Motor cortex stimulation inhibits thalamic sensory neurons and enhances activity of PAG neurons: Possible pathways for antinociception

Motor cortex stimulation is generally suggested as a therapy for patients with chronic and refractory neuropathic pain. However, the mechanisms underlying its analgesic effects are still unknown. In a previous study, we demonstrated that cortical stimulation increases the nociceptive threshold of naive conscious rats with opioid participation. In the present study, we investigated the neurocircuitry involved during the antinociception induced by transdural stimulation of motor cortex in naive rats considering that little is known about the relation between motor cortex and analgesia. The neuronal activation patterns were evaluated in the thalamic nuclei and midbrain periaqueductal gray. Neuronal inactivation in response to motor cortex stimulation was detected in thalamic sites both in terms of immunolabeling (Zif268/Fos) and in the neuronal firing rates in ventral posterolateral nuclei and centromedian-parafascicular thalamic complex. This effect was particularly visible for neurons responsive to nociceptive peripheral stimulation. Furthermore, motor cortex stimulation enhanced neuronal firing rate and Fos immunoreactivity in the ipsilateral periaqueductal gray. We have also observed a decreased Zif268, delta-aminobutyric acid (GABA), and glutamic acid decarboxylase expression within the same region, suggesting an inhibition of GABAergic interneurons of the midbrain periaqueductal gray, consequently activating neurons responsible for the descending pain inhibitory control system. Taken together, the present findings suggest that inhibition of thalamic sensory neurons and disinhibition of the neurons in periaqueductal gray are at least in part responsible for the motor cortex stimulation-induced antinociception. (C) 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Novel functional profiling approach combining reverse phase protein microarrays and human 3-D ex vivo tissue cultures: expression of apoptosis-related proteins in human colon cancer

Cancer is caused by a complex pattern of molecular perturbations. To understand the biology of cancer, it is thus important to look at the activation state of key proteins and signaling networks. The limited amount of available sample material from patients and the complexity of protein expression patterns make the use of traditional protein analysis methods particularly difficult. In addition, the only approach that is currently available for performing functional studies is the use of serial biopsies, which is limited by ethical constraints and patient acceptance. The goal of this work was to establish a 3-D ex vivo culture technique in combination with reverse-phase protein microarrays (RPPM) as a novel experimental tool for use in cancer research. The RPPM platform allows the parallel profiling of large numbers of protein analytes to determine their relative abundance and activation level. Cancer tissue and the respective corresponding normal tissue controls from patients with colorectal cancer were cultured ex vivo. At various time points, the cultured samples were processed into lysates and analyzed on RPPM to assess the expression of carcinoembryonic antigen (CEA) and 24 proteins involved in the regulation of apoptosis. The methodology displayed good robustness and low system noise. As a proof of concept, CEA expression was significantly higher in tumor compared with normal tissue (p<0.0001). The caspase 9 expression signal was lower in tumor tissue than in normal tissue (p<0.001). Cleaved Caspase 8 (p=0.014), Bad (p=0.007), Bim (p=0.007), p73 (p=0.005), PARP (p<0.001), and cleaved PARP (p=0.007) were differentially expressed in normal liver and normal colon tissue. We demonstrate here the feasibility of using RPPM technology with 3-D ex vivo cultured samples. This approach is useful for investigating complex patterns of protein expression and modification over time. It should allow functional proteomics in patient samples with various applications such as pharmacodynamic analyses in drug development.

Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells.

Proteins of the 14-3-3 family can associate with, and/or modulate the activity of, several protooncogene and oncogene products and, thus, are implicated in regulation of signaling pathways. We report that 14-3-3 is associated with another important transducing enzyme, phosphatidylinositol 3-kinase (PI3-K). A recombinant 14-3-3 fusion protein bound several tyrosine-phosphorylated proteins from antigen receptor-stimulated T lymphocytes. PI3-K was identified by immunoblotting and enzymatic assays as one of the 14-3-3-binding proteins in resting or activated cells. Moreover, endogenous 14-3-3 and PI3-K were coimmunoprecipitated from intact T cells. Far-Western blots of gel-purified, immunoprecipitated PI3-K with a recombinant 14-3-3 fusion protein revealed direct binding of 14-3-3 to the catalytic subunit (p110) of PI3-K. Finally, anti-phosphotyrosine immunoprecipitates from activated, 14-3-3-overexpressing cells contained lower PI3-K enzymatic activity than similar immunoprecipitates from control cells. These findings suggest that association of 14-3-3 with PI3-K in hematopoietic (and possibly other) cells regulates the enzymatic activity of PI3-K during receptor-initiated signal transduction.

