Soybean lunasin mediates colon carcinogenesis by inducing apoptosis and preventing outgrowth of metastasis

Colorectal cancer (CRC) is the third most common cancer worldwide. Various factors such as age, lifestyle and dietary patterns affect the risk of having CRC. Epidemiological studies showed a chemopreventive effect of soy consumption against CRC. However, which component(s) of soybean is associated with this reduced risk is not yet fully delineated. The objective of this research was to evaluate the anti-colon cancer potential of lunasin isolated from defatted soybean flour using in vitro and in vivo models of CRC. Lunasin was isolated from defatted soybean flour by a combination of different chromatographic and ultrafiltration techniques. The anti-colon cancer potential of lunasin was determined using different human colon cancer cell lines in vitro and a CRC liver metastasis model in vivo. Lunasin caused cytotoxicity to different human colon cancer cells with an IC50 value of 13.0, 21.6, 26.3 and 61.7 µM for KM12L4, RKO, HCT-116 and HT-29 human colon cancer cells, respectively. This cytotoxicity correlated with the expression of the α5 integrin on human colon cancer cells with a correlation coefficient of 0.78. The mechanism involved in the cytotoxic effect of lunasin was through cell cycle arrest and induction of the mitochondrial pathway of apoptosis. In KM12L4 human colon cancer cells, lunasin caused a G2/M phase arrest increasing the percentage of cells at G2/M phase from 12% (PBS-treated) to 24% (treated with 10 µM lunasin). This arrest was attributed to the capability of lunasin to increase the expression of cyclin dependent kinase inhibitors p21 and p27. At 10 µM, lunasin increased the expression of p21 and p27 in KM12L4 colon cancer cells by 2.2- and 2.3-fold, respectively. Flow cytometric analysis showed that lunasin at 10 µM increased the percentage of cells undergoing apoptosis from 13.6% to 24.7%. This is further supported by fluorescence microscopic analysis of KM12L4 cells treated with 10 µM lunasin showing chromatin condensation and DNA fragmentation. The mechanism involved is through modification of proteins involved in the mitochondrial pathway of apoptosis in KM12L4 cells as 10 µM lunasin reduced the expression of the anti-apoptotic Bcl-2 protein by 2-fold and increased the expression of the pro-apoptotic proteins Bax, cytochrome c and nuclear clusterin by 2.2-, 2.1- and 2.3- fold, respectively. This led to increased expression and activity of the executioner of apoptosis, caspase-3 by 1.8- and 2.3-fold, respectively. This pro-apoptotic property of lunasin can be attributed to its capability to internalize into the cytoplasm and nucleus of colon cancer cells 24 h and 72 h after treatment, respectively. In addition, lunasin mediated metastasis of colon cancer cells in vitro by inhibiting the focal adhesion kinase activation thereby reducing expression of extracellular regulated kinase and nuclear factor kappa B and finally inhibiting migration of colon cancer cells. In KM12L4 colon cancer cells, 10 µM lunasin resulted in the reduction of phosphorylation of focal adhesion kinase and extracellular regulated kinase by 2.5-fold, resulting in the reduced nuclear translocation of p50 and p65 NF-κB subunits by 3.8- and 1.4-fold, respectively. In an in vivo model of CRC liver metastasis, daily intraperitoneal administration of lunasin at 4 mg/kg body weight resulted in the inhibition of KM12L4 liver metastasis as shown by the reduction of the number of liver metastases from 28 (PBS-treated) to 14 (lunasin-treated, P = 0.047) and reduction in tumor burden as measured by liver weight/body weight from 0.13 (PBS-treated) to 0.10 (lunasin-treated, P = 0.039). Moreover, lunasin potentiated the anti-metastatic effect of the chemotherapeutic drug oxaliplatin given at 5 mg/kg body weight twice per week. Lunasin and oxaliplatin combination resulted in a more potent inhibition of outgrowth of KM12L4 cell metastases to the liver reducing the number of liver metastases by 6-fold and reducing the tumor burden in the liver by 3-fold when compared to PBS-treated group. This can be attributed by the capability of lunasin and oxaliplatin to reduce expression of proliferating cell nuclear antigen in liver-tumor tissue as measured by immunohistochemical staining. The results of this research for the first time demonstrated the anti-colon cancer potential of lunasin isolated from defatted soybean flour which might contribute to the chemopreventive effect of soybean in CRC as seen in different epidemiological studies. In conclusion, lunasin isolated from defatted soybean flour mediated colon carcinogenesis by inducing apoptosis and preventing outgrowth of metastasis. We suggest that the results of this research serve as a basis for further study on the chemopreventive effect of lunasin against CRC and a possible adjuvant role for lunasin in therapy of patients with metastatic CRC.

