Cytosolic isocitrate dehydrogenase in humans, mice, and voles and phylogenetic analysis of the enzyme family

In this study, we report cDNA sequences of the cytosolic NADP-dependent isocitrate dehydrogenase for humans, mice, and two species of voles (Microtus mexicanus and Microtus ochrogaster). Inferred amino acid sequences from these taxa display a high level of amino acid sequence conservation, comparable to that of myosin beta heavy chain, and share known structural features. A Caenorhabditis elegans enzyme that was previously identified as a protein similar to isocitrate dehydrogenase is most likely the NADP-dependent cytosolic isocitrate dehydrogenase enzyme equivalent, based on amino acid similarity to mammalian enzymes and phylogenetic analysis. We also suggest that NADP-dependent isocitrate dehydrogenases characterized from alfalfa, soybean, and eucalyptus are most likely cytosolic enzymes. The phylogenetic tree of various isocitrate dehydrogenases from eukaryotic sources revealed that independent gene duplications may have given rise to the cytosolic and mitochondrial forms of NADP-dependent isocitrate dehydrogenase in animals and fungi. There appears to be no statistical support for a hypothesis that the mitochondrial and cytosolic forms of the enzyme are orthologous in these groups. A possible scenario of the evolution of NADP-dependent isocitrate dehydrogenases is proposed.      

Primary prevention of diabetes mellitus type 2 and cardiovascular diseases using a cognitive behavior program aimed at lifestyle changes in people at risk: Design of a randomized controlled trial

Background

Insulin resistance and diabetes are associated with increased oxidative stress and impairment of cellular defence systems. Our purpose was to investigate the interaction between glucose metabolism, antioxidative capacity and heat shock protein (HSP) defence in different skeletal muscle phenotypes among middle-aged obese subjects during a long-term exercise and dietary intervention. As a sub-study of the Finnish Diabetes Prevention Study (DPS), 22 persons with impaired glucose tolerance (IGT) taking part in the intervention volunteered to give samples from the vastus lateralis muscle. Subjects were divided into two sub-groups (IGTslow and IGTfast) on the basis of their baseline myosin heavy chain profile. Glucose metabolism, oxidative stress and HSP expressions were measured before and after the 2-year intervention.

Results

Exercise training, combined with dietary counselling, increased the expression of mitochondrial chaperones HSP60 and glucose-regulated protein 75 (GRP75) in the vastus lateralis muscle in the IGTslow group and that of HSP60 in the IGTfast group. In cytoplasmic chaperones HSP72 or HSP90 no changes took place. In the IGTslow group, a significant positive correlation between the increased muscle content of HSP60 and the oxygen radical absorbing capacity values and, in the IGTfast group, between the improved VO_2max value and the increased protein expression of GRP75 were found. Serum uric acid concentrations decreased in both sub-groups and serum protein carbonyl concentrations decreased in the IGTfast group.

Conclusion

The 2-year intervention up-regulated mitochondrial HSP expressions in middle-aged subjects with impaired glucose tolerance. These improvements, however, were not correlated directly with enhanced glucose tolerance.     

An edit script for taxonomic classifications

Background  

The NCBI taxonomy provides one of the most powerful ways to navigate sequence data bases but currently users are forced to formulate queries according to a single taxonomic classification. Given that there is not universal agreement on the classification of organisms, providing a single classification places constraints on the questions biologists can ask. However, maintaining multiple classifications is burdensome in the face of a constantly growing NCBI classification.     

Mutagenicity of Maillard reaction products from D-glucose-amino acid mixtures and possible roles of active oxygens in the mutagenicity

The mutagenicity for Salmonella typhimurium TA100 without S9 mix of Maillard reaction products (MRP) obtained from equimolar amounts of glucose and amino acids under different pHs was investigated. MRP derived from arginine and lysine exhibited the strongest mutagenicity, and weaker mutagenicity was shown by the mixtures with alanine, serine, threonine and monosodium glutamate. MRP from proline and cysteine had no detectable mutagenicity. Furthermore, glucose-arginine and glucose-lysine reaction mixtures, which presented a marked mutagenicity, showed pH- and browning intensity-dependent expression of their mutagenic activities. The mutagenicity of MRP, especially glucose-arginine and glucose-lysine mixtures, was significantly suppressed by active oxygen scavengers such as cysteine, mannitol, alpha-tocopherol, catalase and superoxide dismutase (SOD) and reducing agents such as sodium bisulfite and glutathione. Among these desmutagenic factors tested, cysteine, catalase, sodium bisulfite and glutathione had higher desmutagenic activities than the others. Accordingly, it is assumed that the mutagenicity of MRP is due to the direct action of low-molecular-weight compounds such as carbonyls and heterocyclics produced by the Maillard reaction and is enhanced by active oxygens, especially singlet oxygen and hydrogen peroxide derived from their autoxidation.     

