Mango starch degradation. II. The binding of alpha-amylase and beta-amylase to the starch granule

During mango ripening, soluble sugars that account for mango sweetening are accumulated through carbon supplied by both photosynthesis and starch degradation. The cultivar Keitt has a characteristic dependence on sugar accumulation during starch degradation, which takes place during ripening, only a few days after detachment from the tree. Most knowledge about starch degradation is based on seeds and leaves currently used as models. However, information about the mango fruit is scarce. This work presents the evaluation of alpha- and beta-amylases in the starch granule surface during fruit development and ripening. Extractable proteins were assayed for amylase activity and detected by immunofluorescence microscopy and correlated to gene expression. The results suggest that both amylases are involved in starch degradation during mango ripening, probably under the dependence of another signal triggered by the detachment from the mother-plant.

Modulation of mucosal immunity in a murine model of food-induced intestinal inflammation

Background Hypersensitivity or uncontrolled responses against dietary antigens can lead to inflammatory disorders like food allergy and current models reflect a variety of causes but do not reveal the detailed modulation of gut immunity in response to food antigens after breakdown in mucosal tolerance. Objective To develop and characterize a murine model for food-induced intestinal inflammation and to demonstrate the modulation of gut immune response by dietary allergenic antigens. Methods C57BL/6 mice were sensitized with peanut proteins, challenged with peanut seeds and their sera and gut segments were collected for subsequent analyses. Results Sensitization and challenged with peanut seeds led to alterations in gut architecture with inflammatory response characterized by oedema in lamina propria and cell infiltrate composed mainly by eosinophils, mast cells, phagocytes, natural killer and plasma cells, together with low percentage of gamma delta(+) and CD4(+)CD25(+)Foxp3(+) cells in Peyer`s patches. These animals also presented high levels of specific IgE and IgG1 in sera and modulation of mucosal immunity was mediated by increased expression of GATA-3, IL-4, IL-13 and TNF-alpha in contrast to low IFN-gamma in the gut. Conclusion A murine model for food-induced intestinal inflammation was characterized in which modulation of gut immunity occurs by peanut antigens in consequence of T-helper type 2 (Th2) allergic response and failure of regulatory mechanisms necessary for mucosa homeostasis, resembling food allergy. This work shed some light on the understanding of the pathogenesis of gastrointestinal disorders and intolerance in the gut and supports the development of therapies for food-related enteropathies like food allergy, focusing on gut-specific immune response.

Monitoring of protein catabolism in neonates and young infants post-cardiac surgery

Aims: To evaluate cell catabolism by balance of nitrogen and phosphate, and creatinine excretion in children post-cardiac surgery; to establish protein and energy requirements to minimize catabolism; and to assess nutritional therapy by following these parameters and serial anthropometric measurements. Methods: A prospective observational study of children with congenital heart disease undergoing cardiac surgery. Blood samples and 24-h urine collections were obtained postoperatively for creatinine measurement and nitrogen and phosphate balance. Anthropometric measurements (weight, mid-arm muscle circumference and triceps skinfold thickness) were obtained preoperatively and at paediatric intensive care unit and hospital discharge. Results: Eleven children were studied for 3-10 postoperative days. Anabolism was associated with higher protein and energy intakes compared to catabolism (1.1 vs. 0.1 g/kg/day and 54 vs. 17 kcal/kg/day, respectively). On days with anabolism, phosphate balance was greater compared with that on days with catabolism. Daily creatinine excretion did not correlate with protein balance. Anthropometric measurements did not change significantly over time. Conclusions: Children with congenital heart disease undergoing cardiac surgery achieved anabolism with > 55 kcal/kg/day and > 1 g/kg/day of protein. Balance of phosphate was useful to monitor cell breakdown. Anthropometric measurements were not valuable to evaluate nutritional therapy in this population.

