Thermodynamics of glutathione binding to the tyrosine 7 to phenylalanine mutant of glutathione S-transferase from Schistosoma japonicum

The binding of glutathione (GSH) to the tyrosine 7 to phenylalanine mutant of Schistosoma japonicum glutathione S-transferase (SjGST-Y7F) has been studied by isothermal titration calorimetry (ITC). At pH 6.5 and 25 °C this mutant shows a higher affinity for glutathione than wild type enzyme despite an almost complete loss of activity in the presence of 1-chloro-2,4-dinitrobenzene (CDNB) as second substrate. The enthalpy change upon binding of GSH is more negative for the mutant than for the wild type GST (SjGST). Changes in accessible solvent areas (ASA) have been calculated based on enthalpy and heat capacity changes. ASA values indicated the burial of apolar surfaces of protein and ligand upon binding. A more negative ΔC_p value has been obtained for the mutant enzyme, suggesting a more hydrophobic interaction, as may be expected from the change of a tyrosine residue to phenylalanine.     

Aromatic trap analysis of free radicals production in experimental collagen-induced arthritis in the rat: protective effect of glycosaminoglycans treatment

Many findings demonstrated that Glycosaminoglycans (GAGs) and Proteoglycans (PGs) possess antioxidant activity. Collagen-induced arthritis (CIA) is an experimental animal model similar to human rheumatoid arthritis (RA) in which free radicals are involved. Sodium salicylate can be used as a chemical trap for hydroxyl radicals (OH •), the most damaging reactive oxygen species (ROS), yielding 2,5-dihydroxybenzoic acid), (2,5-DHBA) and 2,3-dihydroxybenzoic acid (2,3-DHBA). The measurement of these two acids in the plasma allows to indirectly assess the production of OH • radicals. The aim of the study was to investigate the effect of hyaluronic acid (HYA) (30 mg/kg i.p.) or chondroitin-4-sulphate (C4S) (30 mg/kg i.p.), on free radical production in Lewis rats subjected to CIA. After the immunization with bovine collagen type II in complete Freund's adjuvant, rats developed an erosive hind paw arthritis, that produced high plasma OH • levels assayed as 2,3-DHBA and 2,5-DHBA, primed lipid peroxidation, evaluated by analyzing conjugated dienes (CD) in the articular cartilage; decreased the concentration of endogenous vitamin E (VE) and catalase (CA) in the joint cartilage; enhanced macrophage inflammatory protein-2 (MIP-2) serum levels and increased elastase (ELA) evaluated as an index of activated leukocyte polymophonuclear (PMNs) accumulation in the articular joints. The administration of HYA and C4S starting at the onset of arthritis (day 11) for 20 days, limited inflammation and the clinical signs in the knee and paw, reduced OH • production, decreased CD levels, partially restored the endogenous antioxidants VE and CA, reduced MIP-2 serum levels and limited PMNs infiltration. The results indicate that the GAGs HYA and C4S significantly reduce free radical production in CIA and could be used as a tool to investigate the role of antioxidants in RA.     

A (1→3,6)-β-d-galactosyl epitope containing uronic acids associated with bioactive pectins occurs in discrete cell wall domains in hypocotyl and root tissues of flax seedlings

We have used a well-characterized antibody specific for an epitope consisting of (1→3,6)-β-d-galactosyl residues with terminal glucuronic or 4-O-methylglucuronic acids of a bioactive pectin and immunocytochemistry to investigate its secretion and wall distribution in the hypocotyl and root tissues of flax seedlings. Our results show that this antigenic epitope is associated with flax pectins and is expressed by all the cells of the hypocotyl and root tissues. In the hypocotyl, it is abundant in the primary wall of epidermal cells as well as in the secondary wall of fiber cells, and is relatively less abundant in parenchyma cell walls. In contrast, the epitope is not detected in the middle lamellae and cell junction regions. In the root tip cells, immunogold electron microscopy shows that the cell walls of peripheral, columella, meristematic, cortical, and epidermal cells contain significant amounts of this epitope and that the distribution patterns are distinct. Together, these findings show that the antigenic epitope occurs in discrete domains of the wall implying a strict spatial regulation of the epitope-containing molecules. The results also show that, in root cells, the epitope is present within Golgi cisternae and is predominantly assembled in the trans and the trans-Golgi network compartments. Accepted: 21 October 1999     

Small-scale isolation of high molecular weight DNA from Leishmania braziliensis

In this paper, we report a method for isolation of high molecular weight DNA from Leishmania promastigotes. This technique is especially indicated for small-scale purification of DNA suitable for the construction of highly representative genomic libraries. In our protocol, lysis buffer is compatible with RNase treatment, avoiding an additional precipitation step and consequent shearing of DNA. In order to prove the quality of the DNA isolated by this method, a Leishmania braziliensis genomic library was constructed, and an L. braziliensis KMP-11 gene was cloned after screening the library with a heterologous probe.     

