Purification and Biochemical Properties of a Glucose-Stimulated beta-D-Glucosidase Produced by Humicola grisea var. thermoidea Grown on Sugarcane Bagasse

The effect of several carbon sources on the production of mycelial-bound beta-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated beta-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The beta-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50 degrees C, respectively. The purified enzyme was thermostable up to 60 min in water at 55 degrees C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, o-nitrophenyl-beta-D-galactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-beta-D-fucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude beta-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea beta-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

Properties of a purified thermostable glucoamylase from Aspergillus niveus

A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40A degrees C. The enzyme was purified by DEAE-Fractogel and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400 gel filtration, respectively. The pH optimum was 5.0-5.5, and the enzyme remained stable for at least 2 h in the pH range of 4.0-9.5. The temperature optimum was 65A degrees C and retained 100% activity after 240 min at 60A degrees C. The glucoamylase remained completely active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed, confirming that the enzyme was a glucoamylase (1,4-alpha-d-glucan glucohydrolase). The K (m) was calculated as 0.32 mg ml(-1). Circular dichroism spectroscopy estimated a secondary structure content of 33% alpha-helix, 17% beta-sheet and 50% random structure, which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi, A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications.

Production of beta-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of beta-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of beta-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular beta-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 degrees C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 degrees C. beta-Fructofuranosidase activity was slightly activated by Cu(2+), Mn(2+), Mg(2+), and Na(+) at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K(d) values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively. (C) 2008 Elsevier Ltd. All rights reserved.

Mapping changes in coastline geomorphic features using Landsat TM and ETM+ imagery: examples in southeastern Brazil

Multitemporal Landsat Thematic Mapper (TM) and Enhanced Thematic Mapper Plus (ETM+) imagery was used to assess coastline morphological changes in southeastern Brazil. A spectral linear mixing approach (SLMA) was used to estimate fraction imagery representing amounts of vegetation, clean water (a proxy for shade) and soil. Fraction abundances were related to erosive and depositional features. Shoreline, sandy banks (including emerged and submerged banks) and sand spits were highlighted mainly by clean water and soil fraction imagery. To evaluate changes in the coastline geomorphic features, the fraction imagery generated for each data set was classified in a contextual approach using a segmentation technique and ISOSEG, an unsupervised classification. Evaluation of the classifications was performed visually and by an error matrix relating ground-truth data to classification results. Comparison of the classification results revealed an intense transformation in the coastline, and that erosive and depositional features are extremely dynamic and subject to change in short periods of time.

Subduction of the Nazca Ridge and the Inca Plateau: Insights into the formation of ore deposits in Peru

A large number of ore deposits that formed in the Peruvian Andes during the Miocene (15-5 Ma) are related to the subduction of the Nazea plate beneath the South American plate. Here we show that the spatial and temporal distribution of these deposits correspond with the arrival of relatively buoyant topographic anomalies, namely the Nazca Ridge in central Peru and the now-consumed Inca Plateau in northern Peru, at the subduction zone. Plate reconstruction shows a rapid metallogenic response to the arrival of the topographic anomalies at the subduction trench. This is indicated by clusters of ore deposits situated within the proximity of the laterally migrating zones of ridge subduction. It is accordingly suggested that tectonic changes associated with impingement of the aseismic ridge into the subduction zone may trigger the formation of ore deposits in metallogenically fertile suprasubduction environments. (c) 2005 Elsevier B.V All rights reserved.