NO EVIDENCE OF PATHOLOGICAL AUTOIMMUNITY FOLLOWING MYCOBACTERIUM LEPRAE HEAT-SHOCK PROTEIN 65-DNA VACCINATION IN MICE

Heat-shock proteins (HSPs) are currently one of the most promising targets for the development of immunotherapy against tumours and autoimmune disorders. This protein family has the capacity to activate or modulate the function of different immune system cells. They induce the activation of monocytes, macrophages and dendritic cells, and contribute to cross-priming, an important mechanism of presentation of exogenous antigen in the context of MHC class I molecules, These various immunological properties of HSP have encouraged their use in several clinical trials. Nevertheless, an important issue regarding these proteins is whether the high homology among HSPs across different species may trigger the breakdown of immune tolerance and induce autoimmune diseases. We have developed a DNA vaccine codifying the Mycobacterium leprae Hsp65 (DNAhsp65), which showed to be highly immunogenic and protective against experimental tuberculosis. Here, we address the question of whether DNAhsp65 immunization could induce pathological autoimmunity in mice. Our results show that DNAhsp65 vaccination induced antibodies that can recognize the human Hsp60 but did not induce harmful effects in 16 different organs analysed by histopathology up to 210 days after vaccination. We also showed that anti-DNA antibodies were not elicited after DNA vaccination. The results are important for the development of both HSP and DNA-based immunomodulatory agents.

Isoproterenol induces primary loss of dystrophin in rat hearts: correlation with myocardial injury

The mechanism of isoproterenol-induced myocardial damage is unknown, but a mismatch of oxygen supply vs. demand following coronary hypotension and myocardial hyperactivity is the best explanation for the complex morphological alterations observed. Severe alterations in the structural integrity of the sarcolemma of cardiomyocytes have been demonstrated to be caused by isoproterenol. Taking into account that the sarcolemmal integrity is stabilized by the dystrophin-glycoprotein complex (DGC) that connects actin and laminin in contractile machinery and extracellular matrix and by integrins, this study tests the hypothesis that isoproterenol affects sarcolemmal stability through changes in the DGC and integrins. We found different sensitivity of the DGC and integrin to isoproterenol subcutaneous administration. Immunofluorescent staining revealed that dystrophin is the most sensitive among the structures connecting the actin in the cardiomyocyte cytoskeleton and the extracellular matrix. The sarcomeric actin dissolution occurred after the reduction or loss of dystrophin. Subsequently, after lysis of myofilaments, gamma-sarcoglycan, beta-dystroglycan, beta 1-integrin, and laminin alpha-2 expressions were reduced followed by their breakdown, as epiphenomena of the myocytolytic process. In conclusion, administration of isoproterenol to rats results in primary loss of dystrophin, the most sensitive among the structural proteins that form the DGC that connects the extracellular matrix and the cytoskeleton in cardiomyocyte. These changes, related to ischaemic injury, explain the severe alterations in the structural integrity of the sarcolemma of cardiomyocytes and hence severe and irreversible injury induced by isoproterenol.

Tissue diffusion and retention of metalloproteinases in ascending aortic aneurysms and dissections

Histopathological alterations in human aneurysms and dissections of the thoracic ascending aorta include areas of mucoid degeneration within the medial layer, colocalized with areas of cell disappearance and disruption of extracellular matrix elastic and collagen fibers. We studied the presence of matrix metalloproteinases in relation to their capacity to diffuse through the tissue or to be retained in areas of mucoid degeneration in aneurysms and dissections of the ascending aorta. Ascending aortas from 9 controls, 33 patients with aneurysms, and 14 with acute dissections, all collected at surgery, were analyzed. The morphological aspect was similar whatever the etiology or phenotypic expression of the pathological aortas, involving areas of extracellular matrix breakdown and cell rarefaction associated with mucoid degeneration. Release of proMMP-2, constitutively expressed by smooth muscle cells, was not different between controls and aneurysmal aortas, whereas the aneurysmal aortas released more of the active form. Release of pro and active MMP-9 was also similar between controls and aneurysmal aortas. Immunohistochemical staining of MMP-2 and MMP-9 was weak in both control and pathological aortas. In contrast, released MMP-7 (matrilysin) and MMP-3 (stromelysin-1) could not be detected in conditioned media but were present in tissue extracts with no detectable quantitative difference between controls and pathological aortas. Immunohistochemical staining of MMP-7 and MMP-3 revealed their retention in areas of mucoid degeneration, and semiquantitative evaluation of immunostaining showed more MMP-7 in pathological aortas than in controls. In conclusion, areas of mucoid degeneration, the hallmark of aneurysms, and dissections of thoracic ascending aortas, whatever their etiology, are not inert and can retain specific proteases. (c) 2009 Elsevier Inc. All rights reserved.

