Chromatin regulation in schistosomes and histone modifying enzymes as drug targets

Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansiprecludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b.brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansiis alike to the BSF of T. b. bruceiin glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

Sucrose metabolizing enzymes in cell suspension cultures of Bauhinia forficata, Curcuma zedoaria and Phaseolus vulgaris

The objective of this work was to study the activity of sucrose metabolizing enzymes in extracts of cell suspension cultures of Bauhinia forficata Link, Curcuma zedoaria Roscoe and Phaseolus vulgaris L. Invertase pathway was identified in the three studied species. Sucrose synthase pathway was also responsible for sucrose metabolism in Curcuma zedoaria and Phaseolus vulgaris cells. Activity values higher than 300 nmol min-1 mg-1 of protein were found for acid and neutral invertases, UDPglucose pyrophosphorylase and phosphoglucomutase in the cell extract of the three plant species. Sucrose synthase showed low activity in Bauhinia forficata cells. As sucrose concentration in the culture medium decreased, sucrose synthase activity increased in C. zedoaria and P. vulgaris cells. The glycolytic enzymes activity gradually reduced at the end of the culture period, when carbohydrate was limited.

Enzyme activities and pectin breakdown of sapodilla submitted to 1-methylcyclopropene

The objective of this work was to investigate the influence of 1-methylcyclopropene (1-MCP) at 300 nL L-1 on activities of cell wall hidrolytic enzymes and pectin breakdown changes which Sapodilla (Manilkara zapota cv. Itapirema 31) cell wall undergoes during ripening. Sapodilla were treated with ethylene antagonist 1-MCP at 300 nL L-1 for 12 hours and then, stored under a modified atmosphere at 25º C for 23 days. Firmness, total and soluble pectin and cell wall enzymes were monitored during storage. 1-MCP at 300 nL L-1 for 12 hours delayed significantly softening of sapodilla for 11 days at 25º C. 1-MCP postharvest treatment affected the activities of cell wall degrading enzymes pectinmethylesterase and polygalacturonase and completely suppressed increases in beta-galactosidase for 8 days, resulting in less pectin solubilization. Beta-galactosidase seems relevant to softening of sapodilla and is probably responsible for modification of both pectin and xyloglucan-cellulose microfibril network.

Effects of modified atmosphere packaging on ripening of 'Douradão' peach related to pectolytic enzymes activities and chilling injury symptoms

The present study evaluated the effects of modified atmosphere packaging on inhibition of the development of chilling injury symptoms in 'Douradão' peach after cold storage and the possible involvement of cell wall enzymes. Fruits were harvested at the middle stadium of ripening, packed in polypropylene trays and placed inside low density polyethylene (LDPE) bags (30, 50, 60 and 75 µm of thickness) with active modified atmosphere (10 kPa CO2 + 1.5 kPa O2, balance N2). The following treatments were tested: Control: peaches held in nonwrapped trays; MA30: LDPE film - 30 µm; MA50: LDPE film - 50 µm; MA60: LDPE film - 60 µm and MA75: LDPE film - 75 µm. Fruits were kept at 1±1ºC and 90±5% relative humidity (RH) for 28 days. After 14, 21 and 28 days, samples were withdrawn from MAP and kept in air at 25±1ºC and 90±5% RH for ripening. On the day of removal and after 4 days, peaches were evaluated for woolliness incidence, pectolytic enzymes activities. The respiratory rate and ethylene synthesis were monitored during 6 days of ripening. The results showed that MA50 and MA60 treatments had positive effect on the inhibition of the development of woolly texture and reduced pectin methylesterase activity on the ripe fruits, keeping good quality of 'Douradão' peach during 28 days of cold storage. The treatments Control, MA30 and MA75 showed higher woolliness incidence and did not present marketable conditions after 14 days of cold storage.

Modification of a Brazilian smectite clay with different quaternary ammonium salts

In this work, a smectite clay from the State of Paraiba, Brazil, was treated with six different types of ammonium salts, which is an usual method to enhance the affinity between the clay and polymer for the preparation of nanocomposites. The clays, before and after modification, were characterized by X ray diffraction. The conformation of the salts within the platelets of the clay depended on the number of long alkyl chains of the salt. The thermal stability of the clays was also studied. The ammonium salts thermal decomposition was explained in light of their position within the organoclays.

