Identification of products formed by a fructan:fructan fructosyltransferase activity from Lolium rigidum

The products formed by a fructan:fructan fructosyltransferase (FFT) activity purified from Lolium rigidum Gaudin were identified after gas chromatography-mass spectrometry of partially methylated alditol acetates, electrospray ionization-mass spectrometry and reversed-phase high-performance liquid chromatography. The FFT activity synthesized oligofructans up to degree of polymerization (DP) 6, but did not synthesize fructans of DP > 6 even when assayed with (1,1,1)-kestopentaose for up to 10 h. The FFT activity when assayed with 1-kestose or 6(G)-kestose synthesized fructan with fructosyl residues almost exclusively linked by beta-2,1-glycosidic linkages. When assayed with 1-kestose, the FFT activity synthesized tetrasaccharides and pentasaccharides with an internal glucosyl residue. The predominant tetrasaccharide was (1&6(G))-kestotetraose and the predominant pentasaccharide was (1&6(G),1)-kestopentaose. By comparison, tetrasaccharides and pentasaccharides extracted from L. rigidum also contained predominantly beta-2,1-glycosidic linked fructans with an internal glucosyl residue. The only exception was that one of the pentasaccharides contained beta-2,1- and beta-2,6-glycosidic linked fructosyl residues. This pentasaccharide was not synthesized by the FFT activity. The role of this FFT activity in formation of oligofructans in L. rigidum is discussed.

Comparative Analysis of the Performance of Various Crystalloid Cardioplegic Solutions on Myocardial Protection After Prolonged Cold Ischemia

Introduction. The quality and effectiveness of myocardial protection are fundamental problems to expand the use of and consequently good outcomes of donated hearts for transplantation. Objective. The purpose of this investigation was to compare the cardioprotective effects of Krebs-Henseleit, Bretschneider-HTK, St Thomas, and Celsior solutions using a modified nonrecirculating Langendorff column model of isolated perfused rat heart during prolonged cold storage. Materials and Methods. After removal 36 rat hearts underwent isolated perfusion into a Langendorff apparatus using Krebs-Henseleit solution for a 15-minute period of recovery; we excluded organs that did not maintain an aortic pressure above 100 m Hg. Subsequently, we equally distributed the hearts into four groups according to the cardioprotection solution; group 1, Krebs-Henseleit (control); group II, Bretschneider-HTK; group III, St Thomas; and group IV, Celsior. Each heart received the specific cardioplegic solution at 10 C for 2-hour storage at 20 C, before a 15 minutes perfusion with Krebs-Henseleit solution for recovery and stabilization. After 60 additional minutes of perfusion, every 5 minutes we determined heart rate (HR), coronary flow (CF), left ventricular systolic pressure (LVSP), and positive and negative peak of the first derivative of left ventricular pressure (+dP/dt and dP/dt, respectively). Results. Comparative analysis by Turkey`s test showed the following performances among the groups at 60 minutes of reperfusion: HR: II = IV > III > I; CF: II = IV > I = III; LVSP: IV > I = II = III; +dP/dt: IV > I = II = III; and dP/dt: IV = II > I = II. Conclusion. Cardioprotective solutions generally used in clinical practice are not able to avoid hemodynamic alterations in hearts exposed to prolonged ischemia. Celsior solution showed better performance than Bretschneider-HTK, St Thomas, and Krebs-Henseleit.

Synthesis of fructans by partially purified fructosyltransferase activities from Lolium rigidum