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta Aims: Thoracic ascending aortic aneurysms (TAA) are characterized by elastic fibre breakdown and cystic medial degeneration within the aortic media, associated with progressive smooth muscle cell (SMC) rarefaction. The transforming growth factor (TGF)-beta/Smad2 signalling pathway is involved in this process. Because the pericellular fibrinolytic system activation is able to degrade adhesive proteins, activate matrix metalloproteinase (MMP), induce SMC disappearance and increase the bioavailability of TGF-beta, the aim was to investigate the plasminergic system in TAA. Methods and results: Ascending aortas [21 controls and 19 TAAs (of three different aetiologies)] were analysed. Immunohistochemistry showed accumulation of t-PA, u-PA and plasmin in TAAs, associated with residual SMCs. Overexpression of t-PA and u-PA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and zymography on TAA extracts and culture medium conditioned by TAA. Plasminogen was present on the SMC surface and inside cytoplasmic vesicles, but plasminogen mRNA was undetectable in the TAA medial layer. Plasmin-antiplasmin complexes were detected in TAA-conditioned medium and activation of the fibrinolytic system was associated with increased fibronectin turnover. Fibronectin-related material was detected immunohistochamically in dense clumps around SMCs and colocalized with latent TGF-beta binding protein-1. Conclusions: The fibrinolytic pathway could play a critical role in TAA progression, via direct or indirect impact on ECM and consecutive modulation of TGF-beta bioavailability.

Plasminogen Acquisition and Activation at the Surface of Leptospira Species Lead to Fibronectin Degradation

Pathogenic Leptospira species are the etiological agents of leptospirosis, a widespread disease of human and veterinary concern. In this study, we report that Leptospira species are capable of binding plasminogen (PLG) in vitro. The binding to the leptospiral surface was demonstrated by indirect immunofluorescence confocal microscopy with living bacteria. The PLG binding to the bacteria seems to occur via lysine residues because the ligation is inhibited by addition of the lysine analog 6-aminocaproic acid. Exogenously provided urokinase-type PLG activator (uPA) converts surface-bound PLG into enzymatically active plasmin, as evaluated by the reaction with the chromogenic plasmin substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloridein. The PLG activation system on the surface of Leptospira is PLG dose dependent and does not cause injury to the organism, as cellular growth in culture was not impaired. The generation of active plasmin within Leptospira was observed with several nonvirulent high-passage strains and with the nonpathogenic saprophytic organism Leptospira biflexa. Statistically significant higher activation of plasmin was detected with a low-passage infectious strain of Leptospira. Plasmin-coated virulent Leptospira interrogans bacteria were capable of degrading purified extracellular matrix fibronectin. The breakdown of fibronectin was not observed with untreated bacteria. Our data provide for the first time in vitro evidence for the generation of active plasmin on the surface of Leptospira, a step that may contribute to leptospiral invasiveness.

Bovine oocyte vitrification: Effect of ethylene glycol concentrations and meiotic stages

Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0-10, 10-14, and 18-24 h of INK respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 It of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-I or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However, after vitrification in VS-2, blastocyst rates were less at 18 h than 0 h. Both cleavage rates and blastocyst rates were significantly less in all vitrification groups when compared to control group and only control oocytes hatched. In conclusion, both EG concentration and stage of meiotic maturation affect the developmental potential of oocytes after vitrification. (C) 2007 Elsevier B.V. All rights reserved.