Coronary Artery Stenoses: Accuracy of 64-Detector Row CT Angiography in Segments with Mild, Moderate, or Severe Calcification-A Subanalysis of the CORE-64 Trial

Purpose: To evaluate the influence of cross-sectional arc calcification on the diagnostic accuracy of computed tomography (CT) angiography compared with conventional coronary angiography for the detection of obstructive coronary artery disease (CAD). Materials and Methods: Institutional Review Board approval and written informed consent were obtained from all centers and participants for this HIPAA-compliant study. Overall, 4511 segments from 371 symptomatic patients (279 men, 92 women; median age, 61 years [interquartile range, 53-67 years]) with clinical suspicion of CAD from the CORE-64 multi-center study were included in the analysis. Two independent blinded observers evaluated the percentage of diameter stenosis and the circumferential extent of calcium (arc calcium). The accuracy of quantitative multidetector CT angiography to depict substantial (>50%) stenoses was assessed by using quantitative coronary angiography (QCA). Cross-sectional arc calcium was rated on a segment level as follows: noncalcified or mild (<90 degrees), moderate (90 degrees-180 degrees), or severe (>180 degrees) calcification. Univariable and multivariable logistic regression, receiver operation characteristic curve, and clustering methods were used for statistical analyses. Results: A total of 1099 segments had mild calcification, 503 had moderate calcification, 338 had severe calcification, and 2571 segments were noncalcified. Calcified segments were highly associated (P < .001) with disagreement between CTA and QCA in multivariable analysis after controlling for sex, age, heart rate, and image quality. The prevalence of CAD was 5.4% in noncalcified segments, 15.0% in mildly calcified segments, 27.0% in moderately calcified segments, and 43.0% in severely calcified segments. A significant difference was found in area under the receiver operating characteristic curves (noncalcified: 0.86, mildly calcified: 0.85, moderately calcified: 0.82, severely calcified: 0.81; P < .05). Conclusion: In a symptomatic patient population, segment-based coronary artery calcification significantly decreased agreement between multidetector CT angiography and QCA to detect a coronary stenosis of at least 50%.

Biomechanical failure properties and microstructural content of ruptured and unruptured abdominal aortic aneurysms

Purpose: To test the hypothesis that ruptured abdominal aortic aneurysms (AAA) are globally weaker than unruptured ones. Methods: Four ruptured and seven unruptured AAA specimens were harvested whole from fresh cadavers during autopsies performed over an 18-month period. Multiple regionally distributed longitudinally oriented rectangular strips were cut from each AAA specimen for a total of 77 specimen strips. Strips were subjected to uniaxial extension until failure. Sections from approximately the strongest and weakest specimen strips were studied histologically and histochemically. From the load-extension data, failure tension, failure stress and failure strain were calculated. Rupture site characteristics such as location, arc length of rupture and orientation of rupture were also documented. Results: The failure tension, a measure of the tissue mechanical caliber was remarkably similar between ruptured and unruptured AAA (group mean +/- standard deviation of within-subject means: 11.2 +/- 2.3 versus 11.6 +/- 3.6 N/cin; p=0.866 by mixed model ANOVA). In post-hoc analysis, there was little difference between the groups in other measures of tissue mechanical caliber as well such as failure stress (95 +/- 28 versus 98 +/- 23 N/cm(2); p=0.870), failure strain (0.39 +/- 0.09 versus 0.36 +/- 0.09; p=0.705), wall thickness (1.7 +/- 0.4 versus 1.5 +/- 0.4 mm; p=0.470), and % coverage of collagen within tissue cross section (49.6 +/- 12.9% versus 60.8 +/- 9.6%; p=0.133). In the four ruptured AAA, primary rupture sites were on the lateral quadrants (two on left; one on left-posterior; one on right). Remarkably, all rupture lines had a longitudinal orientation and ranged from 1 to 6 cm in length. Conclusion: The findings are not consistent with the hypothesis that ruptured aortic aneurysms are globally weaker than unruptured ones. (C) 2011 Elsevier Ltd. All rights reserved.