Sucrose:sucrose fructosyltransferase (SST) activity was partially purified from whole shoots of Lolium rigidum by a combination of affinity chromatography, gel filtration and anion-exchange chromatography. The SST activity co-eluted with some fructan:fructan fructosyltransferase (FFT) and invertase activities and consequently the partially purified preparation was termed the fructosyltransferase (FT) preparation. The SST-like activity in the FT preparation was purified 214-fold and had an apparent molecular mass of 84 000. The FT preparation contained several peptides with an apparent pI of 4.6-4.7. When assayed with sucrose concentrations up to 600 mM, the FT preparation synthesized 1-kestose at all concentrations, and synthesized 6-kestose at concentrations of 150 mM and greater. The K-m of 1-kestose production was 0.2 M. When the FT preparation was assayed at a concentration of activity approximately half that measured in fresh tissue with 100 mM sucrose, 1-kestose, or 6(G)-kestose as substrates, fructans of degree of polymerization (DP) less than or equal to 5 were synthesized. A partially purified FFT activity, free of SST and invertase activities, which synthesized beta-2,1-glycosidic linked oligofructans of DP less than or equal to 6, was combined in vitro with the FT preparation (FFT-FT preparation) to give a ratio of SST:FFT activities similar to that measured in crude enzyme extracts from L. rigidum. The FFT-FT preparation synthesized oligofructans when assayed with 100 mM concentrations of sucrose, 1-kestose or 6(G)-kestose, but was not able to synthesize fructans of DP greater than or equal to 6 even after extended assays of up to 10 h. The FFT-FT preparation was also assayed with 100 mM sucrose with small amounts of concentrated sucrose added periodically during the assay to maintain the substrate concentration. In this assay, the FFT-FT preparation synthesized fructans up to an apparent DP of 17 or greater. The fructans of DP greater than or equal to 6 synthesized in the assay appeared to form two molecular series containing both beta-2,1- and beta-2,6-glycosidic linked fructosyl residues with terminal or internal glucosyl residues. The apparent rate of SST activity in the assay of the FFT-FT preparation was greater than that measured in a similar assay of the FT preparation alone which did not result in fructans with DP greater than or equal to 6. It was concluded that the FFT-FT preparation, when assayed with a continual supply of sucrose, contained a factor which promoted synthesis of fructans of DP greater than or equal to 6 and synthesis of beta-2,B-glycosidic linkages between fructosyl residues.

Immunoendocrinology of the Thymus in Chagas Disease

During immune response to infectious agents, the host develops an inflammatory response which could fail to eliminate the pathogen or may become dysregulated. In this case, the ongoing response acquires a new status and turns out to be detrimental. The same elements taking part in the establishment and regulation of the inflammatory response (cytokines, chemokines, regulatory T cells and counteracting compounds like glucocorticoids) may also mediate harmful effects. Thymic disturbances seen during Trypanosoma cruzi (T. cruzi) infection fit well with this conceptual framework. After infection, this organ suffers a severe atrophy due to apoptosis-induced thymocyte exhaustion, mainly affecting the immature double-positive (DP) CD4+CD8+ population. Thymus cellularity depletion, which occurs in the absence of main immunological mediators involved in anti-T. cruzi defense, seems to be linked to a systemic cytokine/hormonal imbalance, involving a dysregulated increase in Tumor Necrosis Factor alpha (TNF-alpha) and corticosterone hormone levels. Additionally, we have found an anomalous exit of potentially autoimmune DP cells to the periphery, in parallel to a shrinkage in the compartment of natural regulatory T cells. In this context, our data clearly point to the view that the thymus is a target organ of T. cruzi infection. Preserved thymus may be essential for the development of an effective immune response against T. cruzi, but this organ is severely affected by a dysregulated circuit of proinflammatory cytokines and glucocorticoids. Also, the alterations observed in the DP population might have potential implications for the autoimmune component of human Chagas disease. Copyright (C) 2011 S. Karger AG, Basel

Evaluation of the respiratory muscle strength of cirrhotic patients: Relationship with Child-Turcotte-Pugh scoring system

Pulmonary abnormalities are observed in chronic hepatopathy. The measurement of the maximum inspiratory and expiratory pressure may evaluate lung function and the risks associated with hepatic transplantation. Thus, the present work sought to evaluate the respiratory muscle strength of 29 patients between 17 and 63 years old who were enrolled for liver transplantation. The patients were classified according to Child-Turcotte-Pugh score as A, B, or C, and also according to a physiotherapeutic evaluation, which included measurement of respiratory muscle strength by means of a digital manovactrometer, which determines the maximum inspiratory pressure (MaxIP) and the maximum expiratory pressure (MaxEP). The tests were performed with seated individuals having their nostrils obstructed by a nasal clip. The MaxIP was measured during the effort initiated in the residual volume, whereas the MaxEP was measured during the effort initiated in the total pulmonary capacity, keeping pressures stable for at least 1 second. The statistical analysis was performed through using the Mann-Whitney test with a 5% level of significance. The MaxIP values of Child A 95.5 +/- 40.507 cm H2O (average +/- DP) and Child B 87.2 +/- 35.02 patients were higher than those for Child C patients (34.83 +/- 3.68; P <.05). Similar results were observed for the MaxEP of Child A and B groups (116.25 +/- 31.98 and 97.28 +/- 31.08, respectively; P <.05), versus the Child C group (48.16 +/- 22.60). Between groups A and B, the MaxEP were similar (P >.05). We concluded that Child C patients display muscle weakness significantly greater than that of subjects classified as Child A or B.

