The Neuraxial Effects of Intraspinal Amitriptyline at Low Concentrations

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Apparent digestibility of diets with different concentrations of lysine and energy in piglets with different body weights and post-weaning age

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of Psidium cattleianum leaf extract on Streptococcus mutans viability, protein expression and acid production

Plants naturally produce secondary metabolites that can be used as antimicrobials. The aim of this study was to assess the effects of Psidium cattleianum leaf extract on Streptococcus mutans. The extract (100%) was obtained by decoction of 100 g of leaves in 600 ml of deionized water. To assess killing, S. mutans biofilms were treated with water (negative control) or various extract dilutions [ 100, 50, 25% (v/v) in water] for 5 or 60 min. To evaluate the effect on protein expression, biofilms were exposed to water or 1.6% (v/v) extract for 120 min, proteins were extracted and submitted to 2-dimensional difference gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry. The effect of 1.6% (v/v) extract on acid production was determined by pH measurements and compared to a water control. Viability was similar after 5 min of treatment with the 100% extract or 60 min with the 50% extract (about 0.03% survival). There were no differences in viability between the biofilms exposed to the 25 or 50% extract after 60 min of treatment (about 0.02% survival). Treatment with the 1.6% extract significantly changed protein expression. The abundance of 24 spots was decreased compared to water (p < 0.05). The extract significantly inhibited acid production (p < 0.05). It is concluded that P. cattleianum leaf extract kills S. mutans grown in biofilms when applied at high concentrations. At low concentrations it inhibits S. mutans acid production and reduces the expression of proteins involved in general metabolism, glycolysis and lactic acid production. Copyright (C) 2008 S. Karger AG, Basel.

Chemotherapeutic agents in low noncytotoxic concentrations increase immunogenicity of human colon cancer cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The shielding effect of glycerol against protein ionization in electrospray mass spectrometry

Most commercial recombinant proteins used as molecular biology tools, as well as many academically made preparations, are generally maintained in the presence of high glycerol concentrations after purification to maintain their biological activity. The present study shows that larger proteins containing high concentrations of glycerol are not amenable to analysis using conventional electrospray ionization mass spectrometry (ESI-MS) interfaces. In this investigation the presence of 25% (v/v) glycerol suppressed the signals of Taq DNA polymerase molecules, while 1% (v/v) glycerol suppressed the signal of horse heart myoglobin. The signal suppression was probably caused by the interaction of glycerol molecules with the proteins to create a shielding effect that prevents the ionization of the basic and/or acidic groups in the amino acid side chains. To overcome this difficulty the glycerol concentration was decreased to 5% (v/v) by dialyzing the Taq polymerase solution against water, and the cone voltage in the ESI triple-quadrupole mass spectrometer was set at 80-130 V. This permitted observation of a mass spectrum that contained ions corresponding to protonation of up to 50% of the ionizable basic groups. In the absence of glycerol up to 85% of the basic groups of Taq polymerase became ionized, as observed in the mass spectrum at relatively low cone voltages. An explanation of these and other observations is proposed, based on strong interactions between the protein molecules and glycerol. For purposes of comparison similar experiments were performed on myoglobin, a small protein with 21 basic groups, whose ionization was apparently suppressed in the presence of 1% (v/v) glycerol, since no mass spectrum could be obtained even at high cone voltages. Copyright (C) 2003 John Wiley Sons, Ltd.

Analyzing glycerol-mediated protein oligomerization by electrospray ionization mass spectrometry

Glycerol is widely used as protein stabilizer, in both local and commercial preparations, so it has become necessary to develop methods for mass spectrometric analysis of protein preparations in the presence of glycerol. However, this stabilizing agent may cause signal suppression when present in high concentrations, and is also known to induce protein supercharging even at low concentrations. This work reports the,use of electrospray ionization (ESI) mass spectrometry to characterize glycerol-mediated protein oligomerization. this phenomenon seems to involve the formation of strong non-covalent interactions between protein and glycerol involving close contact between the monomers, leading to formation of protein oligomers adducted with glycerol molecules under the characteristic analytical conditions of the ESI interface. At high orders of oligomerization a lower number of glycerol molecules is required to maintain the high oligomeric states than for the dimers and trimers, and it is possible that for the higher oligomers the monomers become so close to one another that non-covalent bonds between the side chains of the amino acid residues in the proteins may be established. Copyright (C) 2005 John Wiley & Sons, Ltd.

Recovery of rat growth and lipid profiles in adult rats subjected to fetal protein malnutrition with a fructose-rich diet

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Metabolic responses to acute physical exercise in young rats recovered from fetal protein malnutrition with a fructose-rich diet

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Muscle protein metabolism in neonatal alloxan-administered rats: effects of continuous and intermittent swimming training

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Separation and partitioning of Green Fluorescent Protein from Escherichia coli homogenate in poly(ethylene glycol)/sodium-poly(acrylate) aqueous two-phase systems

The partitioning of Green Fluorescent Protein (GFP) in poly(ethylene glycol)/Na-poly(acrylate) aqueous two-phase systems (PEG/NaPA-ATPS) has been investigated. The aqueous two-phase systems are formed by mixing the polymers with a salt and a protein solution. The protein partitioning in the two-phase system was investigated at 25 degrees C. The concentration of the GFP was measured by fluorimetry. It was found that the partitioning of GFP depends on the salt type, pH and concentration of PEG. The data indicates that GFP partitions more strongly to the PEG phase in presence of Na2SO4 relative to NaCl. Furthermore, the GFP partitions more to the PEG phase at higher pH. The partition to the PEG phase is strongly favoured in systems with larger tie-line lengths (i.e. systems with higher polymer concentrations). The molecular weight of PEG is important since the partition coefficient (K) of GFP gradually decreases with increasing PEG size, from K ca. 300-400 for PEG 400 to K equal to 1.19 for PEG 8000. A separation process was developed where GFP was separated from a homogenate in two extraction steps: the GFP is first partitioned to the PEG phase in a PEG 3000/NaPA 8000 system containing 3 wt% Na2SO4, where the K value of GFP was 8. The GFP is then re-extracted to a salt phase formed by mixing the previous top-phase with a Na2SO4 solution. The K-value of GFP in this back-extraction was 0.22. The total recovery based on the start material was 74%. (c) 2008 Elsevier B.V. All rights reserved.