Atividade fisica e saude : estudo do conhecimento de professores de educação fisica do ensino fundamental atraves da educação a distancia

Universidade Estadual de Campinas . Faculdade de Educação Física

Imagem por ressonância magnética: princípios básicos

A ressonância magnética é a propriedade física exibida por núcleos de determinados elementos que, quando submetidos a um campo magnético forte e excitados por ondas de rádio em determinada freqüência (Freqüência de Larmor), emitem rádio sinal, o qual pode ser captado por uma antena e transformado em imagem. A imagem por ressonância magnética (IRM) é o método de diagnóstico por imagem não-invasivo mais sensível para avaliar partes moles, particularmente o encéfalo, porém trata-se de uma técnica onerosa. Ela apresenta grande potencial diagnóstico, poucos efeitos deletérios e muitos benefícios a serem obtidos com o seu uso. Além disso, a IRM fornece informações anatômicas acuradas, imagens em qualquer plano do corpo, bom contraste e resolução espacial e por si só pode sugerir um diagnóstico. Porém, não permite um diagnóstico histológico específico e deve ser interpretada em contexto com outros achados clínicos e patológicos. Esta revisão teve como objetivos mostrar as bases físicas da ressonância magnética e propiciar mais conhecimento aos veterinários.

FBXO25-associated nuclear domains: A novel subnuclear structure

Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.

Genetic parameters for pre-weaning traits in Braunvieh cattle

The objective of this study was to estimate genetic parameters for pre-weaning traits of Braunvieh cattle raised under tropical conditions in Brazil. The weight and weight gain parameters were birth weight (BW, N = 9955), weight at 120 days of age (W120, N = 5901), weaning weight at 205 days (WW, N = 6970), weight gain from birth to 205 days (GAIN205, N = 6013), weight gain from birth to 120 days (GAIN120, N = 5135), and weight gain from 120 to 205 days (GAIN85, N = 4482). Variance components were estimated using the animal model with the MTDFREML software. The relationship matrix included 35,188 animals; phenotypic measures were available for 18,688. Direct and maternal heritability increased from birth to weaning, with estimates of 0.23 +/- 0.037, 0.25 +/- 0.050, 0.41 +/- 0.059 for direct heritability for BW, W120 and WW, respectively, 0.08 +/- 0.012, 0.15 +/- 0.032, 0.22 +/- 0.036 for maternal genetic effects, and 0.18, 0.14 and 0.16 for total heritability estimates. For pre-weaning gains, estimates of heritability were 0.36 +/- 0.059, 0.30 +/- 0.059, 0.12 +/- 0.035 for direct genetic effects of the traits GAIN205, GAIN120 and GAIN85, respectively, 0.23 +/- 0.038, 0.17 +/- 0.037, 0.03 +/- 0.029 for estimates of maternal heritability, and 0.12, 0.13, 0.16 for total heritability, respectively. Genetic correlations between weights were greater between measures taken at shorter intervals. This information can be used to optimize the design of programs for genetic improvement of Braunvieh cattle raised under tropical conditions.