Matrix Metalloproteinase Inhibition Improves Cardiac Dysfunction and Remodeling in 2-Kidney, 1-Clip Hypertension

Background: Enhanced cardiac matrix metalloproteinase activity (MMPs) has been associated with ventricular remodeling and cardiac dysfunction. It is unknown whether MMPs contribute to systolic/diastolic dysfunction and compensatory remodeling in 2-kidney, 1-clip (2K1C) hypertensive rats. To test this hypothesis, we used 2K1C rats after 2 weeks of surgery treated or not with a nonspecific inhibitor of MMPs (doxycycline). Methods and Results: We found that blood pressure and +/-dP/dt increased in 2K1C rats compared with sham groups, and these parameters were attenuated by doxycycline treatment (P < .05). Doxycycline also reversed cardiac hypertrophy observed in 2K1C rats (P < .05). Hypertensive rats showed increased MMP-2 levels in zymograms and in the tissue by immunofluorescence (P < .05) compared with sham groups. Increased total gelatinolytic activity was observed in untreated 2K1C rats when compared with sham groups (P < .05). Doxycycline decreased total gelatinolytic activity in 2K1C rats to control levels (P < .05). Conclusion: An imbalance in gelatinolytic activity, with increased MMP-2 levels and activity underlies the development of morphological and functional alterations found in the compensatory hypertrophy observed in 2K1C hearts. Because function and structure were restored by doxycycline, the inhibition of MMPs or their modulation may provide beneficial effects for therapeutic intervention in cardiac hypertrophy. (J Cardiac Fail 2010;16:599-608)

A randomized controlled trial of homeopathic treatment of weaned piglets in a commercial swine herd

Aim: To evaluate the zootechnic performance and occurrence of diarrhea in piglets in the week post-weaning comparing supplementation with sucrose saline which contained or did not contain added homeopathic medicine. Method. Animals were randomly divided into three groups of 24 piglets each. The control group did not receive any treatment. Another group received sucrose saline, and the third group received sucrose saline with homeopathic medicine added, in the period of zero to seven days post-weaning. The homeopathic treatment consisted of Echinacea angustifolia, Avena sativa, Ignatia amara, Calcarea carbonica, all 6cH. Piglets were weighed daily for weight gain or loss, and observed for diarrhea and feed intake. Results: Animals receiving sucrose saline alone and sucrose saline with homeopathy had less weight loss than control (p = 0.017, p = 0.0001 respectively). There was no statistical difference in relation to overall incidence of diarrhea or food consumption. These data suggest that the supplementation with sucrose saline with added homeopathic medicine in the first seven days post-weaning may be an useful option to reduce weight loss in weaned piglets. Homeopathy (2008) 97, 202-205.

Infiltration effects on sediment mobility under waves

Sediment mobility measurements with a horizontal sand bed under non-breaking waves are reported. Conditions include no seepage and steady downward seepage corresponding to head gradients up to 2.5. The results indicate that infiltration tends to inhibit sediment mobility for a horizontal bcd of 0.2 mm quartz sand exposed to moderated wave induced bed shear stresses. The effect is weak for the parameter range of the present study. The two opposing effects of shear stress increase due to boundary layer thinning and the stabilizing downward drag are successfully accounted for through the modified Shields parameter of Nielsen [Nielsen, P., 1997. Coastal groundwater dynamics. Proc. Coastal Dynamics '97, Plymouth, ASCE, Dp, 546-555] using coefficients derived from independent studies. That is, from the shear stress experiments of Conley [Conley, D.C., 1993. Ventilated oscillatory boundary layers. PhD Thesis, University of California, San Diego, 74 pp.] and the slope stability experiments of Martin and Aral [Martin, C.S. and M.M. Aral, 1971. Seepage force on interfacial bed particles. J. Hydraulics Div., proc. ASCE, Vol. 97, No. Hy7, pp. 1081-1100]. (C) 2001 Elsevier Science B.V. All rights reserved.

