Estudo das interações polifenol-proteína e das reações de escurecimento não-enzimático para o processamento de cajuína

The cashew, a fruit from Brazilian Northeast is used to produce juice due to its flavor and vitamin C richness. However, its acceptance is limited due to its astringency. Cajuína is a derivate product appreciated by its characteristic flavor, freshness and lack of astringency, due to tannin removal. Cajuína is a light yellow beverage made from clarified cashew juice and sterilized after bottling. It differs from the integral and concentrated juice by the clarification and thermal treatment steps. Many problems such as haze and excessive browning could appear if these steps are not controlled. The objective of this work was divided into two stages with the aim to supply process information in order to obtain a good quality product with uniform characteristics (sensory and nutritional). Polyphenol-protein interaction was studied at the clarification step, which is an empirical process, to provide values on the amount of clarifying solution (gelatin) that must be added to achieve a complete juice clarification. Clarification essays were performed with juice dilutions of 1:2 and 1:10 and the effect of metabissulfite and tannic acid addition was evaluated. It was not possible to establish a clarification point. Metabissulfite did not influenced the clarification process however tannic acid addition displaced the clarification point, showing the difficulty visual monitoring of the process. Thermal treatment of clarified juice was studied at 88, 100, 111 e 121 °C. To evaluate the non-enzymatic browning, vitamin C, 5-hidroximetilfurfural (5-HMF) and sugar variation were correlated with color parameters (reflectance spectra, color difference and CIELAB). Kinetic models were obtained for reflectance spectra, ascorbic acid and 5-HMF. It was observed that 5-HMF introduction followed a first order kinetic rate at the beginning of the thermal treatment and a zero order kinetic at later process stages. An inverse correlation was observed between absorbance at 420 nm and ascorbic acid degradation, which indicates that ascorbic acid might be the principal factor on cajuína non-enzymatic browning. Constant sugar concentration showed that this parameter did not contribute directly to the nonenzymatic browning. Optimization techniques showed showed that to obtain a high vitamin C and a low 5-HMF content, the process must be done at 120 ºC. With the water-bath thermal treatment, the 90 °C temperature promoted a lower ascorbic acid degradation at the expense of a higher 5-HMF level

Effect of lentil tannins on albumin hydrolysis by trypsin

Os taninos da casca da semente de lentilha foram extraídos e purificados, levados à interação com albumina isolada de lentilha e com caseína; e estudados por turbidimetria. As interações da albumina e caseína com taninos purificados, a várias relações tanino-proteína, mostraram ser independente e dependente do pH, respectivamente. Hidrólise in vitro com tripsina das proteínas sem taninos indicou que o aquecimento a 99°C/15 min reduzia a susceptibilidade da albumina e aumentava a da caseína à tripsina. A influência de diferentes relações tanino:proteína (1:40; 1:20; 1:5; 1:2,5) na hidrólise mostrou maior inibição para caseína que para albumina de lentilha, independente de aquecimento. Após aquecimento ambas proteínas foram mais hidrolizadas para qualquer das relações tanino proteínas estudadas. A eletroforese em gel de poliacrilamida-dodecilsulfato de sódio do transcurso da hidrólise da interação tanino-albumina nativa mostra a dependência da relação tanino:proteína.

Contribuição ao estudo de redes complexas: modelo de afinidade com métrica

Currently the interest in large-scale systems with a high degree of complexity has been much discussed in the scientific community in various areas of knowledge. As an example, the Internet, protein interaction, collaboration of film actors, among others. To better understand the behavior of interconnected systems, several models in the area of complex networks have been proposed. Barabási and Albert proposed a model in which the connection between the constituents of the system could dynamically and which favors older sites, reproducing a characteristic behavior in some real systems: connectivity distribution of scale invariant. However, this model neglects two factors, among others, observed in real systems: homophily and metrics. Given the importance of these two terms in the global behavior of networks, we propose in this dissertation study a dynamic model of preferential binding to three essential factors that are responsible for competition for links: (i) connectivity (the more connected sites are privileged in the choice of links) (ii) homophily (similar connections between sites are more attractive), (iii) metric (the link is favored by the proximity of the sites). Within this proposal, we analyze the behavior of the distribution of connectivity and dynamic evolution of the network are affected by the metric by A parameter that controls the importance of distance in the preferential binding) and homophily by (characteristic intrinsic site). We realized that the increased importance as the distance in the preferred connection, the connections between sites and become local connectivity distribution is characterized by a typical range. In parallel, we adjust the curves of connectivity distribution, for different values of A, the equation P(k) = P0e

