RCAAP Repository

CHAPTER II: The alpha-amylases (α-1,4-D-glucan-4-glucanohydrolases, EC 3.2.1.1) are enzymes responsible for the digestion of carbohydrates and are found in saliva and in the pancreatic juice. The primary sequences of the salivary (HSA) and pancreatic (HPA) alpha-amylases show structural similarity of 99%. This standart homology suggests that the HSA can be target of experimental studies of the digestion of carbohydrates inhibition, besides studies showed that potential inhibitors have similar behavior to HPA and HSA. Considering the benefits of noninvasive collection of saliva in relation of pancreatic juice, in this present study obtained a fraction called HSA-CTI. Preparations of the crude extract of the bark of Pouteria sp. was tested about the inhibitory effect of enzymatic activity of the HSA-CTI. Samples of saliva were frozen for 48h, centrifugation and the supernatant was subjected to chromatography in Q-Sepharose column (QS-column) equilibrated in 50mM Tris-HCl buffer at pH 8.0 containing 10mM EDTA and 10mM EGTA. The bound proteins were eluted from QS-column with a gradient concentration of NaCl (0-1M). SDS-PAGE, Western blotting assays and amylase activity measured were performed of the obtained fractions. The HSA-CTI fraction not interacted with the column, and thus prepared by lyophilization and stored in the frozen until realization of the inhibition assays. Aqueous preparations for infusion (EAP-C), deccotion (EAP-C), hydroalcoholic (EHP-C) and ethanolic (EEP-C) of the stem bark extract (10mg/mL in DMSO) have assays by pre-incubation at 10 and 30min at 37ºC with the HAS-CTI (1:10). Only the polypeptide of 56Kda, abundant in the fraction, exhibited cross-reaction with anti-HSA antibody. The assay inhibition showed that stem bark extract has effective in to inhibit the HSA-CTI activity. These results indicate that the HSA-CTI can be obtained by a rapid chromatography method can be used in tests of inhibition from plant extracts. The extract of Pouteria sp. presented as a potential candidate for studies of fractionation and discovery of new substances with activity on these enzymes. CHAPTER III: Preliminary studies about extracts from plants of the kind of Pouteria (Sapotaceae) deal their potential to inhibit human alpha-amylase. These studies may involve the discovery of new sources to reduce post-prandial hyperglycemia, important in the treatment of diabetes and obesity. The active components of Pouteria sp. are still mostly unknown, therefore, the present study fractionated and investigated the substances present in the extract of hydroalcoholic Pouteria sp. with the potential inhibitory on the salivary amylase in vitro. The extract has fractionated in Sephadex LH 20 column equilibrated in methanol and washed with water following by acetone. All the fractions (POUN) was assayed to inhibity the alpha-amylase in vitro. POUN 2 and 3 fractions have injected in semipreparative HPLC (Supecolsyl LC-18 column, mobile phase methanol: water) and analyzed in analytical HPLC (Supecolsyl LC-18 Sp column, mobile phase methanol: acetic acid 0.1%). Assay of inhibition showed that fraction POUN7 exhibited inhibition similar of the crude extract with 40.22% and fractions POUN 2 and 3 exibited inhibition with 15% of the amylase activity. The fraction POUN7 has 48.81% of the constitute it in tannin and represent 35% of the composition of extract. The stem bark crude extract Pouteria sp. have composition it rich in tannin, in addition this extract is potential to others studies, due to this substances have involvement in others therapeutic function.