Modificação enzimática da farinha de arroz visando a produção de amido resistente

The aim of this work was to study the enzymatic modification on rice flour using lipase pancreatic and amyloglucosidase to obtain resistant starch. For this, Response Surface Methodology (RSM) was used to determine the best operating conditions for each enzyme. For lypase pancreatic, the highest value for resistant starch (45%) was achieved within 2 h reaction at pH 7 using an enzyme/substrate ratio of 4% (w/w) and Dp= 100/200 tyler. For amyloglucosidase, optima conditions corresponded to an enzyme/substrate ratio of 0,006 mL/g and Dp= 100/200 tyler at 45 ºC, yielding 57% of resistant starch in 2 h reaction. These results show the potential of using both enzymes to modified rice flour, increasing the resistant starch in about 5.7 folds in relation to the flour without treatment (resistant starch=10.6%).

Modification of the performance of WO3-ZrO2 catalysts by metal addition in hydrocarbon reactions

A study of the different hydrocarbon reactions over Ni doped WO3-ZrO2 catalysts was performed. Ni was found as NiO at low Ni concentration while at high Ni concentrations a small fraction was present as a metal. For both cases, Ni strongly modified total acidity and concentration of strong acid sites. In the cyclohexane dehydrogenation reaction, Ni addition promotes both benzene and methyl cyclopentane production. The hydroconversion activity (n-butane and n-octane) increases with the augment of total acidity produced by Ni. The selectivity to reaction products is modified according to the acid strength distribution changes produced by Ni addition.

Enhanced photocatalytic activity of TiO2 films by modification with polyethylene glycol

Titanium dioxide porous thin films on the Anatase phase were deposited onto glass slides by the sol-gel method assisted with polyethylene glycol (PEG). The dip-coated films were characterized using scanning electron microscopy (SEM), thermogravimetric analysis (TGA and DTG), UV-visible spectroscopy and X-ray diffraction (XRD). The photocatalytic activity of the films was determined by means of methyl-orange oxidation tests. The resultant PEG-modified films were crack-free and developed a porous structure after calcination at 500 °C. Photo-oxidation tests showed the dependency of catalytic activity of the films on the number of layers (thickness) and porosity, i.e. of the interfacial area.

Aplicações de enzimas na síntese e na modificação de polímeros

Enzymes are biological catalysts that offer great potential for use in the synthesis and modification of polymers, being more specific and greener than chemical catalysts. In this work, enzymes from the classes of hydrolases (lipase, cutinase and protease) and of oxidoreductases (horseradish peroxidase, manganese peroxidase and laccase) were identified as the main biocatalysts responsible for the synthesis of polymers. Biocatalysis can potentially be part of the life cycle of several polymers, including polyesters, polyurethanes, polycarbonates, polyamides, functionalized polysaccharides and polystyrene, allowing the synthesis of specialty macromolecules for fine applications and with higher added-value than commodity polymers.

Differentiation of Colletotrichum gloeosporioides isolates by using total proteins and esterase electrophoretic patterns and extracellular enzymes production

Isolates of Colletotrichum gloeosporioides (ISO-1, ISO-2, ISO-3, ISO-4, ISO-5 and ISO-6), the causal agent of anthracnose disease on mango fruits, were characterized by electrophoretic patterns of total proteins and esterase in polyacrylamida gel, and also, by production of extracellular enzymes on specific solid substrate. The electrophoretic analysis showed variation in number, intensity of coloration and position of the bands in the gel at each studied system tested. In contrast to the monomorphic behavior to total proteins, high esterase polymorfism was observed indicating difference among isolates. All isolates showed the activity of extracellular enzymes such as amylase, lipase, and protease with some variation among them. The proteolitic activity seemed to be more accentuated than the two other enzymes studied.

Effects of light intensity modification by reflective aluminized screenhouse on sweet pepper growth and yield

Sweet pepper is one of the ten most consumed vegetables in world. Although it develops better under protected environment, the cultivation in tropical countries is practiced in open field due greenhouse structure higher costs. Unfortunately, such practice has compromised the crop to reach either best yield or fruit quality. Since production and cost are the most important criteria for agricultural production, we aimed to evaluate reflective aluminized polypropylene shading net influence on sweet pepper (Capsicum annuum L.) growth and production as intermediary alternative for low/middle income producers from Brazilian tropical regions. Sweet pepper Magali R hybrid was cultivated in two environments: FC - field conditions (control) and RS - reflective shading net with 40% shading rate. RS caused reductions in incident solar radiation (SR) and photosynthetically active radiation (PAR) on the amount of 46.3% and 48.3%, respectively. There were no significant changes in temperature and relative humidity recorded for the two environments. In addition, RS allowed best use efficiency of photosynthetically active radiation since it promoted higher values of plant height, leaf number and area index than those reached on FC on the amount of 29%, 22% and 80 %, respectively. Similarly, plants grown under RS showed higher yield and marketable fruits and promoted less loses by sunscald.