Effects of gonadotropin-exposed medium with high concentrations of progesterone and estradiol-17 beta on in vitro maturation of canine oocytes

During the process of maturation in the oviduct, canine oocytes in the germinal vesicle stage are exposed to decreasing levels of estradiol-17 beta and increasing levels of progesterone. However, hormone concentrations in the microenvironments in which they act are higher than serum concentrations. Therefore, the aim of the present study was to compare the meiotic competence of canine oocytes harvested from anestrous bitches in culture medium containing high concentrations (20 mu g ml(-1)) of estradiol-17 beta and/or progesterone in association to gonadotropins (luteinizing hormone and follicle-stimulating hormone) using three different maturation periods (48, 72, and 96 h). Oocytes were cultured in tissue culture medium (TCM-199) and arranged in four experimental groups: group control, group E2 (estradiol-17 beta), group P4 (progesterone), and group E2 + P4. Regardless of the maturation period, groups P4 and E2 + P4 presented statistically higher rate of germinal vesicle breakdown oocytes compared to the group control and group E2. There were no significant differences among groups on germinal vesicle, metaphase I, metaphase II, and degenerated or unidentifiable oocytes rates. The mean percentage of metaphase II oocytes was higher at 96 h when compared to 72 h. Results of the present research indicate no influence of estradiol-17 beta supplementation, unless in association with progesterone. There is an evidence of the positive effect of progesterone on germinal vesicle breakdown. Results also showed that extended periods of in vitro maturation affect positively maturation rates to metaphase II of low competent oocytes harvested from anestrous bitches, independent of the maturation media. In conclusion, high concentrations of steroids, especially progesterone, have positive effect on in vitro oocyte maturation when the oocytes are derived from the anestrous status.

Substance P regulates the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in cultured human gingival fibroblasts

Background and Objective: Substance P may play a role in the pathogenesis of periodontal disease; however, its mechanisms of modulation are not clear. This study evaluated the effect of two concentrations of Substance P on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in cultured human gingival fibroblasts. Materials and Methods: Fibroblasts were stimulated for 48 h with 10(-4) or 10(-9) m Substance P; untreated fibroblasts served as controls. The expression of MMP-1, -2, -3, -7 and -11 and of TIMP-1 and -2 was evaluated using real-time polymerase chain reaction and western blotting. Resulsts: There was a significant, concentration-dependent stimulatory effect of Substance P on MMP-1, -2, -3 and -7 and TIMP-2 gene expression (p < 0.05), and a probable effect on MMP-11 (p = 0.06). At the higher concentration (10(-4) m Substance P), MMP-1, -2, -3, -7 and -11 and TIMP-2 showed the greatest up-regulation; at the lower concentration (10(-9) (M) Substance P), MMP-1, -3 and -7 and TIMP-2 exhibited diminished up-regulation, with MMP-2 and -11 showing down-regulation (p < 0.05). Expression of TIMP-1 was not affected by Substance P (p > 0.05). Western blotting confirmed that Substance P up-regulated MMP-1, -2, -3 and -11 and TIMP-2. MMP-1, -3 and -11 and TIMP-2 showed greater up-regulation at the higher Substance P concentration and diminished up-regulation at the lower concentration. MMP-2 was up-regulated to a similar degree at both Substance P concentrations. Conclusion: In gingival fibroblast cells, Substance P at the higher concentration (10(-4) m) induced greater up-regulation of MMP-1, -3 and -11 and TIMP-2 expression, but at the lower concentration (10(-9) (M)) induced diminished up-regulation, which may represent a mechanism for modulating periodontal breakdown.

Justice strategy options for increased customer satisfaction in a services recovery setting

This study investigates the influence of justice (procedural, interactional and distributive) on measures of customer satisfaction within a hotel setting. Specifically, the study investigates the levels of satisfaction associated with various combinations of procedural, interactional, and distributive justice related service recovery strategies. Using especially designed video vignettes of a hotel service breakdown, respondents rated their levels of satisfaction for the video vignettes that depicted varying levels of. (a) level of concern shown by the service provider, (b) whether policy was adhered to, (c) degree of 'voice' given to the customer, and (d) type of compensation. Between subject MANOVA analyses revealed a number of main effects and interactions. Results clearly show that satisfaction varied significantly depending on the various combinations of recovery measures. In particular, a two-way interaction between adherence to policy and type of compensation was found. Furthermore, it was found that respondents expressed higher satisfaction with the service when a 50% refund was given, and the provider was seen to be adhering to policy, rather than doing a special favor for the customer. In contrast, when a token measure of compensation is given (i.e. giving away a couple of drink vouchers), respondents expressed higher levels of satisfaction if the service provider was doing a special favor rather than merely adhering to company policy. Implications for managers and scholars are discussed. (C) 2001 Elsevier Science Inc. All rights reserved.