Background: Adenoviruses (AdVs) are important cause of acute respiratory disease (ARD), gastroenteritis, conjunctivitis and urinary infections in humans. Objectives: Detection of AdVs by immunofluorescence assay (IFA) and by polymerase chain reaction (PCR) in nasopharyngeal aspirates of children less than 5 years old presenting ARD in Uberlândia, MG, as well as, comparison of two PCR assays and identification of serotypes that circulated in this region. Study Design: A total of 468 clinical specimens was collected from November 2000 to April 2007 and tested by IFA for adenovirus detection and other viruses. After that, the DNA of the 126 in natura negative/inconclusive samples by IFA which were also negative for rhinovirus by RT-PCR, were extracted by Trizol® and tested by PCRAraújo (Araújo et al, 2001) for adenovirus detection. The positive specimens for adenovirus were inoculated into HEp-2 and A-549 continuous cell lineages. In addition, the DNA of either, in natura samples and cell culture-scrapped samples, were extracted by using the QIAmp DNA Mini Kit (QIAGENTM Valencia, CA) and tested again by PCRAraujo and by PCRAllard (Allard et al., 2001) in order to compare the sensitivity/specificity of both tests. In addition, the serotypes were identified from the nucleotide sequencing of PCR products positive for adenovirus. Results: From the 468 samples, 33 (7.1%) were positive for AdVs, 14 by IFA and 19 by PCRAraujo. From the 32 specimens inoculated in cell culture, it was possible to isolate AdVs in 16. The comparison of the results obtained from the DNA of the 33 in natura samples extracted by the QIAmp DNA Mini Kit (QIAGENTM Valencia, CA) showed that the sensibility of PCRAraujo was a little higher than PCRAllard (92.9% and 90.0%, respectively). However, the first PCR presented a lower specificity than the second one (57.9% and 91.3%, respectively). The serotype AdV2 was detected in almost 60.0% (7/12) of those identified. Conclusions: AdVs were detected in 7.1% of the clinical samples in children with ARD throught the combination of two methods, IFA and PCR. The analysis of the sensibility and specificity of the two PCR assays, showed that PCRAllard presented a little lower sensibility than PCRAraujo and higher specificity. The serotype AdV2 was identified in 7 of the 12 AdVs sequenced samples.

Due to the set of changes and factors corn crop has been subjected in the last few decades, earliness became one of the major characters for addition of genetic value and adaptability to currently cultivated corn hybrids. Thus, selection of characters related to earliness has been of large interest to corn breeding programs. The aim of this work was to estimate genetic parameters related to earliness in a tropical corn population adapted to the central region of Brazil. In this study were evaluated 188 F4 testcrosses in an expanded statistical block design at six locations and plant density of 70,000pl ha-1. Significant differences were detected between the testcrosses (G), Location (L) and GxL interaction. The heritability coefficient for these testcrosses varied from 35.25% for stalk and root lodging (TQA) and 68.91% for pollen shed (FM). Genetic gain estimates showed that it is possible to improve each of these characters individually, via direct selection. However, estimates of indirect effect showed that correlated changes, in general, are unfavorable. The estimated genetic parameters suggest that utilization of selection indexes that, besides grain yield, include characters such as grain moisture, pollen shed, silk, stalk and root lodging to select the testcrosses, under normal development condition the crop, can lead to the development of hybrids with high grain yield and earliness, simultaneously.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Conselho Nacional de Desenvolvimento Científico e Tecnológico

NaCl is a classic osmoregulatory agent that in isotonic conditions with the plasma is able to stabilize the red blood cells (RBC) in a relaxed (R) state, where these cells congregate a certain degree of stability with the capacity of exercise properly all its functions. The ethanol is a classic chaotropic agent, capable of promoting denaturation, but is also an osmolyte, able to promote stabilization of RBC. In 0.9% w/v NaCl and between 0 and 24% v/v ethanol, the RBC suffer lysis, but between 24 and 32%, the RBC are stabilized by the action of ethanol. This stabilization forms compacted or tight (T) RBC, which lost however many cell functions. In this work we studied the effect of concentrations of NaCl at 0.5, 0.9 and 1.1% w/v on the fixed time incubation (30 min) of human blood with concentrations of ethanol between 0 and 32%v/v. The RBC lysis and stabilization transitions by ethanol were described by sigmoids and characterized by their lysis half-point (D50) and stabilization (S50) transitions, respectively, and also by the amplitude (dX) and height (h) of those curves. The average value of D50 increased from 12.61 to 14.29%, but fell back again to 13.97% of ethanol/100 mL of 0.5, 0.9 and 1.1% w/v NaCl, respectively. The average value of S50 fell from 27.26 to 26.22 and 25.99% of ethanol/100 mL of solution with the increase in the concentration of NaCl solution of 0.5 to 0.9 and mL of 1.1% w/v saline, respectively. These results indicate that hypertonic saline solution potentiated the stabilizing effect of ethanol, certainly moving the RBC from of the state R to the state T.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Bovine anaplasmosis is caused by Anaplasma marginale and A. centrale. The most pathogenic and important species for cattle production is A. marginale, and is widely distributed in tropical, subtropical and temperate regions of the world. A. marginale is an intra-erythrocyte rickettsia of susceptible ruminants, biological and mechanically transmitted by ticks and hematophagous insects. The tick Rhipicephalus (Boophilus) microplus is the main vector of A. marginale in Brazil. The congenital form of transmission in cattle may occur, causing the neonatal anaplasmosis. The outer membrane of A. marginale includes six well characterized major surface proteins, MSP1a, MSP1b, MSP2, MSP3, MSP4 and MSP5, which play important role in the development of the immune response of infected animals. In this study, we have used the Phage Display technology to identify specific peptides that were immunoreactive to monoclonal antibodies anti-A. marginale proteins. Peptide selection was performed using a subtractive selection of a peptide library with 12 random amino acids, Ph.D.-12, expressed on the surface of the M13 filamentous phage concurrently against the anti-MSP1a and anti-MSP2. After four rounds of selection and validation by ELISA, the selected peptides have recognized only the anti-MSP1. Analysis of bioinformatics identified 45 peptides, which showed the protein consensus sequence STxS that was represented in 78% of selected phages. Due to the multiple motif repeats found in MSP1 protein, the STSSxL motif may become an important biological target, with potential use in diagnostic tests and vaccine for the control of Anaplasma marginale.