Genetic parameters for postweaning traits in Braunvieh cattle

Genetic parameters for traits related to postweaning growth in Braunvieh cattle, reared under tropical and sub-tropical conditions in Brazil, were studied. Weight traits were weight at 365 days of age (W365, N = 4055), at 450 days (W450, N = 3453), and at 550 days (W550, N = 1946), while weight gains were gain from weaning to 365 days of age (WGW365, N = 3060), from weaning to 450 days (WGW450, N = 2764), from weaning to 550 days (WGW550, N = 1531), from 365 to 550 days of age (WG365550, N = 1528), from 365 to 450 days (WG365450, N = 2401), and from 450 to 550 days (WG450550, N = 1563). A full animal model was used for estimating the variance components, using the MTDFREML software. The dataset contained 18,688 animals with phenotypic measures and 35,188 animals in the relationship matrix. Heritability estimates for postweaning weights decreased with age. For W365, W450 and W550, respectively, the direct heritability estimates were 0.29 +/- 0.061, 0.25 +/- 0.057, 0.16 +/- 0.060, maternal heritability was 0.20 +/- 0.035, 0.18 +/- 0.035, 0.13 +/- 0.052, and total heritability was 0.30, 0.35, 0.26. In this breed, maternal influence was found to be important up to 550 days of age. The greater genetic correlations between weights were observed for weights measured at shorter intervals. A large environmental effect was observed for weight gain between weaning and 550 days; this effect was greater for the gains between 365 and 550 days.

Change in central kinin B2 receptor density after exercise training in rats

Cardiovascular responses elicited by the stimulation of kinin B2 receptors in the IV cerebral ventricle paratrigeminal nucleus or in the thoracic spinal cord are similar to those observed during an exercise bout Considering that the kalikrein-kinin system (KKS) could act on the cardiovascular modulation during behavioral responses as physical exercise or stress this study evaluated the central B2 receptor densities of Wistar (W) and spontani ously hypertensive rats (SHR) after chronic moderate exercise Animals we re exercise-trained for ten weeks on a treadmill Afterwards systolic blood pressure decreased in both trained strains Animals were killed and the medulla and spinal cord extracted for B2 receptor autoradiography Trained animals were compared to their sedentary controls Sedentary groups showed specific binding sites for Hoe-140 (fmol/mg of tissue) in laminas 1 and 2 of the spinal cord nucleus of the solitary tract (NTS) area postrema (AP) spinal trigeminal tract (sp5) and paratrigeminal nucleus (Pa5) In trained W a significant increase (p<0 05) in specific binding was observed in the Pa5 (31 3%) and NTS (28 2%) Trained SHR showed a significant decrease in n ceptor density in lamina 2 (21 9%) of the thoracic spinal cord and an increase in specific binding in Pa5 (36 1%) We suggest that in the medulla chronic exercise could hyper stimulate the KKS enhancing their efficiency through the increase of B2 receptor density involving this receptor in central cardiovascular control during exercise or stress In the lamina 2 B2 receptor might be involved in the exercise-induced hypotension (C) 2010 Elsevier BV All rights reserved

Differentiated dendritic cells expressing nuclear RelB are predominantly located in rheumatoid synovial tissue perivascular mononuclear cell aggregates

Objective. Differentiated dendritic cells (DC) and other antigen-presenting cells are characterized by the nuclear location of RelB, a member of the nuclear factor kappa B/Rel family. To characterize and enumerate differentiated DC in rheumatoid arthritis (RA) peripheral blood (PB), synovial fluid (SF), and synovial tissue (ST), the expression and location of RelB were examined. Methods. RelB protein expression and cellular location were determined in RA PB, SF, and ST by flow cytometry and immunohistochemical analysis of purified cells or formalin-fixed tissue. DNA-binding activity of RelB was determined by electrophoretic: mobility shift-Western immunoblotting assays. Results. Circulating RA PBDC resembled normal immature PBDC in that they did not express intracellular RelB protein. In RA ST serial sections, cells containing nuclear RelB (nRelB) were enriched in perivascular regions. A mean +/- SD of 84 +/- 10% of these cells were DC. The remaining nRelB+,HLA-DR+ cells comprised B cells and macrophages. Only 3% of sorted SFDC contained nRelB, However, RelB present in the nucleus of these SFDC was capable of binding DNA, and therefore capable of transcriptional activity. Conclusion. Circulating DC precursors differentiate and express RelB after entry into rheumatoid ST. Differentiated DC can thus be identified by immunohistochemistry in formalin-fixed ST. Signals for DC maturation may differ between RA ST and SF, resulting in nuclear location of RelB predominantly in ST. This is likely to have functional consequences for the DC in these sites.