Hydraulic tracer studies in a pilot scale subsurface flow constructed wetland

Current design procedures for Subsurface Flow (SSF) Wetlands are based on the simplifying assumptions of plug flow and first order decay of pollutants. These design procedures do yield functional wetlands but result in over-design and inadequate descriptions of the pollutant removal mechanisms which occur within them. Even though these deficiencies are often noted, few authors have attempted to improve modelling of either flow or pollutant removal in such systems. Consequently the Oxley Creek Wetland, a pilot scale SSF wetland designed to enable rigorous monitoring, has recently been constructed in Brisbane, Australia. Tracer studies have been carried out in order to determine the hydraulics of this wetland prior to commissioning it with sealed sewage. The tracer studies will continue during the wetland's commissioning and operational phases. These studies will improve our understanding of the hydraulics of newly built SSF wetlands and the changes brought on by operational factors such as biological films and wetland plant root structures. Results to date indicate that the flow through the gravel beds is not uniform and cannot be adequately modelled by a single parameter, plug flow with dispersion, model. We have developed a multiparameter model, incorporating four plug flow reactors, which provides a better approximation of our experimental data. With further development this model will allow improvements to current SSF wetland design procedures and operational strategies, and will underpin investigations into the pollutant removal mechanisms at the Oxley Creek Wetland. (C) 1997 IAWQ. Published by Elsevier Science Ltd.

Direct visualisation of B-1 inhomogeneity by flip angle dependency

Inhomogeneities in the spatial distribution of the excitatory Radio Frequency (RF) field, are still a dominant source of artifacts and loss of signal to noise ratio in MR imaging experiments, A number of strategies have been proposed to quantify this distribution, However, in this technical note we present a relatively simple MR imaging procedure which can be used to visualise RF inhomogeneities directly either by means of the magnitude or the phase of an image. To visualise the RF field distribution in both the inner and outer volumes of the coil, we have performed experiments in which the entire coil is submerged in a non-conducting fluid, To the best of our knowledge this strategy has not been used previously in order to evaluate coil performance, Finally, we demonstrate that the method is sensitive enough to reveal the effects of the sample properties on the effective RF wavelength of the transmitted field. (C) 1997 Elsevier Science Inc.

Spatial learning ability of rats following differing levels of exposure to alcohol during early postnatal life

Rats exposed to a relatively high dose (7.5 g/kg body weight) of alcohol on either the fifth or tenth postnatal day of age have been reported to have long-lasting deficits in spatial learning ability as tested on the Morris water maze task. The question arises concerning the level of alcohol required to achieve this effect. Wistar rats were exposed to either 2, 4 or 6 g/kg body weight of ethanol administered as a 10% solution. This ethanol was given over an 8-h period on the fifth postnatal day of age by means of an intragastric cannula. Gastrostomy controls received a 5% sucrose solution substituted isocalorically for the ethanol. Another set of pups raised by their mother were used as suckle controls. All surgical procedures were carried out under halothane vapour anaesthesia. After the artificial feeding regimes all pups were returned to lactating dams and weaned at 21 days of age. The spatial learning ability of these rats was tested in the Morris water maze when they were between 61-64 days of age. This task requires the rats to swim in a pool containing water made opaque and locate and climb onto a submerged platform. The time taken to accomplish this is known as the escape latency. Each rat was subjected to 24 trials over 3 days of the test period. Statistical analysis of the escape latency data revealed that the rats given 6 g/kg body weight of ethanol had significant deficits in their spatial learning ability compared with their control groups. However, there was no significant difference in spatial learning ability for the rats given either 2 or 4 g/kg body weight of ethanol compared with their respective gastrostomy or suckle control animals. We concluded that ethanol exposure greater than 4 g/kg over an 8-h period to 5-day-old rats is required for them to develop long-term deficits in spatial learning behaviour. (C) 1998 Elsevier Science Inc.

Circulating levels of brain-derived neurotrophic factor: correlation with mood, cognition and motor function

Brain-derived neurotrophic factor (BDNF) is the most widely distributed neurotrophin in the CNS, where it plays several pivotal roles in synaptic plasticity and neuronal survival. As a consequence, BDNF has become a key target in the physiopathology of several neurological and psychiatric diseases. Recent studies have consistently reported altered levels of BDNF in the circulation (i.e., serum or plasma) of patients with major depression, bipolar disorder, Alzheimer`s disease, Huntington`s disease and Parkinson`s disease. Correlations between serum BDNF levels and affective, cognitive and motor symptoms have also been described. BDNF appears to be an unspecific biomarker of neuropsychiatric disorders characterized by neurodegenerative changes.