At4g24160, a Soluble Acyl-Coenzyme A-Dependent Lysophosphatidic Acid Acyltransferase

Human CGI-58 (for comparative gene identification-58) and YLR099c, encoding Ict1p in Saccharomyces cerevisiae, have recently been identified as acyl-CoA-dependent lysophosphatidic acid acyltransferases. Sequence database searches for CGI-58 like proteins in Arabidopsis (Arabidopsis thaliana) revealed 24 proteins with At4g24160, a member of the alpha/beta-hydrolase family of proteins being the closest homolog. At4g24160 contains three motifs that are conserved across the plant species: a GXSXG lipase motif, a HX4D acyltransferase motif, and V(X)(3)HGF, a probable lipid binding motif. Dendrogram analysis of yeast ICT1, CGI-58, and At4g24160 placed these three polypeptides in the same group. Here, we describe and characterize At4g24160 as, to our knowledge, the first soluble lysophosphatidic acid acyltransferase in plants. A lipidomics approach revealed that At4g24160 has additional triacylglycerol lipase and phosphatidylcholine hydrolyzing enzymatic activities. These data establish At4g24160, a protein with a previously unknown function, as an enzyme that might play a pivotal role in maintaining the lipid homeostasis in plants by regulating both phospholipid and neutral lipid levels.

Étude de l'activité physique de suppression tumorale de la protéine KBP2 dans les lignées de cellules de carcinome du sein MCF7

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Étude de l'activité physique de suppression tumorale de la protéine KBP2 dans les lignées de cellules de carcinome du sein MCF7

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Avaliação de alguns parâmetros da qualidade da anchoita (Engraulis anchoita) enlatada em óleo de girassol e em molho de tomate

O consumo de pescado no Brasil cresceu 40% nos últimos 7 anos passando de 6,46 para 9,03 kg/habitante/ano, valor que se aproxima do recomendado pela Organização Mundial de Saúde. A razão está relacionada com a subutilização de determinadas espécies e a falta de diversificação da indústria processadora para a produção de alimentos com maior valor agregado. Considerando o esgotamento de determinadas espécies com a utilização da sobrepesca, é possível o emprego da anchoita na forma de conservas através da utilização de meios de cobertura como molho de tomate e óleo comestível. Este trabalho teve como objetivo elaborar conservas de anchoita (Engraulis anchoita) com a utilização de 2 meios de cobertura: molho de tomate e óleo de girassol, submetidas a tempos de salmouragem diferenciados, 2 e 5 min e com o emprego ou não de pré-cozimento. De acordo com os padrões estabelecidos, as amostras de pescado fresco utilizadas para a execução dos enlatados apresentaram resultados físico químicos adequados comprovando o frescor do pescado envolvido no processo, ou sejam: 16,29 mg/100 g amostra para N-BVT, 7,90 mg/100 g amostra para N-TMA e pH 6,5. As conservas em molho de tomate submetidas a operação de salmouragem durante 2 min, com e sem pré-cozimento apresentaram, respectivamente, 16,57 e 16,24% proteínas, 3,94 e 4,66% lipídios, 73,0 e 73,28% umidade, 3,22 e 3,67% cinzas, 0,17 e 0,19% de cloretos (NaCl). As conservas com molho de tomate, utilizando anchoita eviscerada salmourada por 5 min, com e sem pré-cozimento, apresentaram respectivamente, 15,94 e 15,31% proteínas, 3,15 e 4,90 lipídios%, 73,70 e 73,98% umidade, 3,80 e 4,10% cinzas, 0,21 e 0,24% cloretos (NaCl). Para as conservas de anchoita em óleo de girassol, utilizando tempo de salmouragem de 2 min, com e sem pré-cozimento, apresentaram 16,97 e 16,76% proteínas, 7,68 e 5,70% lipídios, 65,87 e 68,74% umidade, 3,16 e 3,28% cinzas, 0,10 e 0,12% cloretos (NaCl), respectivamente. As conservas utilizando o pescado previamente submetido a salmouragem por 5 min e posteriormente enlatado com a adição de óleo de girassol, com e sem pré-cozimento apresentaram respectivamente, 15,97 e 15,89% proteínas, 7,91 e 5,19% lipídios, 66,26 e 68,23% umidade, 3,78 e 3,87% cinzas, 0,13 e 0,21% NaCl. As análises microbiológicas das conservas submetidas aos diferentes tratamentos mostraram ausência de Salmonella spp, Staphylococcus coagulase positiva e Clostridium sulfito-redutor, resultados estes, de acordo com o estabelecido pela legislação higiênico-sanitária brasileira. Nos testes de esterilidade comercial não foram constatadas alterações visíveis nos enlatados submetidos a incubação por 5 dias a 36 ± 1°C (determinação de micro-organismos aeróbios viáveis) e a 7-10 dias a 55 ± 1°C (termófilos). Considerando as quantidades de pescado enlatado (80, 90 e 100g), o rendimento para todas as amostras apresentaram, no mínimo, 50% de pescado em relação ao peso líquido. A avaliação sensorial realizada por teste de ordenação para a avaliação da preferência não apresentou diferenças significativas entre as amostras.