The trophic interactions of young Arctic grayling (Thymallus arcticus) in an Arctic tundra stream

1. Young (0+) Arctic grayling (Thymallus arcticus) have the potential to control the trophic structure of Arctic tundra streams through consumption, nutrient excretion and the modification of prey behaviour. The effect of young grayling on three trophic levels (algae, invertebrates and fish) was investigated by manipulating fish density and by fertilizing the river with phosphorus (P). 2. Nutrients, epilithic chlorophyll a, benthic invertebrates and fish biomass were measured within each fish density treatment (0, 4, and 40 fish m^–2) within the P-limited reference zone and the P-enriched fertilized zone of the Kuparuk River, Alaska. 3. Epilithic chlorophyll a increased with increased fish density in both reference and fertilized zones, while mayfly density decreased with increased fish density in the fertilized zone only. Final mean mass of young grayling in the 40 fish m^–2 cages was lower than mean mass in the 4 fish m^–2 cages. 4. Young grayling may produce a top-down cascading trophic effect in areas where nutrients are not limited. 5. River nutrient status and river discharge may modify the strength of top-down control by young grayling.     

Effects of Blood Transfusion on Exercise Capacity in Thalassemia Major Patients

Anemia has an important role in exercise performance. However, the direct link between rapid changes of hemoglobin and exercise performance is still unknown.To find out more on this topic, we studied 18 beta-thalassemia major patients free of relevant cardiac dysfunction (age 33.5±7.2 years,males = 10). Patients performed a maximal cardiopulmolmonary exercise test (cycloergometer, personalized ramp protocol, breath-by-breath measurements of expired gases) before and the day after blood transfusion (500 cc of red cell concentrates). After blood transfusion, hemoglobin increased from 10.5±0.8 g/dL to 12.1±1.2 (p<0.001), peak VO₂ from 1408 to 1546mL/min (p<0.05), and VO₂ at anaerobic threshold from 965 to 1024mL/min (p<0.05). No major changes were observed as regards heart and respiratory rates either at peak exercise or at anaerobic threshold. Similarly, no relevant changes were observed in ventilation efficiency, as evaluated by the ventilation vs. carbon dioxide production relationship, or in O₂ delivery to the periphery as analyzed by the VO₂ vs. workload relationship. The relationship between hemoglobin and VO₂ changes showed, for each g/dL of hemoglobin increase, a VO₂ increase = 82.5 mL/min and 35 mL/min, at peak exercise and at anaerobic threshold, respectively. In beta-thalassemia major patients, an acute albeit partial anemia correction by blood transfusion determinates a relevant increase of exercise performance, observed both at peak exercise and at anaerobic threshold.     

Hypertriglyceridemia Is Independently Associated with Renal,but Not Retinal Complications in Subjects with Type 2 Diabetes: A Cross-Sectional Analysis of the Renal Insufficiency And Cardiovascular Events (RIACE) Italian Multicenter Study

Objective

Atherogenic dyslipidemia seems to play a major role in microvascular complications and in residual microvascular risk after statin therapy, which reduces triglycerides up to 40%. We assessed whether raised TG levels are associated with an increased burden from microvascular complications in patients with type 2 diabetes.

Methods

Subjects from the Renal Insufficiency And Cardiovascular Events (RIACE) Italian Multicentre Study (n=15,773) were divided in 4 groups depending on whether they had plasma triglycerides below (NTG, 67.8%) or above (HTG, 32.2%) 1.7 mmol/L and were (42.4%) or not on (57.6%) statin therapy. Estimated GFR (eGFR) was calculated from serum creatinine, albuminuria was measured by immunonephelometry or immunoturbidimetry, and retinopathy was evaluated by fundus examination.

Results

HTG subjects, either with or without statin, had higher prevalence of albuminuria, reduced eGFR and chronic kidney disease (CKD), especially the albuminuric forms, but not of retinopathy, than NTG subjects. In contrast, cardiovascular disease and advanced DR were more prevalent in subjects on statin than in those not, independently of triglyceride levels. Logistic regression analysis confirmed that HTG, without or with statin, was independently associated with micro and macroalbuminuria, mildly to severely reduced eGFR, and all CKD phenotypes, but not with retinopathy. The adjusted odd ratios for CKD increased linearly for every 0.26 mmol/L increase (approximately one decile) in triglyceride levels. The increase was higher with increasing severity of albuminuria, eGFR loss and CKD phenotype as well as in subjects receiving than in those not receiving statin treatment.

Conclusions

Triglycerides are associated with CKD, but not retinopathy in subjects with type 2 diabetes, independently of statin treatment. These data point to a possible role of hypertriglyceridemia in the development of CKD, though it remains to be demonstrated that diabetic individuals might benefit from triglyceride reduction with statins and eventually with combination therapy with fibrates.

Trial Registration

www.ClinicalTrials.gov NCT00715481