose and the irradiated region are correct. The Fricke gel (FXG) is the main dosimeter that supplies visualization of the three-dimensional (3D) dose distribution. In this work the dosimetric characteristics of the modified Fricke dosimeter produced at the Radiation Metrology Centre of the Institute of Energetic and Nuclear Research (IPEN) such as gel concentration dose response dependence, xylenol orange addition influence, dose response between 5 and 50Gy and signal stability were evaluated by magnetic resonance imaging (MRI). Using the same gel solution, breast simulators (phantoms) were shaped and absorbed dose distributions were imaged by MRI at the Nuclear Resonance Laboratory of the Physics Institute of Sao Paulo University. (C) 2007 Elsevier Ltd. All rights reserved.