Generalized quasi mode theory of macroscopic canonical quantization in cavity quantum electrodynamics and quantum optics I. Theory

The quasi mode theory of macroscopic quantization in quantum optics and cavity QED developed by Dalton, Barnett and Knight is generalized. This generalization allows for cases in which two or more quasi permittivities, along with their associated mode functions, are needed to describe the classical optics device. It brings problems such as reflection and refraction at a dielectric boundary, the linear coupler, and the coupling of two optical cavities within the scope of the theory. For the most part, the results that are obtained here are simple generalizations of those obtained in previous work. However the coupling constants, which are of great importance in applications of the theory, are shown to contain significant additional terms which cannot be 'guessed' from the simpler forms. The expressions for the coupling constants suggest that the critical factor in determining the strength of coupling between a pair of quasi modes is their degree of spatial overlap. In an accompanying paper a fully quantum theoretic derivation of the laws of reflection and refraction at a boundary is given as an illustration of the generalized theory. The quasi mode picture of this process involves the annihilation of a photon travelling in the incident region quasi mode, and the subsequent creation of a photon in either the incident region or transmitted region quasi modes.

Long-term metabolic and skeletal muscle adaptations to short-sprint training: Implications for sprint training and tapering

The adaptations of muscle to sprint training can be separated into metabolic and morphological changes. Enzyme adaptations represent a major metabolic adaptation to sprint training, with the enzymes of all three energy systems showing signs of adaptation to training and some evidence of a return to baseline levels with detraining. Myokinase and creatine phosphokinase have shown small increases as a result of short-sprint training in some studies and elite sprinters appear better able to rapidly breakdown phosphocreatine (PCr) than the sub-elite. No changes in these enzyme levels have been reported as a result of detraining. Similarly, glycolytic enzyme activity (notably lactate dehydrogenase, phosphofructokinase and glycogen phosphorylase) has been shown to increase after training consisting of either long (> 10-second) or short (< 10-second) sprints. Evidence suggests that these enzymes return to pre-training levels after somewhere between 7 weeks and 6 months of detraining. Mitochondrial enzyme activity also increases after sprint training, particularly when long sprints or short recovery between short sprints are used as the training stimulus. Morphological adaptations to sprint training include changes in muscle fibre type, sarcoplasmic reticulum, and fibre cross-sectional area. An appropriate sprint training programme could be expected to induce a shift toward type Ha muscle, increase muscle cross-sectional area and increase the sarcoplasmic reticulum volume to aid release of Ca2+. Training volume and/or frequency of sprint training in excess of what is optimal for an individual, however, will induce a shift toward slower muscle contractile characteristics. In contrast, detraining appears to shift the contractile characteristics towards type IIb, although muscle atrophy is also likely to occur. Muscle conduction velocity appears to be a potential non-invasive method of monitoring contractile changes in response to sprint training and detraining. In summary, adaptation to sprint training is clearly dependent on the duration of sprinting, recovery between repetitions, total volume and frequency of training bouts. These variables have profound effects on the metabolic, structural and performance adaptations from a sprint-training programme and these changes take a considerable period of time to return to baseline after a period of detraining. However, the complexity of the interaction between the aforementioned variables and training adaptation combined with individual differences is clearly disruptive to the transfer of knowledge and advice from laboratory to coach to athlete.

Delineation of sulphide ore-zones by borehole radar tomography at Hellyer Mine, Australia

Velocity and absorption tomograms are the two most common forms of presentation of radar tomographic data. However, mining personnel, geophysicists included, are often unfamiliar with radar velocity and absorption. In this paper, general formulae are introduced, relating velocity and attenuation coefficient to conductivity and dielectric constant. The formulae are valid for lossy media as well as high-resistivity materials. The transformation of velocity and absorption to conductivity and dielectric constant is illustrated via application of the formulae to radar tomograms from the Hellyer zinc-lead-silver mine, Tasmania, Australia. The resulting conductivity and dielectric constant tomograms constructed at Hellyer demonstrated the potential of radar tomography to delineate sulphide ore zones. (C) 2001 Elsevier Science B.V. All rights reserved.