Characterisation of protease activity in extracellular products secreted by Giardia duodenalis trophozoites treated with propolis

Results from our laboratory revealed propolis activity on Giardia trophozoites proliferation. Since therapeutic agents can inhibit the activity of proteases related to relevant biologic and physiologic processes of parasites, this study was undertaken to characterise the proteolytic activity of excretory/secretory products (ESP) of trophozoites treated with propolis. ESP was obtained from culture supernatants of trophozoites exposed to 250 and 500 mu g mL(-1) of propolis. ESP were tested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the protein profiles and the protease activity was assayed in gelatin-containing gels. Synthetic inhibitors were used to characterise the protease classes. Treated and non-treated ESP showed a similar protein and hydrolysis pattern. A simple pattern of protein composed by five evident bands of approximately 167, 132, 79, 61 and 51 kDa was found, and the zymograms comprised hydrolysis zones distributed from > 170 to 23 kDa. No inhibition was seen on protease activity of propolis-treated trophozoites, whose hydrolysis pattern was similar to control. One may conclude that both ESP degraded gelatin and the activity was predominantly due to cysteine proteases. Although propolis had no effect on the proteolytic activity, further studies could identify the active constituents responsible for propolis antigiardial activity and their mechanisms of action.

Prostate carcinogenesis induced by N-methyl-N-nitrosourea (mnu) in gerbils: Histopathological diagnosis and potential invasiveness mediated by extracellular matrix components

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

Morphological and enzymatic analysis of the midgut of Anopheles darlingi during blood digestion

The midgut of adult female Anopheles darlingi is comprised of narrow anterior and dilated posterior regions, with a single layered epithelium composed by cuboidal digestive cells. Densely packed apical microvilli and an intricate basal labyrinth characterize each cell pole. Before blood feeding, apical cytoplasm contains numerous round granules and whorled profiles of rough endoplasmic reticulum. Engorgement causes a great distension of midgut. This provokes the flattening of digestive cells and their nuclei. Simultaneously, apical granules disappear, the whorls of endoplasmic reticulum disassemble and 3 h post bloodmeal (PBM), nucleoli enlarge manyfold. An intense absorptive process takes place during the first 24h PBM, with the formation of large glycogen inclusions, which persist after the end of the digestive process. Endoproteases activities are induced after bloodmeal and attain their maximum values between 10 and 36 h PBM. At least two different aminopeptidases seem to participate in the digestive process, with their maximum activity values at 36 and 48 h PBM, respectively. Coarse electrondense aggregates, possibly debris from digested erythrocytes, begin to appear on the luminal face of the peritrophic membrane from 18 h PBM and persist during all the digestive process, and are excreted at its end. We suggest that these aggregates could contain some kind of insoluble form of haem, in order of neutralize its toxicity. (c) 2005 Elsevier Ltd. All rights reserved.

Functional morphology of adult female Culex quinquefasciatus midgut during blood digestion

The adult female Culex quinquefasciatus midgut comprises a narrow anterior and a dilated posterior region, with epithelia composed of a monolayer of adjacent epithelial cells joined at the apical portion by septate junctions. Densely packed apical microvilli and an intricate basal labyrinth characterise each cell pole. Our morphological studies suggest that, during blood digestion, the anterior midgut region also participates in an initial absorptive stage which is probably related to the intake of water, salts and other small molecules. This activity peaked by 6 h after bloodmeal feeding (ABF) and ended approximately 18 h ABF, when the peritrophic membrane was already formed. After this time, absorption only occurred in the posterior region, with morphologic and biochemical evidence of high synthetic activity related to the secretion of proteases. Chymotrypsin, elastase, aminopeptidase, and trypsin reached their maximum activity at around 36 h ABF. Digestion products were apparently absorbed and transported to the basal labyrinth, from where they should be released to the hemolymph. At 72 h ABF, proteolysis had already ended and protein levels had returned to those observed before blood meal. The epithelium of the posterior region, however, did not return to its initial morphology, appearing quite disorganised. Additionally, from 48 h ABF onwards some epithelial cells showed morphological signals of apoptosis. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Sugar digestion in mosquitoes: Identification and characterization of three midgut alpha-glucosidases of the neo-tropical malaria vector Anopheles aquasalis (Diptera : Culicidae)