HLA class II antigens positively and negatively associated with hepatosplenic schistosomiasis in a Chinese population

To identify possible associations between host genetic factors and the onset of liver fibrosis following Schistosoma japonicum infection, the major histocompatibility class II alleles of 84 individuals living on an island (Jishan) endemic for schistosomiasis japonica in the Poyang Lake Region of Southern China were determined. Forty patients exhibiting advanced schistosomiasis, characterised by extensive liver fibrosis, and 44 age and sex-matched control subjects were assessed for the class II haplotypes HLA-DRBI and HLA-DQB1. Two HLA-DRB1 alleles, HLA-DRB1*0901 (P = 0.012) and *1302 (P = 0.039), and two HLA-DQB1 alleles, HLA-DQB1*0303 (P = 0.012) and *0609 (P = 0.037), were found to be significantly associated with susceptibility to fibrosis. These associated DRB1 and DQB1 alleles are in very strong linkage disequilibrium, with DRB1*0901-DQB1*0303 and DRB1*1302-DQB1*0609 found as: common haplotypes in this population. In contrast, the alleles HLA-DRB1*1501 (P = 0.025) and HLA-DQB 1*0601 (P = 0.022) were found to be associated with resistance to hepatosplenic disease. Moreover, the alleles DQB1*0303 and DRB1*0901 did not increase susceptibility in the presence of DQB1*0601, indicating that DQB1*0601 is dominant over DQB1*0303 and DRB1*0901. The study has thus identified both positive and negative associations between HLA class II alleles and the risk of individuals developing moderate to severe liver fibrosis following schistosome infection. (C) 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Macrocycles mimic the extended peptide conformation recognized by aspartic, serine, cysteine and metallo proteases

It has been previously demonstrated that aspartic, serine, metallo and cysteine proteases bind to their inhibitors and substrate analogues in a single conformation, the saw-tooth or extended beta-strand. Consequently a generic approach to the development of protease inhibitors is the use of constraints that conformationally restrict putative inhibitor molecules to an extended form. In this way the inhibitor is pre-organized for binding to a protease and does not need to rearrange its structure. One constraining device that has proven to be effective for such pre-organization is macrocyclization. This article illustrates the general principle that macrocycles, especially those composed of 3-4 amino acids and usually 13-17 ring atoms, can effectively mimic the extended conformation of short peptide sequences. Such structure-stabilising macrocycles are stable to degradation by proteases, valuable components of potent protease inhibitors, and in many cases they are also bioavailable.

Characterization of two HKT1 homologues from Eucalyptus camaldulensis that display intrinsic osmosensing capability

Plants have multiple potassium (K+) uptake and efflux mechanisms that are expressed throughout plant tissues to fulfill different physiological functions. Several different classes of K+ channels and carriers have been identified at the molecular level in plants. K+ transporters of the HKT1 superfamily have been cloned from wheat (Triticum aestivum), Arabidopsis, and Eucalyptus camaldulensis. The functional characteristics as well as the primary structure of these transporters are diverse with orthologues found in bacterial and fungal genomes. In this report, we provide a detailed characterization of the functional characteristics, as expressed in Xenopus laevis oocytes, of two cDNAs isolated from E. camaldulensis that encode proteins belonging to the HKT1 superfamily of K+/Na+ transporters. The transport of K+ in EcHKT-expressing oocytes is enhanced by Na+, but K+ was also transported in the absence of Na+. Na+ is transported in the absence of K+ as has been demonstrated for HKT1 and AtHKT1. Overall, the E. camaldulensis transporters show some similarities and differences in ionic selectivity to HKT1 and AtHKT1. One striking difference between HKT1 and EcHKT is the sensitivity to changes in the external osmolarity of the solution. Hypotonic solutions increased EcHKT induced currents in oocytes by 100% as compared with no increased current in HKT1 expressing or uninjected oocytes. These osmotically sensitive currents were not enhanced by voltage and may mediate water flux. The physiological function of these osmotically induced increases in currents may be related to the ecological niches that E. camaldulensis inhabits, which are periodically flooded. Therefore, the osmosensing function of EcHKT may provide this species with a competitive advantage in maintaining K+ homeostasis under certain conditions.