A framework for conservation area designation utilizing an ecotope concept and its application to a mountainous national park in Korea

This study focused on establishing a framework for the designation of a conservation area reflecting an ecotope concept, and its application to Korea’s Seoraksan National Park. Taking into consideration the mountainous topography of the study area, the watershed was chosen as the planning unit, and ecotope mapping was carried out to identify topographic and biological features. For the evaluation of the state of recent use, the trail index and the distribution status of tour and management facilities were used. These features were processed using Marxan with Zones. As a result, the optimal solution of scenario 1 was selected as the final one following detailed verification of the area ratio and grouping of the Park Nature Conservation Area (zone A, 76.7%) and the Park Nature Environment Area (I) (zone B, 14.3%), the inclusion ratio of biotope grade I (95.2%) and the satisfied shortfall value of the conservation indicators. In the overlap analysis between the optimal solution and recent zonation, most of the features included in the suggested scenario were found to be correctly distributed according to the intensity of conservation. Furthermore, the northern parts of the watershed which include sub-alpine vegetation with the Baekdudaegan ridge as its ecological axis was newly separated into the Park Conservation Area. This verification confirmed the improvement of recent zonation through the concrete criteria of ecotope mapping.     

Effects of PM10 in human peripheral blood monocytes and J774 macrophages

Background

Asthma is characterized by type 2 T-helper cell (Th2) inflammation, goblet cell hyperplasia, airway hyperreactivity, and airway fibrosis. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and its receptor, CCR2, have been shown to play important roles in the development of Th2 inflammation. CCR2-deficient mice have been found to have altered inflammatory and physiologic responses in some models of experimental allergic asthma, but the role of CCR2 in contributing to inflammation and airway hyperreactivity appears to vary considerably between models. Furthermore, MCP-1-deficient mice have not previously been studied in models of experimental allergic asthma.

Methods

To test whether MCP-1 and CCR2 are each required for the development of experimental allergic asthma, we applied an Aspergillus antigen-induced model of Th2 cytokine-driven allergic asthma associated with airway fibrosis to mice deficient in either MCP-1 or CCR2. Previous studies with live Aspergillus conidia instilled into the lung revealed that MCP-1 and CCR2 play a role in anti-fungal responses; in contrast, we used a non-viable Aspergillus antigen preparation known to induce a robust eosinophilic inflammatory response.

Results

We found that wild-type C57BL/6 mice developed eosinophilic airway inflammation, goblet cell hyperplasia, airway hyperreactivity, elevations in serum IgE, and airway fibrosis in response to airway challenge with Aspergillus antigen. Surprisingly, mice deficient in either MCP-1 or CCR2 had responses to Aspergillus antigen similar to those seen in wild-type mice, including production of Th2 cytokines.

Conclusion

We conclude that robust Th2-mediated lung pathology can occur even in the complete absence of MCP-1 or CCR2.     

Oxidative stress status of highly prolific sows during gestation and lactation

Elevated oxidative stress is reported to be associated with pregnancy complications in highly prolific sows. Oxidative DNA damage and the antioxidant status were determined in blood samples collected during the course of gestation and lactation in multiparous sows. Blood samples were drawn from sows (n = 5) on days 30, 60, 90 and 110 of gestation (G30, G60, G90 and G110, respectively), on day 3, 10 and 18 of lactation (L3, L10 and L18, respectively) and on day 5 of postweaning (W5). Lymphocytes were isolated from the fresh blood and cryopreserved in each time point. Lymphocyte DNA damage was analyzed by alkaline single-cell gel electrophoresis (comet assay) to determine the single- and double-strand brakes and endogenous antioxidant concentrations using an HPLC system with UV detection. The comet assay showed elevated (P < 0.05) DNA damage (between 38% and 47%) throughout the gestational and lactational periods than during early gestation (G30; 21%). Plasma retinol concentration was reduced (P < 0.05) at the end of gestation (G110) compared with G30. Plasma α-tocopherol concentrations also showed a similar trend as to retinol. This study indicates that there is an increased systemic oxidative stress during late gestation and lactation, which are not fully recovered until the weaning compared with the G30, and that antioxidant nutrients in circulation substantially reduced in the mother pig at G110.     