Dietary carbohydrates provide an important source of energy for flight, and contribute to longevity and fecundity of mosquitoes. The most common sugar mosquitoes ingest is sucrose, and digestion of this substance is carried out mainly by alpha-glucosidases. In the current work, we tested the efficiency of sucrose on Anopheles aquasalis female diet. The best longevity (days) was reached when sugar was available in the diet, whereas most only blood fed females were dead 6 days after emergence. Three alpha-glucosidase isoforms were detected in the adult female midgut, named alpha Glu1, alpha Glu2 and alpha Glu3. These are acidic alpha-glucosidases with optima pH around pH 5.5. alpha Glu1 and alpha Glu2 are present in both secreted and membrane-bound forms, whereas alpha Glu3 only in anchored to membranes. The alpha-glucosidase activity is concentrated mainly in the posterior midgut (70%), both in non-fed or 10% sucrose fed females. The single form of these a-glucosidases seemed to be similar to 70 kDa polypeptides, although alpha Glu2 is presented in >= 600 kDa self-aggregates. K, values of alpha Glu1, alpha Glu2 and alpha Glu3 differed significantly from each other, supporting the statement that three alpha-glucosidases are produced in the female midgut. Together, all data suggest that sugar is an essential component of A. aquasalis female diet. In addition, alpha-glucosidases are synthesized in the same place where sucrose is digested and absorbed, the midgut. (c) 2007 Elsevier B.V. All rights reserved.

On the Apterous Line of the Termite Velocitermes heteropterus (Isoptera: Termitidae): Developmental Pathways and Cellulose Digestion

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

Production of extracellular acid proteases by Aspergillus clavatus

The production of extracellular acid proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on haemoglobin pH 5.0 at 37 degreesC. The highest acid proteolytic activity (80 U/ml) was observed in culture medium containing glucose and gelatin at 1% (w/v) at 30 degreesC at the third day of incubation. Cultures developed in Vogel medium with glucose at 2% (w/v) showed at about 45% of proteolytic activity when compared to the cultures with 1% of the same sugar. The optimum pH of enzymatic activity was 2.0 and the enzyme was stable at pH values ranging from 2.0 to 4.0. The optimum temperature was 40 degreesC and the half-lives at 40, 45 and 50 degreesC were 30, 10 and 5 min, respectively.

Purification and some properties of an extracellular acid protease from Aspergillus clavatus

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

Purification and Characterization of Two Extracellular Xylanases from Penicillium sclerotiorum: A Novel Acidophilic Xylanase

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Autonomic control of heart rate during forced activity and digestion in the snake Boa constrictor

Reptiles, particularly snakes, exhibit large and quantitatively similar increments in metabolic rate during muscular exercise and following a meal, when they are apparently inactive. The cardiovascular responses are similar during these two states, but the underlying autonomic control of the heart remains unknown. We describe both adrenergic and cholinergic tonus on the heart during rest, during enforced activity and during digestion (24-36h after ingestion of 30% of their body mass) in the snake Boa constrictor. The snakes were equipped with an arterial catheter for measurements of blood pressure and heart rate, and autonomic tonus was determined following infusion of the beta -adrenergic antagonist propranolol (3mg kg(-1)) and the muscarinic cholinoceptor antagonist atropine (3 mg kg-1).The mean heart rate of fasting animals at rest was 26.4 +/- 1.4 min(-1), and this increased to 36.1 +/- 1.4 min(-1) (means +/- S.E.M.; N=8) following double autonomic block (atropine and propranolol). The calculated cholinergic and adrenergic tones were 60.1 +/- 0.3% and 19.8 +/- 2.2%, respectively. Heart rate increased to 61.4 +/- 1.5 min(-1) during enforced activity, and this response was significantly reduced by propranolol (maximum values of 35.8 +/-1.6 min(-1)), but unaffected by atropine. The cholinergic and adrenergic tones were 2.6 +/- 2.2 and 41.3 +/- 1.9 % during activity, respectively. Double autonomic block virtually abolished tachycardia associated with enforced activity (heart rate increased significantly from 36.1 +/- 1.4 to 37.6 +/- 1.3 min(-1)), indicating that non-adrenergic, non-cholinergic effectors are not involved in regulating heart rate during activity. Blood pressure also increased during activity.Digestion was accompanied by an increase in heart rate from 25.6 +/- 1.3 to 47.7 +/- 2.2 min(-1) (N=8). In these animals, heart rate decreased to 44.2 +/- 2.7 min-1 following propranolol infusion and increased to 53.9 +/- 1.8 min-1 after infusion of atropine, resulting in small cholinergic and adrenergic tones (6.0 +/- 3.5 and 11.1 +/- 1.1 %, respectively). The heart rate of digesting snakes was 47.0 +/- 1.0 min(-1) after double autonomic blockade, which is significantly higher than the value of 36.1 1.4 min-1 in double-blocked fasting animals at rest. Therefore, it appears that some other factor exerts a positive chronotropic effect during digestion, and we propose that this factor may be a circulating regulatory peptide, possibly liberated from the gastrointestinal system in response to the presence of food.

