Is there an inter-organ glutathione redox cycle?

Intracellular glutathione redox status is a function of the flux through glutathione peroxidase-glutathione reductase system. Specific activities of these two enzymes in rat liver cytosol and erythrocyte hemolysates were determined. Relative to glutathione peroxidase levels, glutathione reductase activity was about 15-fold more abundant in the rat liver than in erythrocytes. This is suggestive of greater capacity of the liver to reduce oxidised glutathione (GSSG). Based on these results and from the pattern of glutathione efflux from different cells and tissues [Sies, H. & Akerboom, T.P.M. (1984) Methods Enzymol. 105, 445-451], it is speculated that an interorgan glutathione redox cycle may be operative wherein liver is central to the reduction of GSSG and other disulphides.     

Origin and diversification of Indian Ceropegieae (Apocynaceae) and its possible relation to the Indian monsoon

The Indian subcontinent has experienced a major shift in climatic regime from a wet tropical regime to increased seasonal rainfall, since the late Miocene. This shift has been attributed to the intensification of monsoons, which led to opening up of dry habitats in humid forests and formation of deciduous forests. We explored the role of this climatic shift in the origin and diversification of dry‐adapted plant genera Ceropegia and Brachystelma (Ceropegiae, Asclepiadoideae, Apocynaceae). We sampled Ceropegia and Brachystelma from across India and used five markers (two nuclear and three plastid regions) to reconstruct a global phylogeny of this group. Indian members of the tribe Ceropegiae were derived from Africa through at least four independent dispersal events. All dispersal events occurred in late Miocene after establishment of a monsoon climate. One of these early dispersing lineages underwent rapid radiation in peninsular India, giving rise to around 50 species. Thus, both dispersal and diversification events coincided with the intensification of monsoons and concomitant aridification. The role of environment in the evolution of floral characteristics and root type in the Indian radiation is also discussed. This is one of the first reports on a dry‐adapted endemic radiation of plants in India.     

Energetic Pathway Sampling in a Protein Interaction Domain

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Metabolic fate of glutamate and evaluation of flux through the 4-aminobutyrate (GABA) shunt in Aspergillus niger

Accumulation of GABA and a concurrent block in the Krebs cycle suggest a functional GABA bypass in the acidogenic Aspergillus niger. Apart from the demonstration of enzyme machinery required, a direct measurement of flux through this glutamate decarboxylation loop was attempted. The distribution of carbon from glucose and glutamate was studied using A. niger mycelia grown on different media. The uptake and incorporation of (14)C label into organic acids and amino acids was followed by paper chromatography. Flow of label from glucose into citrate, glutamate and GABA increased in cells harvested at later stages of acidogenic growth. Very little citrate was derived from glutamate while ten times more label reached GABA from labeled glutamate. Radioactivity from L-[U-(14)C]glutamate and not from L-[1-(14)C]glutamate was recovered in GABA. This demonstrated that alpha-decarboxylation of L-glutamate was the source of GABA. Unless grown on GABA, A. niger mycelia did not take up externally supplied GABA. A direct measure of GABA shunt flux was thus not feasible. Therefore a combination of metabolite balance technique and the kinetic approach was applied to evaluate flux from glutamate to succinate in normal and acidogenic A. niger. The flux relative to TCA cycle was estimated by using uptake rate for radiolabeled glutamate, rate of accumulation of certain metabolites and the reactions of GABA metabolism. The analysis indicated that GABA shunt is operative in A. niger and its operation is enhanced during acidogenic growth of the fungus. This is the first report of an estimation of the flux through GABA shunt in a fungus.     

Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase

Agmatine, a significant polyamine in bacteria and plants, mostly arises from the decarboxylation of arginine. The functional importance of agmatine in fungi is poorly understood. The metabolism of agmatine and related guanidinium group-containing compounds in Aspergillus niger was explored through growth, metabolite, and enzyme studies. The fungus was able to metabolize and grow on l-arginine, agmatine, or 4-guanidinobutyrate as the sole nitrogen source. Whereas arginase defined the only route for arginine catabolism, biochemical and bioinformatics approaches suggested the absence of arginine decarboxylase in A. niger. Efficient utilization by the parent strain and also by its arginase knockout implied an arginase-independent catabolic route for agmatine. Urea and 4-guanidinobutyrate were detected in the spent medium during growth on agmatine. The agmatine-grown A. niger mycelia contained significant levels of amine oxidase, 4-guanidinobutyraldehyde dehydrogenase, 4-guanidinobutyrase (GBase), and succinic semialdehyde dehydrogenase, but no agmatinase activity was detected. Taken together, the results support a novel route for agmatine utilization in A. niger. The catabolism of agmatine by way of 4-guanidinobutyrate to 4-aminobutyrate into the Krebs cycle is the first report of such a pathway in any organism. A. niger GBase peptide fragments were identified by tandem mass spectrometry analysis. The corresponding open reading frame from the A. niger NCIM 565 genome was located and cloned. Subsequent expression of GBase in both Escherichia coli and A. niger along with its disruption in A. niger functionally defined the GBase locus (gbu) in the A. niger genome.     

Crystal structure of RlmM, the 2��O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA

RlmM (YgdE) catalyzes the S-adenosyl methionine (AdoMet)-dependent 2′O methylation of C2498 in 23S ribosomal RNA (rRNA) of Escherichia coli. Previous experiments have shown that RlmM is active on 23S rRNA from an RlmM knockout strain but not on mature 50S subunits from the same strain. Here, we demonstrate RlmM methyltransferase (MTase) activity on in vitro transcribed 23S rRNA and its domain V. We have solved crystal structures of E. coli RlmM at 1.9 Å resolution and of an RlmM–AdoMet complex at 2.6 Å resolution. RlmM consists of an N-terminal THUMP domain and a C-terminal catalytic Rossmann-like fold MTase domain in a novel arrangement. The catalytic domain of RlmM is closely related to YiiB, TlyA and fibrillarins, with the second K of the catalytic tetrad KDKE shifted by two residues at the C-terminal end of a beta strand compared with most 2′O MTases. The AdoMet-binding site is open and shallow, suggesting that RNA substrate binding may be required to form a conformation needed for catalysis. A continuous surface of conserved positive charge indicates that RlmM uses one side of the two domains and the inter-domain linker to recognize its RNA substrate.     

Continuing to Confront COPD International Patient Survey: Economic Impact of COPD in 12 Countries

BackgroundThe Continuing to Confront COPD International Patient Survey estimated the prevalence and burden of COPD across 12 countries. Using data from this survey we evaluated the economic impact of COPD.MethodsThis cross-sectional, population-based survey questioned 4,343 subjects aged 40 years and older, fulfilling a case definition of COPD based on self-reported physician diagnosis or symptomatology. Direct cost measures were based on exacerbations of COPD (treated and those requiring emergency department visits and/or hospitalisation), contacts with healthcare professionals, and COPD medications. Indirect costs were calculated from work loss values using the Work Productivity and Activity Impairment scale. Combined direct and indirect costs estimated the total societal costs per patient.ResultsThe annual direct costs of COPD ranged from $504 (South Korea) to $9,981 (USA), with inpatient hospitalisations (5 countries) and home oxygen therapy (3 countries) being the key drivers of direct costs. The proportion of patients completely prevented from working due to their COPD ranged from 6% (Italy) to 52% (USA and UK) with 8 countries reporting this to be ≥20%. Total societal costs per patient varied widely from $1,721 (Russia) to $30,826 (USA) but a consistent pattern across countries showed greater costs among those with increased burden of COPD (symptoms, health status and more severe disease) and a greater number of comorbidities.ConclusionsThe economic burden of COPD is considerable across countries, and requires targeted resources to optimise COPD management encompassing the control of symptoms, prevention of exacerbations and effective treatment of comorbidities. Strategies to allow COPD patients to remain in work are important for addressing the substantial wider societal costs.     

Cloning and use of sC as homologous marker for Aspergillus niger transformation

The sC sequence from Aspergillus niger was cloned and developed into a homologous marker system for genetic transformation. The coding region of the sC gene amplified by PCR from the A. niger genome was provided with Aspergillus nidulans expression signals (gpdA promoter and trpC terminator). This chimeric construct was used to successfully transform a spontaneous sC- isolate of A. niger to prototrophy. The transformants analyzed by Southern analysis showed integration of multiple copies of the transforming DNA. They also exhibited much higher ATP sulfurylase activity than the wild-type A. niger strain reinforcing the molecular data. This demonstrates the usefulness of the sCniger construct, driven by PgpdA, as a marker for A. niger transformation.