RCAAP Repository

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Centro Universitário de Patos de Minas

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

CHAPTER II: A protease, which we designate Eumiliin, was isolated from the latex of Euphorbia milii var. hislopii by a combination of ion-exchange chromatography using DEAE-Sephacel and gel filtration with Sephadex G-75. Eumiliin is a monomeric protein with an apparent molecular mass of 30 kDa by SDS PAGE under reducing conditions and gave one main peak at 29814 kDa in MALDITOF/ TOF mass spectrometry. Eumiliin has caseinolytic and fibrinogenolytic activities, but no hemorrhagic or defibrinating activities. The enzyme readily hydrolyzes the Aα-chain of fibrinogen and, more slowly, the Bß-chain. Its fibrinogenolytic activity is inhibited by b-mercaptoethanol and leupeptin. In contrast, EDTA and benzamidine did not affect the activity of Eumiliin. The caseinolytic activity of Eumiliin had a pH optimum of 8.0 and was stable in solution at up to 40 °C; activity was completely lost at 80 °C. Intraplantar injection of Eumiliin (1 25 μg/paw) caused a dose- and time-dependent hyperalgesia, which peaked 1 - 5 h after enzyme injection. Intraplantar injection of Eumiliin (1 25 μg/paw) also caused an oedematogenic response that was maximal after 1 h. Morphological analyses indicated that Eumiliin induced an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 h after injection. CHAPTER III: A protease, which we designate Eumiliin, was isolated from the latex of Euphorbia milii var. hislopii by a combination of ion-exchange chromatography using DEAE-Sephacel and gel filtration with Sephadex G-75. Eumiliin is a monomeric protein with an apparent molecular mass of 30 kDa by SDS PAGE under reducing conditions and gave one main peak at 29814 kDa in MALDITOF/ TOF mass spectrometry. Eumiliin has caseinolytic and fibrinogenolytic activities, but no hemorrhagic or defibrinating activities. The enzyme readily hydrolyzes the A-chain of fibrinogen and, more slowly, the B-chain. Its fibrinogenolytic activity is inhibited by b-mercaptoethanol and leupeptin. In contrast, EDTA and benzamidine did not affect the activity of Eumiliin. The caseinolytic activity of Eumiliin had a pH optimum of 8.0 and was stable in solution at up to 40 °C; activity was completely lost at 80 °C. Intraplantar injection of Eumiliin (1 25 μg/paw) caused a dose- and time-dependent hyperalgesia, which peaked 1 - 5 h after enzyme injection. Intraplantar injection of Eumiliin (1 25 μg/paw) also caused an oedematogenic response that was maximal after 1 h. Morphological analyses indicated that Eumiliin induced an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 h after injection. CHAPTER IV: Bhalternin and Eumiliin are proteases extracted from the venom of Bothrops alternatus and latex of Euphorbia milli var. hislopii, respectively. The stabilities of Bhalternin and Eumiliin against denaturation by heat and urea were determined and compared. The plant protease proved to be tougher when heated at high temperatures and also when subjected to the action of urea as denaturant. Further studies are needed to better characterize the conditions for stabilization of these enzymes, especially those related to the strategies needed to protect the transport and storage in the commercial, therapeutic and biotechnological processes.

Universidade Federal de Uberlândia

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Concentration of ammonia in blood increases during endurance exercise and can be toxic for muscle cells and brain metabolism, potentially leading to both peripheral and central fatigue. Intense exercise can promote failure in replace ATP leading to greater deamination of AMP and consequent increase in blood ammonia. High concentration of ammonia is toxic and harms the performance. Keto acids has been proposed to capture the blood nitrogen compounds. Here we describe the protective effects of acute and chronic keto acids supplementation on nitrogen metabolism and physical performance in male rats submitted to a resistance training protocol. This study investigated the protective effect of supplementation keto analogues against acute nitrogen compounds and physical performance in rats submitted to exercise.

Ammonia (here used as a synonym for NH3 + NH4+) is a toxic metabolite with deleterious effects on the central nervous system. Exercise can be used as a model to study ammonia metabolism and hyperammonemia. The temporary disturbances in the central nerve system caused by exercise are similar to the observed in hepatic disease and neurodegenerative disorders. Increase of adenosine monophosphate (AMP) during prolonged exercise leads to an production of inosine monophosphate (IMP). Furthermore, amino acids are used as carbon donors for the tricarboxylic acid cycle to maintain the ATP concentration in the cell. Both metabolic pathways lead to an increase in intracellular and ammonemia concentration. In these events, the blood ammonia concentration can raise up to 400% the resting levels. Changes in ammonia levels in response to exercise can be managed through the use of amino acids or carbohydrates that interfere with the metabolism of ammonia. Low carbohydrates diet (called here as ketogenic diet) combined with physical exercise can reduce glycogen stores, inducing early states of hyperammonemia. We explored the ability of a ketogenic diet to enhance the effect of exercise on ammonia production. Though, keto analogues can serve as a nutritional supplement to provide amino acids of high biological value, as well as a tool for ammonia sequestering. In the present study, we used exercise stress to investigate ammonia metabolism. We studied a low-carbohydrate ketogenic diet and exercise as a hyperammonemia model in order to understand the role of the association of keto analogues and amino acid (KAAA) supplementation in ammonia metabolism.