Ventilatory compensation of the alkaline tide during digestion in the snake Boa constrictor

The increased metabolic rate during digestion is associated with changes in arterial acid-base parameters that are caused by gastric acid secretion (the 'alkaline tide'). Net transfer of HCl to the stomach lumen causes an increase in plasma HCO3- levels, but arterial pH does not change because of a ventilatory compensation that counters the metabolic alkalosis. It seems, therefore, that ventilation is controlled to preserve pH and not P-CO2, during the postprandial period. To investigate this possibility, we determined arterial acid-base parameters and the metabolic response to digestion in the snake Boa constrictor, where gastric acid secretion was inhibited pharmacologically by oral administration of omeprazole. The increase in oxygen consumption of omeprazole-treated snakes after ingestion of 30% of their own body mass was quantitatively similar to the response in untreated snakes, although the peak of the metabolic response occurred later (36 h versus 24 h). Untreated control animals exhibited a large increase in arterial plasma HCO3- concentration of approximately 12 mmol 1(-1), but arterial pH only increased by 0.12 pH units because of a simultaneous increase in arterial P-CO2 by about 10 mmHg. Omeprazole virtually abolished the changes in arterial pH and plasma HCO3- concentration during digestion and there was no increase in arterial P-CO2. The increased arterial P-CO2 during digestion is not caused, therefore, by the increased metabolism during digestion or a lower ventilatory responsiveness to ventilatory stimuli during a presumably relaxed state in digestion. Furthermore, the constant arterial P-CO2, in the absence of an alkaline tide, of omeprazole-treated snakes strongly suggests that pH rather than P-CO2 normally affects chemoreceptor activity and ventilatory drive.

Effects of inhibition gastric acid secretion on arterial acid-base status during digestion in the toad Bufo marinus

Digestion affects acid-base status, because the net transfer of HCl from the blood to the stomach lumen leads to an increase in HCO3- levels in both extra- and intracellular compartments. The increase in plasma [HCO3-], the alkaline tide, is particularly pronounced in amphibians and reptiles, but is not associated with an increased arterial pH, because of a concomitant rise in arterial Pco(2) caused by a relative hypoventilation. In this study, we investigate whether the postprandial increase in Paco(2) of the toad Bufo marinus represents a compensatory response to the increased plasma [HCO3-] or a state-dependent change in the control of pulmonary ventilation. To this end, we successfully prevented the alkaline tide, by inhibiting gastric acid secretion with omeprazole, and compared the response to that of untreated toads determined in our laboratory during the same period. In addition, we used vascular infusions of bicarbonate to mimic the alkaline tide in fasting animals. Omeprazole did not affect blood gases, acid-base and haematological parameters in fasting toads, but abolished the postprandial increase in plasma [HCO3-] and the rise in arterial Pco(2) that normally peaks 48 h into the digestive period. Vascular infusion of HCO3-, that mimicked the postprandial rise in plasma [HCO3-], led to a progressive respiratory compensation of arterial pH through increased arterial Pco(2) Thus, irrespective of whether the metabolic alkalosis is caused by gastric acid secretion in response to a meal or experimental infusion of bicarbonate, arterial pH is being maintained by an increased arterial Pco(2). It seems, therefore, that the elevated Pco(2), occuring during the postprandial period, constitutes of a regulated response to maintain pH rather than a state-dependent change in ventilatory control. (C) 2003 Elsevier B.V. All rights reserved.