The role of integrated information acquisition and management in the analysis of coastal ecosystem change

This book chapter represents a synthesis of the work which started in my PhD and which has been the conceptual basis for all of my research since 1993. The chapter presents a method for scientists and managers to use for selecting the type of remotely sensed data to use to meet their information needs associated with a mapping, monitoring or modelling application. The work draws on results from several of my ARC projects, CRC Rainforest and Coastal projects and theses of P.Scarth , K.Joyce and C.Roelfsema.

Hypophagia induced by glucocorticoid deficiency is associated with an increased activation of satiety-related responses

Uchoa ET, Sabino HA, Ruginsk SG, Antunes-Rodrigues J, Elias LL. Hypophagia induced by glucocorticoid deficiency is associated with an increased activation of satiety-related responses. J Appl Physiol 106: 596-604, 2009. First published November 20, 2008; doi: 10.1152/japplphysiol.90865.2008.-Glucocorticoids have major effects on food intake, demonstrated by the decrease of food intake following adrenalectomy. Satiety signals are relayed to the nucleus of the solitary tract (NTS), which has reciprocal projections with the arcuate nucleus (ARC) and paraventricular nucleus (PVN) of the hypothalamus. We evaluated the effects of glucocorticoids on the activation of hypothalamic and NTS neurons induced by food intake in rats subjected to adrenalectomy (ADX) or sham surgery 7 days before the experiments. One-half of ADX animals received corticosterone (ADX + B) in the drinking water (B: 25 mg/l). Fos/tyrosine hydroxylase (TH), Fos/corticotrophin-releasing factor (CRF) and Fos immunoreactivity were assessed in the NTS, PVN, and ARC, respectively. Food intake and body weight were reduced in the ADX group compared with sham and ADX + B groups. Fos and Fos/TH in the NTS, Fos, and Fos/CRF immunoreactive neurons in the PVN and Fos in the ARC were increased after refeeding, with higher number in the ADX group, compared with sham and ADX + B groups. CCK administration showed no hypophagic effect on ADX group despite a similar increase of Fos/TH immunoreactive neurons in the NTS compared with sham and ADX + B groups, suggesting that CCK alone cannot further increase the anorexigenic effect induced by glucocorticoid deficiency. The present data indicate that glucocorticoid withdrawal reduced food intake, which was associated with higher activation of ARC, CRF neurons of the PVN, and catecholaminergic neurons of the NTS. In the absence of glucocorticoids, satiety signals elicited during a meal lead to an augmented activation of brain stem and hypothalamic pathways.

Proposal for a Trigonometric Method to Evaluate the Abduction Angle of the Lower Limbs in Neonates

It is difficult to precisely measure articular arc movement in newborns using a goniometer. This article proposes an objective method based on trigonometry for the evaluation of lower limb abduction. With the newborn aligned in the dorsal decubitus position, 2 points are marked at the level of the medial malleolus, one on the sagittal line and the other at the end of the abduction. Using the right-sided line between these 2 points and a line from the medial malleolus to the reference point at the anterior superior iliac spine or umbilical scar, an isosceles triangle is drawn, and half of the inferential abduction angle is obtained by calculating the sine. Twenty healthy full-term newborns comprise the study cohort. Intersubject and intrasubject variability among the abduction angle values (mean [SD], 37 degrees [4]degrees) is low. This method is advantageous because the measurement is precise and because the sine can be used without approximation.

CHARACTERIZATION OF Kiss1 NEURONS USING TRANSGENIC MOUSE MODELS

Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptor (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or beta-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Me lanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed melanocortin receptors type 4 (MC4R). Specifically in the preoptic area, 18-28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.