Buffalo cheese whey proteins, identification of a 24 kda protein and characterization of their hydrolysates: in vitro gastrointestinal digestion

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Transcription and expression of Plasmodium falciparum histidine-rich proteins in different stages and strains : implications for rapid diagnostic tests

Background: Although rapid diagnostic tests (RDTs) for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates. Methods: Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle. Results: Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed. Conclusions: The levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs.

CLXXX. Vegetable Proteins. I. The Proteins of Dolichos Lab Lab

The field bean (Dolichos lab lab ; Tamil name, Mochai ; Kanarese, Avarai) is a legume which is widely cultivated in South India often as a mixed crop with cereals. The kernel of the seed enters into the diet of may South Indian households, and in the Mysore State the seed are used as a delicacy when they are green for over four months in the year. The haulm, husk and pods are commonly used a fodder. As the kernel which is widely used as an article of food and considered to be very nutritious, contains about 24% of protein hitherto uninvestigated and as the quality of protein plays an important role in nutrition, the present work was undertaken.

NMR Spectroscopy of Multi-domain Proteins : Immunoglobulin-like Domains of Human Filamin A

Proteins are complex biomacromolecules playing fundamental roles in the physiological processes of all living organisms. They function as structural units, enzymes, transporters, process regulators, and signal transducers. Defects in protein functions often derive from genetic mutations altering the protein structure, and impairment of essential protein functions manifests itself as pathological conditions. Proteins operate through interactions, and all protein functions depend on protein structure. In order to understand biological mechanisms at the molecular level, one has to know the structures of the proteins involved. This thesis covers structural and functional characterization of human filamins. Filamins are actin-binding and -bundling proteins that have numerous interaction partners. In addition to their actin-organizing functions, filamins are also known to have roles in cell adhesion and locomotion, and to participate in the logistics of cell membrane receptors, and in the coordination of intracellular signaling pathways. Filamin mutations in humans induce severe pathological conditions affecting the brain, bones, limbs, and the cardiovascular system. Filamins are large modular proteins composed of an N-terminal actin-binding domain and 24 consecutive immunoglobulin-like domains (IgFLNs). Nuclear magnetic resonance (NMR) spectroscopy is a versatile method of gaining insight into protein structure, dynamics and interactions. NMR spectroscopy was employed in this thesis to study the atomic structure and interaction mechanisms of C-terminal IgFLNs, which are known to house the majority of the filamin interaction sites. The structures of IgFLN single-domains 17 and 23 and IgFLN domain pairs 16-17 and 18-19 were determined using NMR spectroscopy. The structures of domain pairs 16 17 and 18 19 both revealed novel domain domain interaction modes of IgFLNs. NMR titrations were employed to characterize the interactions of filamins with glycoprotein Ibα, FilGAP, integrin β7 and dopamine receptors. Domain packing of IgFLN domain sextet 16 21 was further characterized using residual dipolar couplings and NMR relaxation analysis. This thesis demonstrates the versatility and potential of NMR spectroscopy in structural and functional studies of multi-domain proteins.