Arterial acid-base status during digestion and following vascular infusion of NaHCO3 and HCl in the South American rattlesnake, Crotalus durissus

Digestion is associated with gastric secretion that leads to an alkalinisation of the blood, termed the alkaline tide. Numerous studies on different reptiles and amphibians show that while plasma bicarbonate concentration ([HCO3-](pl)) increases substantially during digestion, arterial pH (pHa) remains virtually unchanged, due to a concurrent rise in arterial PCO2 (PaCO2) caused by a relative hypoventilation. This has led to the suggestion that postprandial amphibians and reptiles regulate pHa rather than PaCO2.Here we characterize blood gases in the South American rattlesnake (Crotalus durissus) during digestion and following systemic infusions of NaHCO3 and HCl in fasting animals to induce a metabolic alkalosis or acidosis in fasting animals. The magnitude of these acid-base disturbances were similar in magnitude to that mediated by digestion and exercise. Plasma [HCOT] increased from 18.4+/-1.5 to 23.7+/-1.0 mmol L-1 during digestion and was accompanied by a respiratory compensation where PaCO2 increased from 13.0+/-0.7 to 19.1+/-1.4 mm Hg at 24 h. As a result, pHa decreased slightly, but were significantly below fasting levels 36 h into digestion. Infusion of NaHCO3 (7 mmol kg(-1)) resulted in a 10 mmol L-1 increase in plasma [HCO3-] within 1 h and was accompanied by a rapid elevation of pHa (from 7.58+/-0.01 to 7.78+/-0.02). PaCO2, however, did not change following HCO3- infusion, which indicates a lack of respiratory compensation. Following infusion of HCl (4 mmol kg(-1)), plasma pHa decreased by 0.07 units and [HCO3-](pl) was reduced by 4.6 mmol L-1 within the first 3 h. PaCO2, however, was not affected and there was no evidence for respiratory compensation.Our data show that digesting rattlesnakes exhibit respiratory compensations to the alkaline tide, whereas artificially induced metabolic acid-base disturbances of same magnitude remain uncompensated. It seems difficult to envision that the central and peripheral chemoreceptors would experience different stimuli during these conditions. One explanation for the different ventilatory responses could be that digestion induces a more relaxed state with low responsiveness to ventilatory stimuli. (C) 2005 Elsevier B.V. All rights reserved.

Cellular and extracellular behavior in the gerbil (Meriones unguiculatus) ventral prostate following different types of castration and the consequences of testosterone replacement

Mongolian gerbils (Meriones unguiculatus) were grouped into two experimental groups: GEx.01 suffered orchiectomy and after 30 days received doses of testosterone cipionate (T), while GEx.02 received weekly and alternated doses of the anti-androgens cyproterone acetate and flutamide for 30 days, and the animals were then euthanized. Structural evaluation reveals a more intense reduction in epithelial height in GEx.02. Smooth muscle cells (SMC) presented a star-shaped aspect after 30 days of hormonal ablation and basal membrane was shown to be more intensely grooved in GEx.01. In both groups, after hormonal replacement, recovery in epithelial height could be noted and the SMC presented its phenotypes, but an increase in RER was seen, characterizing a modulation from its contractile to secreting phenotype. In conclusion, the prostate presented involution capacity after androgen ablation and the ability to reorganize after hormonal replacement, but events resulting from orchiectomy and subsequent T replacement were shown to be more aggressive to the prostate. (c) 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

In situ hybridization of bat chromosomes with human (TTAGGG)n probe, after previous digestion with Alu I

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