Reversal of cardiovascular remodelling with candesart

Cardiovascular remodelling, defined as ventricular and vascular hypertrophy together with fibrosis, characterises hypertension following inhibition of the production of the endogenous vasodilator, nitric oxide (NO). This study has determined whether the cardiovascular remodelling following chronic NO synthase inhibition can e reversed by administration of the selective angiotensin II AT(1)-receptor antagonist, candesartan. Male Wistar rats were treated with L-nitroarginine methyl ester (L-NAME, 400 mg/l in drinking water) for eight weeks and with candesartan cilexetil (2 mg/kg/day by oral gavage) for the last four weeks. L-NAME-treated rats became hypertensive with systolic blood pressure increasing from 110 +/- 4 mmHg (control) to 170 +/- 10 mmHg. Rats developed left ventricular hypertrophy (control 1.70 +/- 0.06; L-NAME 2.10 +/- 0.04 mg/kg body wt) with markedly increased deposition of perivascular and interstitial collagen. Candesartan returned blood pressure, left ventricular weights and collagen deposition to control values. Echo cardiographic assessment showed concentric hypertrophy with an increased fractional shortening; this was reversed by candesartan treatment. Heart failure was not evident. In the isolated Langendorff heart, diastolic stiffness increased in L-NAME-treated rats while the rate of increase in pressure (+dP/dt) increased after eight weeks only; candesartan reduced collagen deposition and normalised +dP/dt. In isolated left ventricular papillary muscles, the potency (negative log EC50) of noradrenaline as a positive inotropic compound was unchanged, (control 6.56 +/- 0.14); maximal increase in force before ectopic beats was reduced from 5.0 +/- 0.4 mN to 2.0 +/- 0.2 mN. Noradrenaline potency as a vasoconstrictor in thoracic aortic rings was unchanged, but maximal contraction was markedly reduced from 25.2 +/- 2.0 mN to 3.0 +/- 0.3 mN; this was partially reversed by candesartan treatment. Thus, chronic inhibition of NO production with L-NAME induces hypertension, hypertrophy and fibrosis with increased toxicity and significant decreases in vascular responses to noradrenaline. These changes were at least partially reversible by treatment with candesartan, implying a significant role of AT(1)-receptors in L-NAME-induced cardiovascular changes.

Reversal of cardiac and renal fibrosis by pirfenidone and spironolactone in streptozotocin-diabetic rats

1 Fibrosis leads to chronic impairment of cardiac and renal function and thus reversal of existing fibrosis may improve function and survival. This project has determined whether pirfenidone, a new antifibrotic compound, and spironolactone, an aldosterone antagonist, reverse both deposition of the major extracellular matrix proteins, collagen and fibronectin, and functional changes in the streptozotocin(STZ)-diabetic rat. 2 Streptozotocin (65 mg kg(-1) i.v.)-treated rats given pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone; approximately-200 mg kg(-1) day(-1) as 0.2-2g l(-1) drinking water) or spironolactone (50 mg kg(-1) day(-1) s.c.) for 4 weeks starting 4 weeks after STZ showed no attenuation of the increased blood glucose concentrations and increased food and water intakes which characterize diabetes in this model. 3 STZ-treatment increased perivascular and interstitial collagen deposition in the left ventricle and kidney, and surrounding the aorta. Cardiac, renal and plasma fibronectin concentrations increased in STZ-diabetic rats. Passive diastolic stiffness increased in isolated hearts from STZ-diabetic rats. Both pirfenidone and spironolactone treatment attenuated these increases without normalizing the decreased + dP/dt(max) of STZ-diabetic hearts. 4 Left ventricular papillary muscles from STZ-treated rats showed decreased maximal positive inotropic responses to noradrenaline, EMD 57033 (calcium sensitizer) and calcium chloride; this was not reversed by pirfenidone or spironolactone treatment. STZ-treatment transiently decreased GFR and urine flow rates in isolated perfused kidneys; pirfenidone but not spironolactone prevented the return to control values. 5 Thus, short-term pirfenidone and spironolactone treatment reversed cardiac and renal fibrosis and attenuated the increased diastolic stiffness without normalizing cardiac contractility or renal function in STZ-diabetic rats.

Familial Paget's disease of bone: Nonlinkage to the PDB1 and PDB2 loci on chromosomes 6p and 18q in a large pedigree

Paget's disease of bone is a common condition characterized by bone pain, deformity, pathological fracture, and an increased incidence of osteosarcoma. Genetic factors play a role in the pathogenesis of Paget's disease but the molecular basis remains largely unknown. Susceptibility loci for Paget's disease of bone have been mapped to chromosome 6p21.3 (PDB1) and 18q121.1-q22 (PDB2) in different pedigrees, We have identified a large pedigree of over 250 individuals with 49 informative individuals affected with Paget's disease of bone; 31 of whom are available for genotypic analysis. The disease is inherited as an autosomal dominant trait in the pedigree with high penetrance by the sixth decade. Linkage analysis has been performed with markers at PDB1; these data show significant exclusion of linkage with log,, of the odds ratio (LOD) scores < -2 in this region. Linkage analysis of microsatellite markers from the PDB2 region has excluded linkage with this region, with a 30 cM exclusion region (LOD score < -2.0) centered on D18S42, These data confirm the genetic heterogeneity of Paget's disease of bone. Our hypothesis is that a novel susceptibility gene relevant to the pathogenesis of Paget's disease of bone lies elsewhere in the genome in the affected members of this pedigree and will be identified using a microsatellite genomewide scan followed by positional cloning.