Characterization of two novel polyfunctional mastoparan peptides from the venom of the social wasp Polybia paulista

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monitoring the positioning of short polycationic peptides in model lipid bilayers by combining hydrogen/deuterium exchange and electrospray ionization mass spectrometry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Study of the mechanism of action of anoplin, a helical antimicrobial decapeptide with ion channel-like activity, and the role of the amidated C-terminus

Anoplin, an antimicrobial, helical decapeptide from wasp venom, looses its biological activities by mere deamidation of its C-terminus. Secondary structure determination, by circular dichroism spectroscopy in amphipathic environments, and lytic activity in zwitterionic and anionic vesicles showed quite similar results for the amidated and the carboxylated forms of the peptide. The deamidation of the C-terminus introduced a negative charge at an all-positive charged peptide, causing a loss of amphipathicity, as indicated by molecular dynamics simulations in TFE/water mixtures and this subtle modification in a peptide's primary structure disturbed the interaction with bilayers and biological membranes. Although being poorly lytic, the amidated form, but not the carboxylated, presented ion channel-like activity on anionic bilayers with a well-defined conductance step; at approximately the same concentration it showed antimicrobial activity. The pores remain open at trans-negative potentials, preferentially conducting cations, and this situation is equivalent to the interaction of the peptide with bacterial membranes that also maintain a high negative potential inside. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Restriction endonuclease banding and digestion resistant heterochromatin in triatomines, genus Panstrongylus

The present work realized a comparative study in meiosis of two triatomines, Panstrongylus herreri and P. megistus, by cytogenetic techniques involving the restriction endonucleases Hae III and Alu I and C-banding. The system of sex chromosomes in Panstrongylus is of the X1X2Y type, and experiments corroborated the common origin hypothesis of the X chromosomes by fragmentation of single X. In both species the restriction endonucleases (RE) presented banding patterns in part similar to C-banding. However, in some early meiotic phases it was possible to verify differentiation of the heterochromatic pattern. This work suggests that other elements besides presence of recognition sites, such as chromatin packing degree and DNA-protein interaction, act in RE results, since digestion patterns occur in early spermatogenesis. However, metaphase chromosomes were practically inaccessible to the endonucleases.

Papel das proteínas Yops de Yersinia pseudotuberculosis na ativação dos linfócitos B

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effects of nitric oxide and arginine vasopressin on sodium intake induced by central angiotensin II. Part 2

We study the effects of angiotensin receptors antagonists, arginine vasopressin receptor antagonist, L-arginine and L-NAME, injected into supraoptic nucleus of the hypothalamus (SON) on sodium intake induced by the injection of angiotensin II (ANGII). Holtzman rats weighing 200-250 g with canulae implanted into the SON were used. The drugs were injected in 0.5 μL over 30-60 sec. Sodium intake after injection of saline SAL+SAL 0.15 M NaCl was 0.10±00.1 mL 2 h -1; SAL+ANGII injected into SON increased sodium intake. Losartan injected prior to ANGII into SON decreased sodium intake induced by ANGII. PD123319 injected prior to ANGII produced no changes in sodium intake induced by ANGII. AVPA receptor V 1 antagonist injected prior to ANGII reduced sodium intake with a less intensity than losartan. L-arginine injected prior to ANGII decreases sodium intake at a same intensity than losartan. L-NAME injected prior to ANGII potentiated sodium intake induced by ANGII. Losartan injected simultaneously with L-arginine prior to ANGII blocked the natriorexigenic effect of ANGII. These results confirm the importance of SON in the control of sodium intake. Also suggest that both AT 1 and arginine vasopressin V 1 receptors interact with nitrergic pathways within the SON influencing the sodium metabolism by changing sodium appetite induced by ANGII. © 2007 Asian Network for Scientific Information.

Orphan nuclear receptor NGFI-B forms dimers with nonclassical interface

The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen-deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B-dependent transcription exercised by GR in cells. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society.