The lateral habenula regulates defensive behaviors through changes in 5-HT-mediated neurotransmission in the dorsal periaqueductal gray matter

Chemical stimulation of the lateral nucleus of the habenula (LHb), an area implicated in the regulation of serotonergic activity in raphe nuclei, affects the acquisition of inhibitory avoidance and escape expression of rats submitted to the elevated T-maze test of anxiety. Here, we investigated whether facilitation of 5-HT-mediated neurotransmission in the dorsal periaqueductal gray (dPAG) accounts for the behavioral consequences in the elevated T-maze induced by chemical stimulation of the LHb. The dPAG in the midbrain, which is innervated by 5-HT fibers originating from the dorsal raphe nucleus (DRN), has been consistently implicated in the genesis/regulation of anxiety- and fear-related defensive responses. The results showed that intra-dPAG injection of WAY-100635 or ketanserin, 5-HT(1A) and 5-HT(2A/2C) receptor antagonists, respectively, counteracted the anti-escape effect caused by bilateral intra-LHb injection of kainic acid (60 pmol/0.2 mu l). Ketanserin, but not WAY-100635, blocked kainic acid`s facilitatory effect on inhibitory avoidance acquisition. Overall, the results suggest that the pathway connecting the LHb to the DRN is involved in the control of 5-HT release in the dPAG, and facilitation of 5-HT-mediated neurotransmission in the latter area distinctively impacts upon the expression of anxiety- and fear-related defensive behaviors. While stimulation of 5-HT(1A) receptors selectively affects escape performance, 5-HT(2A/2C) receptors modulate both inhibitory avoidance and escape. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

A novel role for MNTB neuron dendrites in regulating action potential amplitude and cell excitability during repetitive firing

Principal cells of the medial nucleus of the trapezoid body (MNTB) are simple round neurons that receive a large excitatory synapse (the calyx of Held) and many small inhibitory synapses on the soma. Strangely, these neurons also possess one or two short tufted dendrites, whose function is unknown. Here we assess the role of these MNTB cell dendrites using patch-clamp recordings, imaging and immunohistochemistry techniques. Using outside-out patches and immunohistochemistry, we demonstrate the presence of dendritic Na(+) channels. Current-clamp recordings show that tetrodotoxin applied onto dendrites impairs action potential (AP) firing. Using Na(+) imaging, we show that the dendrite may serve to maintain AP amplitudes during high-frequency firing, as Na(+) clearance in dendritic compartments is faster than axonal compartments. Prolonged high-frequency firing can diminish Na(+) gradients in the axon while the dendritic gradient remains closer to resting conditions; therefore, the dendrite can provide additional inward current during prolonged firing. Using electron microscopy, we demonstrate that there are small excitatory synaptic boutons on dendrites. Multi-compartment MNTB cell simulations show that, with an active dendrite, dendritic excitatory postsynaptic currents (EPSCs) elicit delayed APs compared with calyceal EPSCs. Together with high- and low-threshold voltage-gated K(+) currents, we suggest that the function of the MNTB dendrite is to improve high-fidelity firing, and our modelling results indicate that an active dendrite could contribute to a `dual` firing mode for MNTB cells (an instantaneous response to calyceal inputs and a delayed response to non-calyceal dendritic excitatory postsynaptic potentials).

Blocking systemic nitric oxide production alters neuronal activation in brain structures involved in cardiovascular regulation during polymicrobial sepsis