Effects of environmental modification on mastitis occurrence and hormonal changes in Holstein cows

The purpose of this research was to evaluate the effects of evaporative cooling in freestall on mastitis occurrence, milk production, and composition, as well as cortisol, T3 (triiodothyronine), and T4 (thyroxin) levels in lactating dairy cows. Twenty-eight multiparous cows averaging 70 ± 10 day postpartum were used in four treatments from January to March 2003. The treatments were: Day (cooling from 7:00 a.m. to 7:00 p.m.); Night (cooling from 7:00 p.m. to 7:00 a.m.); 24-hour (cooling 24-hour); and Control (no cooling). Wired cup test was used for clinical mastitis diagnosis, and the California Mastitis Test (CMT) was used to identify subclinical mastitis. Blood and milk samples were taken weekly for microbiological and hormonal analyses. The cortisol levels were higher than normal values in all treatment groups, suggesting stress conditions, but T3 and T4 levels remained normal in all groups. The occurrence of subclinical mastitis was lower in Day and Night groups than in Control and 24-hour groups. Regarding the microbiological analyses, in all groups the isolation of Corynebacterium sp. from milk samples increased while negative coagulase staphylococci (CNS) declined as etiological agents of subclinical mastitis. However, in Day and 24-hour groups, coagulase positive staphylococci (CPS) increased mainly Staphylococcus aureus (49.8% and 47.7% respectively). The Night group showed a decrease in subclinical mastitis occurrences. Our data indicate that all animals subjected to treatments presented high levels of cortisol, indicating a stress condition. The Night treatment presented a reduction in microbial isolation, suggesting a reduced susceptibility to mastitis.

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

Abstract In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.

Effects of Cinnamon extract on biochemical enzymes, TNF-α and NF-κB gene expression levels in liver of broiler chickens inoculated with Escherichia coli

Abstract: Infection with Escherichia coli (E. coli) is a common disease in poultry industry. The use of antibiotics to treat diseases is facing serious criticism and concerns. The medicinal plants may be effective alternatives because of their multiplex activities. The aim of this study was to investigate the effects of cinnamon extract on the levels of liver enzymes, tumor necrosis factor-alpha (TNF-α) and nuclear factor-kappa B (NF-κB) gene expressions in liver of broiler chickens infected with E. coli. Ninety Ross-308 broilers were divided into healthy or E. coli-infected groups, receiving normal or cinnamon extract (in concentrations of 100 or 200mg/kg of food) supplemented diets. E. coli suspension (108cfu) was injected subcutaneously after 12 days cinnamon administration. Seventy-two hours after E. coli injection, the blood samples were taken for biochemical analysis of liver enzymes in serum (spectrophotometrically), and liver tissue samples were obtained for detection of gene expression of inflammatory markers TNF-α and NF-κB, using real-time PCR. Infection with E. coli significantly increased the levels of TNF-α and NF-κB gene expressions as well as some liver enzymes including creatine-kinase (CK), lactate-dehydrogenase (LDH), alanine-transferase (ALT) and aspartate-transferase (AST) as compared with control group (P<0.05). Pre-administration of cinnamon extract in broilers diet (in both concentrations) significantly reduced the tissue levels of TNF-α and NF-κB gene expressions and enzymes CK and ALT in serum of broiler chickens inoculated with E. coli in comparison with E. coli group (P<0.05 and P<0.01). The levels of LDH and AST were significantly decreased only by 200mg/kg cinnamon extract in infected broilers. The level of alkaline-phosphatase (ALP) was not affected in any groups. Pre-administration of cinnamon extract in diets of broiler chickens inoculated with E. coli could significantly reduce the gene expression levels of pro-inflammatory mediators and liver enzymes activities, thereby protecting the liver against this pathologic condition.