Developing new radiotherapy techniques using linac based gamma radiation sources

A major challenge in cancer radiotherapy is to deliver a lethal dose of radiation to the target volume while minimizing damage to the surrounding normal tissue. We have proposed a model on how treatment efficacy might be improved by interfering with biological responses to DNA damage using exogenous electric fields as a strategy to drastically reduce radiation doses in cancer therapy. This approach is demonstrated at this Laboratory through case studies with prokaryotes (bacteria) and eukaryotes (yeast) cells, in which cellkilling rates induced by both gamma radiation and exogenous electric fields were measured. It was found that when cells exposed to gamma radiation are immediately submitted to a weak electric field, cell death increases more than an order of magnitude compared to the effect of radiation alone. This finding suggests, although does not prove, that DNA damage sites are reached and recognized by means of long-range electric DNA-protein interaction, and that exogenous electric fields could destructively interfere with this process. As a consequence, DNA repair is avoided leading to massive cell death. Here we are proposing the use this new technique for the design and construction of novel radiotherapy facilities associated with linac generated gamma beams under controlled conditions of dose and beam intensity.

Fibronectin induces MMP2 expression in human prostate cancer cells

High-grade prostate cancers express high levels of matrix metalloproteinases (MMPs), major enzymes involved in tumor invasion and metastasis. However, the tumor cell lines commonly employed for prostate cancer research express only small amounts of MMPs when cultivated as monolayer cultures, in common culture media. The present study was conducted to ascertain whether culture conditions that include fibronectin can alter MMP2 and MMP9 expression by the human prostatic epithelial cell lines RWPE-1, LNCaP and PC-3. These cells were individually seeded at 2×104cells/cm2, cultivated until they reached 80% confluence, and then exposed for 4h to fibronectin, after which the conditioned medium was analyzed by gelatin zymography. Untreated cells were given common medium. Only RWPE-1 cells express detectable amounts of MMP9 when cultivated in common medium, whereas the addition of fibronectin induced high expression levels of pro and active forms of MMP2 in all tested cell lines. Our findings demonstrate that normal and tumor prostate cell lines express MMP2 activity when in contact with extracellular matrix components or blood plasma proteins such as fibronectin. Future studies of transcriptomes and proteomes in prostate cancer research using these cell lines should not neglect these important conclusions. © 2012 Elsevier Inc..

Cooperative RNA Polymerase Molecules Behavior on a Stochastic Sequence-Dependent Model for Transcription Elongation

The transcription process is crucial to life and the enzyme RNA polymerase (RNAP) is the major component of the transcription machinery. The development of single-molecule techniques, such as magnetic and optical tweezers, atomic-force microscopy and single-molecule fluorescence, increased our understanding of the transcription process and complements traditional biochemical studies. Based on these studies, theoretical models have been proposed to explain and predict the kinetics of the RNAP during the polymerization, highlighting the results achieved by models based on the thermodynamic stability of the transcription elongation complex. However, experiments showed that if more than one RNAP initiates from the same promoter, the transcription behavior slightly changes and new phenomenona are observed. We proposed and implemented a theoretical model that considers collisions between RNAPs and predicts their cooperative behavior during multi-round transcription generalizing the Bai et al. stochastic sequence-dependent model. In our approach, collisions between elongating enzymes modify their transcription rate values. We performed the simulations in Mathematica® and compared the results of the single and the multiple-molecule transcription with experimental results and other theoretical models. Our multi-round approach can recover several expected behaviors, showing that the transcription process for the studied sequences can be accelerated up to 48% when collisions are allowed: the dwell times on pause sites are reduced as well as the distance that the RNAPs backtracked from backtracking sites. © 2013 Costa et al.

An Integrative Genomic and Transcriptomic Analysis Reveals Potential Targets Associated with Cell Proliferation in Uterine Leiomyomas

Background: Uterine Leiomyomas (ULs) are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40-50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs). Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs. Methodology: We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC) and gene expression microarrays (SAM). The CONEXIC algorithm was applied to integrate the data. Principal Findings: The integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively) and IGFBP5 (P = 0.0002 and P = 0.006, respectively) were up-regulated in the tumours when compared with the adjacent normal myometrium. Conclusions: The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs. © 2013 Cirilo et al.

The Deoxyhypusine Synthase Mutant dys1-1 Reveals the Association of eIF5A and Asc1 with Cell Wall Integrity

The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1) catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A) or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of mRNAs associated with cell integrity. © 2013 Galvão et al.