Synthesis of Silica Vesicles with Controlled Entrance Size for High Loading, Sustained Release, and Cellular Delivery of Therapeutical Proteins

A rationally designed two-step synthesis of silica vesicles is developed with the formation of vesicular structure in the first step and fine control over the entrance size by tuning the temperature in the second step. The silica vesicles have a uniform size of ≈50 nm with excellent cellular uptake performance. When the entrance size is equal to the wall thickness, silica vesicles after hydrophobic modification show the highest loading amount (563 mg/g) towards Ribonuclease A with a sustained release behavior. Consequently, the silica vesicles are excellent nano-carriers for cellular delivery applications of therapeutical biomolecules.

Chemotherapy pretreatment sensitizes solid tumor-derived cell lines to Vα24+ NKT cell-mediated cytotoxicity

There is an increasing awareness of the therapeutic potential for combining immune-based therapies with chemotherapy in the treatment of malignant diseases, but few published studies evaluate possible cytotoxic synergies between chemotherapy and cytotoxic immune cells. Human Vα24 +/Vβ11+ NKT cells are being evaluated for use in cell-based immunotherapy of malignancy because of their immune regulatory functions and potent cytotoxic potential. In this study, we evaluated the cytotoxicity of combinations of chemotherapy and NKT cells to determine whether there is a potential to combine these treatment modalities for human cancer therapy. The cytotoxicity of NKT cells was tested against solid-tumor derived cell lines NCI-H358, DLD-1, HT-29, DU-145, TSU-Pr1 and MDA-MB231, with or without prior treatment of these target cells, with a range of chemotherapy agents. Low concentrations of chemotherapeutic agents led to sensitization of cell lines to NKT-mediated cytotoxicity, with the greatest effect being observed for prostate cancer cells. Synergistic cytotoxicity occurred in an NKT cell in a dose-dependent manner. Chemotherapy agents induced upregulation of cell surface TRAIL-R2 (DR5) and Fas (CD95) expression, increasing the capacity for NKT cells to recognize and kill via TRAIL- and FasL-mediated pathways. We conclude that administration of cytotoxic immune cells after chemotherapy may increase antitumor activities in comparison with the use of either treatment alone.

Probiotics modify tight junction proteins in an animal model of non-alcoholic fatty liver disease

Objectives We have investigated the effects of a multi–species probiotic preparation containing a combination of probiotic bacterial genera that included Bifidobacteria, Lactobacilli and a Streptococcus in a mouse model of high fat diet/obesity induced liver steatosis. Methods Three groups of C57B1/6J mice were fed either a standard chow or a high fat diet for 20 weeks, while a third group was fed a high fat diet for 10 weeks and then concomitantly administered probiotics for a further 10 weeks. Serum, liver and large bowel samples were collected for analysis. Results The expression of the tight junction proteins ZO-1 and ZO-2 was reduced (p < 0.05) in high fat diet fed mice compared to chow fed mice. Probiotic supplementation helped to maintain tight ZO-1 and ZO-2 expression compared with the high fat diet group (p < 0.05), but did not restore ZO-1 or ZO-2 expression compared with chow fed mice. Mice fed a high fat diet ± probiotics had significant steatosis development compared to chow fed mice (p < 0.05); steatosis was less severe in the probiotics group compared to the high fat diet group. Hepatic triglycerides concentration was higher in mice fed a high fat diet ± probiotics compared to the chow group (p < 0.05), and was lower in the probiotics group compared to the high fat diet group (p < 0.05). Compared to chow fed mice, serum glucose and cholesterol concentrations, and the activity of alanine transaminase were higher (p < 0.05), whereas serum triglyceride concentration was lower (p < 0.05) in mice fed a high fat diet ± probiotics. Conclusions Supplementation with a multi-species probiotic formulation helped to maintain tight junction proteins ZO-1 and ZO-2, and reduced hepatic triglyceride concentrations compared with a HFD alone.