24 serum constituents were dosed from 102 Brazilian donkeys, to compare with values obtained for other authors to other races of equines and to verify if the age and the sex influence in these values. Of the animals, 77 were male and 25 females; It had been also grouped by age: 0-6 months (38 animals), 7-12 months (29 animals), 13-18 months (28 animals) and more than 18 months (13 animals). After the dosages, were made the statistical analysis by the test of Tukey 5% and compared the values of the different groups. The majority of the values was in accordance with the results considered for other authors, few differences had been found. Statistical differences between males and females had been found, only for magnesium, GGT (gamma glutamiltransferase) and urea; however, in relation to the age, significant differences for the majority of the constituent had been found, mainly for the young, enclosed animals in the age band of 0 the 6 months. Amongst the 24 analyzed constituent, the only ones that they had not presented variations related to the age had been the urea and the ionized calcium. The results that were different from the other authors can be related to the race, ambient influences, amongst others. The values of the sanguine constituent had not been differed significantly between the sex; however, it is noticed that the age intervenes considerably with the values of total proteins, albumen, globulin, relation albumen: globulin, creatinine, urates, cholesterol, triglicéris, match, total calcium, relation calcium: match, Mg (magnesium), chlorides, sodium, potassium, AST (aspartate aminotransferase), ALT (alanine aminotransferase), GGT and CK (creatine kinase). The observed variations are given that they can serve of base for accomplishment of new research involving Brazilian donkeys and contribute for the creation of exclusive values of reference to this race.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Patients with subclinical hypothyroidism treated with sodium L-thyroxine realized test of physical effort in cycle ergometer to investigate the alterations in the concentrations of blood lactate; total protein, immunoglobulin A, nitric oxide, alpha-amylase activity, present in the saliva; and tolerance to the physical effort. We selected eight female volunteers aged 51.8 ± 4.9 years, TSH > 4.4 mU/dL, total T3 and free T4 in the plasma and without clinical signs of hypothyroidism. The values of blood lactate and salivary total protein was more raised (p<0.05) when compared before and after the administration of L-thyroxine in the realization of the effort test. The displacement for right of the point of threshold of blood lactate, salivary total protein and the increase of a stage of training in the incremental test reflected a greater adaptation to the exercise after the treatment. The values of blood lactate, salivary total protein and salivary nitric oxide, before and after the test, was greater when the volunteers was treatment with L-thyroxine (p<0.05). The values of salivary alpha-amylase activity and concentration of salivary immunoglobulin A, after and before the effort test, hadn t difference (p<0.05) in relation to the treatment with L-thyroxine. Some of biomarkers analyzed in this study had not alterations that indicated one better adaptation after the exercise. However, the treatment with L-thyroxine showed alterations of the salivary nitric oxide and the anaerobic threshold in the test of physical effort when evaluated by the measure of blood lactate and salivary total protein. These data showed that the replacement of L-thyroxine increases the tolerance to the physical effort.