Effects of nitrate and phosphate availabilities on growth, photosynthesis and pigment and protein contents in colour strains of Hypnea musciformis (Wulfen in Jacqu.) J.V. Lamour. (Gigartinales, Rhodophyta)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

beta(2)-Agonists and cAMP inhibit protein degradation in isolated chick (Gallus domesticus) skeletal muscle

1. The role of beta(2)-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours.2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective beta(2)-agonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP.3. The decrease in the rate of total protein degradation induced by 10(-5) M clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10(-5) M ICI 118.551, a selective beta(2)-antagonist.4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10(-4) M dibutyryl-cAMP or isobutylmethylxanthine.5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.

LOW-PROTEIN DIET IMPAIRS GLUCOSE-INDUCED INSULIN-SECRETION FROM AND CA-45 UPTAKE BY PANCREATIC RAT ISLETS

Glucose-induced insulin secretion rom and Ca-45 uptake by isolated pancreatic islets, derived from rats fed with normal (NPD) or low protein diet (LPD), were studied. Insulin secretion from both types of islets in response to increasing concentrations of glucose followed an S-shaped pattern. However, basal secretion observed at substimulatory concentrations of glucose (0-5.6 mM), as well as maximal release, obtained at 16.7 mM or higher glucose concentrations were significantly reduced in islets from LPD. Furthermore, in LPD rat islets, the dose-response curve to glucose was clearly shifted to the right compared with NPD islets, with the half-maximal response occurring at 8.5 and 14.4 mM glucose for NPD and LPD islets, respectively. In islets from NPD rats, the Ca-45 content, after 5 or 90 min in the presence of 8.3 mM glucose, was higher than that observed for islets kept at 2.8 mM glucose and increased further at 16.7 mM glucose. After 5 min of incubation, the Ca-45 uptake by LPD islets in the presence of 8.3 mM glucose was slightly higher than basal values (2.8 mM glucose); however, no further increase in the Ca-45 uptake was noticed at 16.7 mM glucose. In LPD islets a significant increase in Ca-45 uptake over basal values was registered only at 16.7 mM glucose, after 90 min of incubation. These data indicate that the poor secretary response to glucose observed in islets from LPD rats may be related to a defect in the ability of glucose to increase Ca2+ uptake and/or to reduce Ca2+ efflux from beta-cells.

Intestinal starch disappearance increased in steers abomasally infused with starch and protein

Steers (379 +/- 10 kg) with ruminal, duodenal, and ileal cannulas were used in a 5 x 5 Latin square digestion trial to quantify and evaluate the relationship between intestinal protein supply and intestinal starch disappearance. Treatments were infusions of 0, 50, 100, 150, or 200 g/d of casein along with 1,042 g/d of raw cornstarch. Abomasal infusions were accomplished by passing tubing and a pliable retaining washer through the reticular-omasal orifice into the abomasum. Steers were fed a 93% corn silage, 7% supplement diet that contained 12% crude protein at 1.65% body weight in 12 equal portions/d. Periods lasted 17 d (12 d for adaptation, 2 d of collections, and 3 d of rest). The quantity and percentage of organic matter and protein disappearance from the small intestine increased linearly (P < 0.03) with infused casein. Greater quantities of starch disappeared with increased casein infusion (P < 0.01). The infusion of 200 g/d of casein increased small intestinal starch disappearance by 226 g/d over the control. Casein infusion did not affect the quantity or percent of organic matter, starch, or protein disappearance in the large intestine. Treatments did not change ruminal ammonia N, ruminal pH, or plasma glucose concentrations. Starch disappearance from the small intestine was increased with greater protein flow to the duodenum of steers.

Serum iron and plasma fibrinogen concentrations as indicators of systemic inflammatory diseases in horses

Background: Detection of systemic inflammation, which is important for proper diagnosis and prompt treatment, can be challenging.Hypothesis: Measurement of plasma iron concentration is a sensitive method for detecting systemic inflammation in horses compared with measurements of plasma Fibrinogen concentration, a traditional marker for inflammation in the horse.Animals: Ninety-seven horses hospitalized with diseases causing systemic inflammation, 22 horses with localized inflammation, and 12 clinically normal horses were included in this study.Methods: A retrospective study was made on hospitalized horses that had both plasma iron and fibrinogen concentrations measured on hospital admission.Results: Plasma iron concentration was lower in horses with systemic inflammation (64 +/- 45 mu g/dL) than the reference interval minimum (105 mu g/dL) and were significantly lower (P = .001) than the value in a group of horses with local inflammation (123 +/- 45 mu g/dL) and in healthy transported horses (143 +/- 29 mu g/dL). Low plasma iron and high fibrinogen concentrations were both sensitive indicators of systemic inflammation in horses with sensitivity of 90 and 82%, respectively. There was a similar correlation between either continued decreases in iron concentration (R-sp of 0.239) or increases in fibrinogen concentration (R-sp of 0.280) during hospitalization and a worse prognosis.Conclusions and Clinical Importance: Measurement of plasma iron concentration better reflected acute inflammation than did fibrinogen concentration.