Universidade Federal de Uberlândia

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

CHAPTER II: The observation that the membrane fluidity must remain within a critical interval outside which the stability and functionality of the cell tends to decrease, shows that the stability, fluidity and function are related and that the measure of erythrocytes stability allows inferences about the fluidity or functionality of these cells. The test of the FOE provides three parameters to measure stability: dX, H50 and dX/H50. These parameters enable us to relate various hematological and biochemical variables with erythrocyte fluidity or functionality and, consequently, to associate these variables to the risk of disease. This study determines the biochemical and hematological variables that are directly or indirectly related to the stability of erythrocyte through those parameters and how they relate to each other, besides knowing what are the most significant variables. For this, we measured the erythrocytes stability by the FOE method and correlated them with various hematologic and biochemical variables for 71 patients. These data were treated mathematically by simple correlation and multivariate statistics. The erythrocytes stability showed a greater association with hematologic variables than the biochemical variables. The RDW stands out for their strong and significant correlation, and freedom of influences. For the biochemical variables the erythrocytes stability was more sensible to LDLC. Erythrocyte stability is significantly associated with RDW and LDL-C. Thus, the level of LDL-C is a consistent link between stability and functionality, allowing a measure of stability is more one indirect parameter for assessing the risk of vascular diseases.

Universidade Federal do Acre

CHAPTER II: Objective: Our aim was to compare the performance of three serological assays in leprosy patients and their household contacts utilizing two quantitative ELISA tests using native PGL-I (PGL-1 ELISA), synthetic ND-O-HSA (ND-O-HSA ELISA), and the semi-quantitative lateral flow test (ML-Flow). Methods: Compare the performance of three immunological assays, PGL-I ELISA, ND-O-HSA ELISA, and ML-Flow were evaluated in 156 leprosy patients and 191 household contacts. Results: The sensitivity results of the PGL-1, ND-O-HSA, and ML-Flow were 68.83%, 63.65%, and 60.65%, respectively. The native and synthetic PGL-I ELISA assays detected antibodies in 22.73%, 31.82% of the paucibacillary (PB) patients, and the ML Flow test did not detect antibodies in this group. The ML-Flow test was able to discriminate patients into PB and multibacillary (MB) forms, while the native PGL-I and ND-O-HSA correlated with the bacillary load and the Ridley-Jopling clinical forms. In household contacts, the native PGL-I, ND-O-HSA, and ML-Flow assays detected seropositivity of 25%, 17%, and 10%, respectively. Conclusions: The use of ELISA and ML-Flow tests are thus recommended as additional tools in the diagnosis and classification of the clinical forms, aiding in prescribing the correct treatment regimen to prevent subsequent nerve damage and disability. CHAPTER III: Host pathogen interactions are mainly mediated by specialized molecules of the cell envelope. One of these essential mycobacterial cell wall components is the lipoarabinomannan (LAM). LAM has immunomodulatory roles, but its heterogeneity may be responsible for the differential immune response in leprosy patients and contacts. The research to structural motifs that could contribute as virulence factors and/or protective epitopes, and thereby derive effective biomarkers for diagnosis, drugs and/ or vaccines against leprosy has been very developed. Therefore, our aim was to develop specific mimetic peptides to this lipoglycan by using Phage Display of a random heptamer peptide library that may recognize a differential response in patients and contacts. We have used the anti-LAM CS-35 monoclonal antibody as a target for three rounds of selection. After sequencing and translation, peptides were pre-validated by ELISA and compared to the synthetic LAM-BSA antigen. The most reactive and repetitive peptide motif (A9) was subsequently tested against serum from 54 leprosy patients and 27 endemic controls by ELISA. The A9 phage-displayed peptide clone presented high levels of IgG antibodies in paucibacillary patients, from which 50% of them presented highly reactive sera. This reactivity has also been detected in tuberculoid, borderline-borderline and lepromatous patients. High levels of IgG1 were most frequent in endemic controls and reactional patients. On the other hand, the IgG profile and its subclasses in patients presented high levels of IgG and IgG2 and low levels of IgG1. The A9 clone presented a significant correlation with the synthetic LAM-BSA, for the IgG1 response. The highly reactive IgG response against the A9 clone was associated with the tuberculoid clinical form diagnosis, and detection of both IgG1 and IgG3 against this clone was associated with protection in endemic controls. CHAPTER IV: Heat Shock Proteins (HSPs), GroES and GroEL, are targets of strong human T-cell response, and a third of the cells responsive to M. leprae, recognize these proteins. Monoclonal antibodies mAbs CS-01 and CS-44 selected mimetic peptides that are ligands of their Fab portions, by phage display technique. Sera from 54 patients, 48 household contacts and 27 endemic controls were submitted to ELISA with B2 and A1 mimetic clones of the GroES and GroEL proteins, respectively, for detection of IgG and its subclasses. Using the mimetic clone of B2 GroES, the ELISA detected IgG antibodies present in sera of patients, contacts and endemic controls. The IgG antibodies were abundant in sera from multibacillary patients, especially in lepromatous (LL). A decline of IgG1 was found in patients and household contacts that became sick with leprosy and a raise of this subclass was present in sera of household contacts that did not develop the disease. With the mimetic clone of GroEL A1, the reactive antibodies were abundant in multibacillary patients, with a correlation with the bacillary load. In this study we observed that IgG antibodies against GroEL and GroES can be detected in the diagnosis of leprosy in serological tests produced with clones mimetics of these proteins. And the subclasses of IgG antibodies to GroES can demonstrate a targeting of antigenic molecules that induce the production of protective antibodies.

