Effect of inducers on metabolism of benzo(a)pyrene in vivo and in vitro: analysis by high pressure liquid chromatography

The characterisation of metabolites formed from benzo(a)pyrene (BP) by Aspergillus ochraceus TS and effect of inducers on BP metabolism are reported. The high pressure liquid chromatographic profile of BP metabolites was similar to that of mammalian microsomes furnishing diols, quinones and phenols. The production of BP-4,5-dihydrodiol (K-region diol) by Aspergillus ochraceus TS seems to be novel and provides first report on BP metabolism by eukaryotic fungi. In control, phenols and quinones were produced in excess over dihydrodiols while the induced preparation showed the reverse order. Presumably the induction effecting production of excess dihydrodiols influenced the synthesis of epoxide hydrolase. In addition, a differential increase in BP metabolism was observed with inducers of narrow and broad specificity.     

Evidence for clustered mannose as a new ligand for hyaluronan- binding protein (HABP1) from human fibroblasts

We have earlier reported that overexpression of the gene encoding human hyaluronan-binding protein (HABP1) is functionally active, as it binds specifically with hyaluronan (HA). In this communication, we confirm the collapse of the filamentous and branched structure of HA by interaction with increasing concentrations of recombinant-HABP1 (rHABP1). HA is the reported ligand of rHABP1. Here, we show the affinity of rHABP1 towards D-mannosylated albumin (DMA) by overlay assay and purification using a DMA affinity column. Our data suggests that DMA is another ligand for HABP1. Furthermore, we have observed that DMA inhibits the binding of HA in a concentration-dependent manner, suggesting its multiligand affinity amongst carbohydrates. rHABP1 shows differential affinity towards HA and DMA which depends on pH and ionic strength. These data suggest that affinity of rHABP1 towards different ligands is regulated by the microenvironment.     

RankAggreg, an R package for weighted rank aggregation

Background

Determining the parameters of a mathematical model from quantitative measurements is the main bottleneck of modelling biological systems. Parameter values can be estimated from steady-state data or from dynamic data. The nature of suitable data for these two types of estimation is rather different. For instance, estimations of parameter values in pathway models, such as kinetic orders, rate constants, flux control coefficients or elasticities, from steady-state data are generally based on experiments that measure how a biochemical system responds to small perturbations around the steady state. In contrast, parameter estimation from dynamic data requires time series measurements for all dependent variables. Almost no literature has so far discussed the combined use of both steady-state and transient data for estimating parameter values of biochemical systems.

Results

In this study we introduce a constrained optimization method for estimating parameter values of biochemical pathway models using steady-state information and transient measurements. The constraints are derived from the flux connectivity relationships of the system at the steady state. Two case studies demonstrate the estimation results with and without flux connectivity constraints. The unconstrained optimal estimates from dynamic data may fit the experiments well, but they do not necessarily maintain the connectivity relationships. As a consequence, individual fluxes may be misrepresented, which may cause problems in later extrapolations. By contrast, the constrained estimation accounting for flux connectivity information reduces this misrepresentation and thereby yields improved model parameters.

Conclusion

The method combines transient metabolic profiles and steady-state information and leads to the formulation of an inverse parameter estimation task as a constrained optimization problem. Parameter estimation and model selection are simultaneously carried out on the constrained optimization problem and yield realistic model parameters that are more likely to hold up in extrapolations with the model.     

Global Wild Annual Lens Collection: A Potential Resource for Lentil Genetic Base Broadening and Yield Enhancement

Crop wild relatives (CWRs) are invaluable gene sources for various traits of interest, yet these potential resources are themselves increasingly threatened by the impact of climate change as well as other anthropogenic and socio-economic factors. The prime goal of our research was to cover all aspects of wild Lens genetic resource management like species characterization, agro-morphological evaluation, diversity assessment, and development of representative sets for its enhanced utilization in lentil base broadening and yield improvement initiatives. We characterized and evaluated extensively, the global wild annual Lens taxa, originating from twenty seven counties under two agro-climatic conditions of India consecutively for three cropping seasons. Results on various qualitative and quantitative characters including two foliar diseases showed wide variations for almost all yield attributing traits including multiple disease resistance in the wild species, L. nigricans and L. ervoides accessions. The core set developed from the entire Lens taxa had maximum representation from Turkey and Syria, indicating rich diversity in accessions originating from these regions. Diversity analysis also indicated wide geographical variations across genepool as was reflected in the core set. Potential use of core set, as an initial starting material, for genetic base broadening of cultivated lentil was also suggested.     