CHAPTER II: The effects of the association of nandrolone with metoprolol on striated muscle were investigated. Forty male Wistar rats were randomly distributed into four groups: control, treated with nandrolone 20mg/kg/week, treated with metoprolol 1mg/kg/day, and treated with both nandrolone and metoprolol. Left ventricle, soleus, and gastrocnemius sections were cut on a cryostat (5&#956;m), and stained with hematoxylin and eosin, or picrosirius red. Digital images were captured and analyzed by software. Cross-sectional area (CSA), diameter, number of myonuclei per fiber, central myonuclei, splitting cells, myonuclear domain, percentage of non-contractile tissue, and serum testosterone were measured. Glucocorticoid (GR) and androgen receptor (AR) were analyzed by immunodetection. An increase was seen in the morphometric parameters analyzed in both cardiac and striated fibers from animal treated with nandrolone. Metoprolol partially restored the cardiac hypertrophy caused by nandrolone without reducing the final percentage of non-contractile tissue in the ventricle. However, the anabolic effect of nandrolone was not reestablished by metoprolol in the striated fiber. Nandrolone administration increased serum testosterone levels and up-regulated the expression of AR whereas down-regulated the expression of GR (P<0.05). Our results suggest that the effects of the association of nandrolone with metoprolol are different in cardiac and muscle fibers. CHAPTER III: The effects of the association of nandrolone and metoprolol on the reproductive system, serum testosterone, total protein, and glutamate and glutamine concentrations of rats were investigated. Forty male Wistar rats were randomly distributed into 4 groups: control, treated with metoprolol 1mg/kg/day, treated with nandrolone 20mg/kg/week, and treated with both metoprolol and nandrolone. Testes and spermatozoa were histologically processed and analyzed to determine: volume, mass, size, width of the seminiferous tubules, epithelial height, tubular lumen, and volumetric proportion. Head images from spermatozoa were captured and analyzed by software to assess the difference between coloration and structure. Nandrolone treatment caused a decrease in mass, diameter and testicular size, as well as width of the seminiferous tubules, epithelial height, tubular lumen and volumetric proportion. On the other hand, serum testosterone, total protein and glutamine and glutamate concentrations were higher in these animals (P<0.05). Metoprolol did not cause any significant effect on the morphological parameters investigated. The alterations caused by nandrolone were significantly lower when analyzed in association with metoprolol, which could indicate a possible protective effect of the beta-blocker on the testes.

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Multiple sclerosis (MS), degenerative disease characterized by defects in the myelin membrane of neural cells, takes place probably by oxidative, inflammatory and autoimmune mechanisms in genetically vulnerable individuals, under the influence of environmental factors associated with feeding. [Hypothesis] As the structural composition of biological membranes is largely affected by diet, physical activity and use of medicines, the interference of environmental factors in the homeostasis of structural membranes of neural cells in carriers of MS must also be shared by other post-mitotic cells of the body, such as erythrocytes. [Objective] This study aimed to investigate the influence of statin use on the stability of erythrocytes membranes against hypotonic shock and chaotropic action of ethanol in patients with MS. [Methods] The population was composed of 16 women suffering from MS (26-58 years) who were being monitored at the Clinical Hospital of the Federal University of Uberlândia. They were divided into two groups, one group treated with 20 mg/day of simvastatin for 2.66 ± 1.03 years (n = 6) and another group without the use of statin (n = 10). A control group consisted of 6 healthy women with BMI and age (28-56 years) similar to problem group, all without the use of medications and without chronic consumption of alcohol. We evaluated the scores of patients in the Expanded Disability Status Scale (EDSS), hematological and biochemical variables, in addition to the stability of erythrocytes against hypotonic shock and ethanol. The stability of erythrocytes was evaluated by the half-transition points, H50 and D50, obtained from the curves of hemolysis induced by hypotonic shock and chaotropic action of ethanol, respectively. Statistical comparisons of results between groups were made by ANOVA and Tukey post-hoc test. [Results] There were no statistically significant differences in EDSS scores or the hematological variables between groups, but the MS patients under use of statin showed levels of total cholesterol and LDL-cholesterol significantly lower than those without use of that drug. The values of H50 of the MS patients without use of statins (0.472 ± 0.013 g.dL-1 NaCl) were significantly higher than those of volunteers in the control group (0.427 ± 0.015 g.dL-1 NaCl). The H50 values of the carriers of MS with use of statins (0.457 ± 0.013 g.dL-1 NaCl) declined (although not in a statistically significant basis) compared to carriers of MS without the use of statins, but they were not equal to the values of H50 of volunteers in the control group. Moreover, the values of D50 of MS patients without use of statin (13.87 ± 0.74% v/v ethanol) were significantly lower than those of volunteers in the control group (15.38 ± 0.24 g.dL-1 NaCl), but the values of D50 in the MS patients under statin therapy (15.20 ± 0.15 g.dL-1 NaCl) were not significantly different from those of volunteers in the control group, although they had been significantly higher than those of MS patients without use of statins. [Conclusions] The carriers of MS without use of statin showed erythrocytes less stable against hypotonic chock and action of ethanol than the control group. The use of statins increased the stability of erythrocytes against ethanol in the MS patients, making it similar to the RBC stability of the volunteers without the disease. But the use of statins did not increase osmotic stability of the erythrocyte membrane against hypotonicity in such a way to make it similar to the membrane stability of the volunteers without the disease. These findings are important and reflect the homeostatic conditions in the erythrocytes membranes by the moment of their evaluation. This could be merely a consequence of changes in lifestyle associated with the development of the disease, but could also be associated with a lifestyle that may have implications in the development of MS. The reasons for believing in the validity of the last hypothesis are discussed.