Universidade Federal de Uberlândia

We investigated biomarkers of injury, inflammation and oxidative stress in the blood after high intensity training. Nineteen male athletes performed a combination of high-intensity aerobic and anaerobic training. Samples were acquired immediately before and from 3 h to 72 h after the exercise. CK was elevated 200% at 3 h post-exercise, reaching a 300% peak increase at 12 h and returning to pre-exercise levels within 48 h. LDH activity was 25% higher 3 h after exercise, increasing to 56% higher 6 h after exercise and returning to pre-exercise levels within 12 h. Leukocyte levels were 50% higher and neutrophil levels were 70% higher 3 h after exercise than at baseline, while lymphocyte levels increased by up to 55% after 12 h. MCP-1 was elevated by 40% after 6 h and decreased by 37% 72 h after exercise. TNF-alpha levels were lower in all post-exercise samples. IL-6 and CRP levels remained stable throughout the entire recovery period. The levels of oxidative stress markers remained stable during the experiment. CK and LDH blood appearance and clearance are faster than classic described, coaches and physicians must respect these windows to accurately estimate muscle damage. The neutrophil/lymphocyte ratio summarizes the mobilization of two leukocyte subpopulations in a single marker and may be used to predict the end of the post-exercise recovery period. Further analysis of the immune response using serum cytokines indicated that the high-intensity exercise performed by highly trained athletes only generates inflammation localized to the skeletal muscle.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Universidade Federal de Uberlândia

Universidade Federal de Uberlândia

Anaplasmosis, a persistent intraerythrocytic infection of cattle by Anaplasma marginale, causes severe anemia and a higher rate of abortion, resulting in significant loss to both dairy and beef industries. Clinical diagnosis is based on symptoms and confirmatory laboratory tests are required. Currently, all the diagnostic assays have been developed with whole antigens with indirect ELISA based on multiple epitopes. In a pioneer investigation we demonstrated the use of critical motifs of an epitope as biomarkers for immunosensor applications. Mimotopes of the MSP1a protein functional epitope were obtained through Phage Display after three cycles of selection of a 12-mer random peptide library against the neutralizing monoclonal antibody 15D2. Thirty-nine clones were randomly selected, sequenced, translated and aligned with the native sequence. The consensus sequences SxSSQSEASTSSQLGA was obtained, which is located in C-terminal end of the 28-aa repetitive motif of the MSP1a protein, but the alignment and sequences variation among mimotopes allowed us to map the critical motif STSSxL within the consensus sequence. Based on these results, two peptides were chemically synthesized; one based on the critical motif (STSSQL, Am1) and the other based on the consensus sequence aligned with the native epitope (SEASTSSQLGA, Am2). Sera from 24 infected and 52 healthy animals were tested by ELISA for reactivity against Am1 and Am2, which presented sensitivities of 96% and 100%, respectively. The Am1 peptide was incorporated onto a biolectrode (graphite modified with poly-3-hydroxyphenylacetic acid) and direct serum detection was demonstrated by impedance, differential pulse voltammetry, and atomic force microscopy. The electrochemical sensor system proved to be highly effective in discriminating sera from positive and negative animals. These immunosensors were highly sensitive and selective for positive IgG, contaminants did not affect measurements, and were based on a simple, fast and reproducible electrochemical system.