In a previous study, we concluded that overproduction of nitric oxide (NO) by inducible nitric Oxide synthase (iNOS) in the late phase of sepsis prevents hypothalamic activation, blunts vasopressin secretion and contributes to hypotension, irreversible shock and death. The aim of this follow-up study was to evaluate if the same neuronal activation pattern happens in brain structures related to cardiovascular functions. Male Wistar rats received intraperitoneal injections of aminoguanidine, an iNOS inhibitor, or saline 30 min before cecal ligation and puncture (CLP) or sham surgeries. The animals were perfused 6 or 24 h after the surgeries and the brains were removed and processed for Fos immunocytochemistry We observed an increase (P < 0.001) in c-fos expression 6 h after CLP in the area postrema (AP), nucleus of he tractus solitarius (NTS), ventral lateral medulla (VLM), locus coeruleus (LC) and parabrachial nucleus (PB). At 24 h after CLP, however, c-fos expression was strongly decreased in all these nuclei (P < 0.05), except for the VLM. Aminoguanidine reduced c-fos expression in the AP and NTS at 6 h after CLR but showed an opposite effect at 24 h, with an increase in the AP, NTS, and also in the VLM. No such effect was observed in the LC and PB at 6 or 24 h. In all control animals, c-fos expression was minimal or absent. We conclude that in the early phase of sepsis iNOS-derived NO may be partially responsible for the activation of brain structures related to cardiovascular regulation. During the late phase, however, this activation is reduced or abolished. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Peripheral withdrawal recruits distinct central nuclei in morphine-dependent rats

This study examined if brain pathways in morphine-dependent rats are activated by opioid withdrawal precipitated outside the central nervous system. Withdrawal precipitated with a peripherally acting quaternary opioid antagonist (naloxone methiodide) increased Fos expression but caused a more restricted pattern of neuronal activation than systemic withdrawal (precipitated with naloxone which enters the brain). There was no effect on locus coeruleus and significantly smaller increases in Fos neurons were produced in most other areas. However in the ventrolateral medulla (A1/C1 catecholamine neurons), nucleus of the solitary tract (A2/C2 catecholamine neurons), lateral parabrachial nucleus, supramamillary nucleus, bed nucleus of the stria terminalis. accumbens core and medial prefrontal cortex no differences in the withdrawal treatments were detected. We have shown that peripheral opioid withdrawal can affect central nervous system pathways. Crown Copyright (C) 2001 Published by Elsevier Science Ltd. All rights reserved.

As formas simbólicas espaciais e a dinâmica da centralidade em Vitória : um esforço de análise

O presente trabalho aborda, sob a perspectiva da geografia cultural renovada, a temática das formas simbólicas no espaço urbano, tento como problematização o estudo de caso das formas simbólicas espaciais mais representativas da centralidade na Área Central de Vitória (ES). O interesse sobre o tema está associado o quadro relativamente recente no qual a Área Central de Vitória, sobretudo nas últimas duas décadas do século XX, passa a sofrer com um intenso processo de descentralização apresentando como alguns efeitos dessa dinâmica não só aparecimento de núcleos secundários de comércio e serviços afastados da Área Central, quanto também exerce um papel fundamental no que se ficou consagrado como “crise da área central”. Entretanto, se o quadro acima descrito é muito recente, por outro lado, durante o amplo período da produção do espaço urbano da capital capixaba em que a organização interna da cidade foi tributária, fundamentalmente, do processo de centralização, o Centro de Vitória foi o lócus privilegiado de materialização de formas simbólicas espaciais tipicamente associadas à centralidade intra-urbana tais como edificações que são símbolos dos poderes político, jurídico e econômico; igrejas; os monumentos, dentre outras formas simbólicas espaciais. Deste modo, a dissertação pretende investigar o advento da descentralização sobre as formas simbólicas espaciais tipicamente associadas à Área Central da capital capixaba, questionando em que medida essas formas permanecem enquanto signos de resistência simbólica a importância da área, bem como a natureza da eventual requalificação de significado ao qual foram, eventualmente, submetidas.

Ciclo testicular e espermatogênese de Devario aequipinnatus (cypriniformes, cyprinidae) submetido a diferentes temperaturas

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Estratigrafia do Caloviano do Algarve oriental

The comparative study of the Callovian sections in eastern Algarve allowed us to demonstrate that the discontinuity surface at the base of Malm lies always over the Lower-Callovian, as opposite to what happens in western Algarve; the Bathonian-Callovian transition, continuous in western Algarve (Mareta beach) is marked, in eastern Algarve, by a generalised discontinuity of variable vertical extention. It is verified that, in eastern Algarve, the Callovian formations are always or at the nucleus of anticlinal structures, probably linked to halokinetic tectonic activity, or in large radins folds derived from compressive phases.