Hemoglobin glutathionylation can occur through cysteine sulfenic acid intermediate: Electrospray ionization LTQ-Orbitrap hybrid mass spectrometry studies

Glutathionylated hemoglobin (Hb-SSG) is now recognized as a promising biomarker of systemic oxidative stress. Aim of this study is to gain a mechanistic insight into its formation. The ability of GSSG to form Hb-SSG through a thiol-disulfide exchange mechanism was firstly examined. For this purpose, GSSG (ranging from 0.23 to 230 μmol/g Hb, 15 μM–15 mM final concentrations) was incubated with 1 mM Hb and the relative content of Hb-SSG determined by direct infusion mass spectrometry (Orbitrap as analyzer). No detectable Hb-SSG was observed at a GSSG concentration range found in physiopathological conditions (0.13–0.23 μmol/g Hb). To reach a detectable Hb-SSG signal, the GSSG concentration was raised to 2.3 μmol/g Hb (0.5% relative abundance). The relative content of Hb-GSSG dose-dependently increased to 6% and 11% at 77 and 153 μmol/g Hb, respectively. The second step was to demonstrate whether Hb-SSG is formed through a sulfenic acid intermediate, a well-recognized mechanism of S-protein glutathionylation. Cys β93 sulfenic acid was found to be formed by oxidizing Hb with 1 mM H₂O₂, as demonstrated by direct infusion and LC–ESI-MS/MS experiments and using dimedone as derivatazing agent. When H₂O₂-treated Hb was incubated with physiological concentrations of GSH (9 μmol/g Hb), the corresponding Hb-SSG form was detected, reaching 15% of relative abundance. In summary, we here demonstrate that Hb glutathionylation can occur through a Cys sulfenic acid intermediate which is formed in oxidizing conditions. Hb glutathionylation is also mediated by a thiol-disulfide transfer mechanism, but this requires a concentration of GSSG which is far to be achieved in physiopathological conditions.     

Monitoring ungulates in steep non-forest habitat: a comparison of faecal pellet and helicopter counts

Faecal pellet counts have been widely used to monitor the abundances of introduced ungulates in New Zealand, but ground-based sampling cannot be conducted safely in the steep non-forest habitats that are common in New Zealand's Southern Alps. Helicopter counts may be an effective technique for monitoring ungulates in steep non-forest habitat. We evaluated the relationship between faecal pellet and helicopter counts of ungulates (primarily feral goat Capra hircus) at 12 non-forest sites in the Southern Alps. Within each site we counted the numbers of ungulates from a helicopter on three occasions and the number of intact faecal pellets along 30 transects. Mean observed densities of feral goats derived from helicopter counts ranged from 0.0 to 20.2 km^?2. There was a positive curvilinear (concave down) relationship between faecal pellet and helicopter counts. Compared with faecal pellet counts, helicopter counts were cheaper, could identify ungulate species and provided estimates of absolute density. Helicopter counts are a cost-effective method for monitoring ungulates in the steep non-forest habitats of New Zealand's Southern Alps.     

Mutagenic and carcinogenic heterocyclic amines as affected by muscle types/skin and cooking in pan-roasted mackerel

The dependence on muscle types/skin and on degrees of cooking in the formation of heterocyclic amines (HCAs) of pan-roasted mackerel was studied. High levels of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were found in very well done skin and ordinary muscle, being 4.2 and 5.3 ng/g, followed by 2-amino-3,4-dimethylimidazo[4,5-f] quinoxaline (MeIQx), being 1.8 and 2.1 ng/g and 2-amino-9H-pyrido[2,3-b]indole (AalphaC), being 1.2 and 2.8 ng/g, respectively. In pan-roasted mackerel, ordinary muscles contributed much more greatly to the formation of HCAs than skins due to its higher HCA contents and composition (76.1%).