Intraspecific genetic variability analysis of Neovossia indica causing Karnal bunt of wheat using repetitive elements

Neovossia indica (Tilletia indica), causing Karnal bunt of wheat, affects major wheat growing regions all over the world. Karnal bunt ranks as one of the major diseases of wheat causing quality losses and monetary losses due to international quarantine regulations. The present work is the first report of a genetic diversity analysis of Indian isolates of N. indica. A library of N. indica isolate Ni7 was constructed in a λZAPII system, and three repetitive elements were identified for molecular analysis. These repetitive elements generated complex hybridization profiles producing fingerprint patterns of all seven isolates. Copy-number estimation of these three elements, pNiR9, pNiR12 and pNiR16, indicated the presence of 32, 61 and 64 copies, respectively. Cluster analysis based on hybridization patterns grouped together moderately virulent isolates Ni1, Ni7 and Ni8, thus suggesting a positive correlation between virulence typing and cluster analysis based on molecular data. Variability analysis of N. indica isolates will aid in checking new resistant sources in host germplasm. Received: 20 April 1999 / Accepted: 29 July 1999     

Excess generation of endogenous heme inhibits L-alanine:4,5-dioxovalerate transaminase in rat liver mitochondria

In the present study, we examined the possibility that the excess heme generation within mitochondria may provide a local concentration, sufficient to inhibit the activity of L-alanine:4,5-dioxovalerate transaminase, the enzyme proposed for an alternate route of delta-aminolevulinic acid biosynthesis in mammalian system. This was accomplished by assaying together L-alanine:4,5-dioxovalerate transaminase and heme synthetase activities in intact mitochondria isolated from rat liver. Endogenous heme in intact mitochondria has been generated in excess, by increasing the concentration of the substrate of heme synthetase. Our studies showed that the activity of L-alanine:4,5-dioxovalerate transaminase decreased as the rate of heme formation increased. In intact mitochondria, almost 50% inhibition of alanine:4,5-dioxovalerate transaminase was obtained with 4.0 mumole of heme generation. We conclude that end product inhibition of L-alanine:4,5-dioxovalerate transaminase by hemin, which was proposed in earlier report by us (FEBS Letter (1985), 189, 129), is an important physiological mechanism for the regulation of hepatic heme biosynthesis.     

Autoproteolysis of rat p67 generates several peptide fragments: the N-terminal fragment, p26, is required for the protection of eIF2alpha from phosphorylation

Eukaryotic initiation factor 2-associated glycoprotein, p67, plays important roles in the regulation of eIF2alpha phosphorylation and thus maintains cell growth and proliferation. The p67 sequence can be divided into two segments, the N-terminal segment of amino acids 1-107 (p26) and the downstream segment of amino acids 108-480 (p52). Comparison of the amino acid sequences of p67 from lower to higher organisms suggests that there is a progressive addition of several unique domains at the N-terminus of p67, and these unique domains, which are present in p26, play important roles in the modulation of eIF2alpha phosphorylation in mammalian cells. To test the hypothesis that the p26 segment is generated from p67 due to its autoproteolysis and whether p26 is required for the protection of eIF2alpha from phosphorylation, we have analyzed the time-dependent cleavage of functionally active rat recombinant p67 purified from either baculovirus-infected insect cells or transiently transfected mammalian cells. We noticed a regulated cleavage of p67 that generates several peptides along with the most stable p26 and p52 fragments. The p52 fragment has a low level of autoproteolysis activity that possibly increases the autoproteolysis of full-length p67. This activity could not be inhibited by a serine protease inhibitor, PMSF, but could be inhibited by a cocktail of protease inhibitors that includes bestatin, leupeptin, E64, AEBSF, and aprotinin. To provide evidence that the fragmentation of p67 is not due to the presence of any contaminant protease(s), we fractionated purified rat p67 with molecular sieve, anion exchange, and cation exchange chromatographic steps performed in the presence of different K+ ion concentrations. Our data show that the extent of cleavage of p67 into different fragments is higher in the presence of 0.75 M K+ ion and in samples stored at -80 degrees C. Under parallel conditions, p67's mutants, D251A and D262A, exhibited very little to no cleavage, whereas the H231E mutant exhibited extensive cleavage that generated a large amount of p26 fragment. The p26 fragment exhibited protection of eIF2alpha phosphorylation both in vivo and in vitro. Altogether, our data provide evidence that rat p67 has autoproteolytic activity that generates p26, which is required to block eIF2alpha from phosphorylation.