Universidade Federal de Uberlândia

The green tea is considered a functional food with antioxidant activity, and this delays the process of old age and helps in the prevention of chronicle and degenerative diseases, such as cancer and heart affections. Of catechins, present in green tea, may is extremely important in the inactivation of free radicals, which provoke damages to the DNA molecules and to the cell membranes prevent the cytotoxicity induced by the oxidative stress in different tissues, by actuating in the process of chemoprevention. In this study two crosses, ST and HB aimed at evaluating the protective effects of green tea in three different concentrations (25, 50 e 75mg/mL), combined with the antitumor agent, doxorrubicin (DXR), of the genotoxic action; by the of wing spot test (SMART) of Drosophila melanogaster. The results obtained demonstrated that in MH descendants there was not a statistically significant increase in the frequencies of spots, in the individuals treated with green tea, in standard cross (ST) as well as in the high bioactivation cross (HB - characterized by a high constitutive level of cytochrome P450) . However we could observe a statistically significant decrease in the sum of mutant spots, in MH descendants of both crossings (ST and HB), in all concentrations of green tea associated to DXR. These results suggest that green tea does not present genotoxic effect, but presents protective affect against the mutagenic and recombinagenic action of DXR in the somatic cells of D. melanogaster, in the experimental concentrations used in this study.

The sugarcane borer, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae), is the most important insect pest of sugarcane grown in Brazil and others localities in the Americas, causing stalk damage to sugarcane plants which results in loss of efficiency in both sugar and alcohol production. The extensive damage caused by this pest in the sugarcane cultivation and its difficult control by conventional insecticides justify the search for new strategies that can help to reduce the use of harmful chemicals to humans and the environment. In order to generate new molecules with insecticidal activity against the sugarcane borer, the present work aimed to build a scFv antibody fragment library through Phage Display technology for selection and characterization of molecules with binding properties against D. saccharalis larval midgut proteins. The methodology used was efficient, allowing the selection of a scFv with immunoreactivity for the insect intestinal epithelium and with potential insecticidal activity.

Breast carcinoma (BC) is currently regarded as a complex and heterogeneous disease presenting abnormalities in several molecular pathways. Although BC is the leading female cancer in incidence and the second one in mortality worldwide, the prognostic and predictive factors known are incapable to predict the outcome of all patients as well as to avoid unnecessary treatment. Applicability of Phage Display technology allows the construction of antibody combinatorial libraries identifying human monoclonal antibodies to clinical and therapeutic practice. In this work was constructed a recombinant Fab library which aimed recognized tumor antigens in patients with breast cancer. Transcripts from twenty patients with diagnosed invasive ductal carcinoma were used to construct a library with 1,7x106 different clones, which was selected against breast tissues. ELISA assays demonstrated reactivity of 22 clones, of which was selected the one with the biggest difference between the study groups. Imunohistochemistry experiments confirmed the specificity of FabC-4 clone to tumor antigens. This work provided significant biotechnological results with a specific Fab recombinant library, which indicated its great potential in selecting antibodies for breast carcinoma, opening new possibilities in diagnostic and therapeutics areas.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Fundação de Amparo a Pesquisa do Estado de Minas Gerais