Conjugation of phosphonoacetic acid to nucleobase promotes a mechanism-based inhibition

Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity constraint and may substantially limit the efficacy of therapy. Phosphonoacetic acid has long been known to possess a restricted potential to block DNA biosynthesis. In order to achieve a better affinity, this compound has been linked with natural nucleotide at different positions. The structural context of the resulted conjugates has been found to be crucial for the acquisition by DNA polymerases. We show that nucleobase-conjugated phosphonoacetic acid is being accepted, but this alters the processivity of DNA polymerases. The data presented here not only provide a mechanistic rationale for a switch in the mode of DNA synthesis, but also highlight the nucleobase-targeted nucleotide functionalization as a route for enhancing the specificity of small molecule inhibitors.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.      

FAIR: A server for internal sequence repeats

An Internet computing server has been developed to identify all the occurrences of the internal sequence repeats in a protein and DNA sequences. Further, an option is provided for the users to check the occurrence(s) of the resultant sequence repeats in the other sequence and structure (Protein Data Bank) databases. The databases deployed in the proposed computing engine are up-to-date and thus the users will get the latest information available in the respective databases. The server is freely accessible over the World Wide Web (WWW). AVAILABILITY: http://bioserver1.physics.iisc.ernet.in/fair/     

Global Kinetic Analysis of Mammalian E3 Reveals pH-dependent NAD+/NADH Regulation,Physiological Kinetic Reversibility,and Catalytic Optimum

Mammalian E3 is an essential mitochondrial enzyme responsible for catalyzing the terminal reaction in the oxidative catabolism of several metabolites. E3 is a key regulator of metabolic fuel selection as a component of the pyruvate dehydrogenase complex (PDHc). E3 regulates PDHc activity by altering the affinity of pyruvate dehydrogenase kinase, an inhibitor of the enzyme complex, through changes in reduction and acetylation state of lipoamide moieties set by the NAD⁺/NADH ratio. Thus, an accurate kinetic model of E3 is needed to predict overall mammalian PDHc activity. Here, we have combined numerous literature data sets and new equilibrium spectroscopic experiments with a multitude of independently collected forward and reverse steady-state kinetic assays using pig heart E3. The latter kinetic assays demonstrate a pH-dependent transition of NAD⁺ activation to inhibition, shown here, to our knowledge, for the first time in a single consistent data set. Experimental data were analyzed to yield a thermodynamically constrained four-redox-state model of E3 that simulates pH-dependent activation/inhibition and active site redox states for various conditions. The developed model was used to determine substrate/product conditions that give maximal E3 rates and show that, due to non-Michaelis-Menten behavior, the maximal flux is different compared with the classically defined k_cat.     

A bioenergetic model of the mitochondrial population undergoing permeability transition

Mitochondrial permeability transition (MPT) is a highly regulated complex phenomenon that is a type of ischemia/reperfusion injury that can lead to cell death and ultimately organ dysfunction. A novel population transition and detailed permeability transition pore regulation model were integrated with an existing bioenergetics model to describe MPT induction under a variety of conditions. The framework of the MPT induction model includes the potential states of the mitochondria (aggregated, orthodox and post-transition), their transitions from one state to another as well as their interaction with the extra-mitochondrial environment. The model encodes the three basic necessary conditions for MPT: a high calcium load, alkaline matrix pH and circumstances which favor de-energization. The MPT induction model was able to reproduce the expected bioenergetic trends observed in a population of mitochondria subjected to conditions that favor MPT. The model was corroborated and used to predict that MPT in an acidic environment is mitigated by an increase in activity of the mitochondrial potassium/hydrogen exchanger. The model was also used to present the beneficial impact of reducing the duration mitochondria spend in the orthodox state on preserving the extra-mitochondrial ATP levels. The model serves as a tool for investigators to use to understand the MPT induction phenomenon, explore alternative hypotheses for PTP regulation, as well as identify endogenous pharmacological targets and evaluate potential therapeutics for MPT mitigation.     

A minimal model for the mitochondrial rapid mode of Ca²+ uptake mechanism

Mitochondria possess a remarkable ability to rapidly accumulate and sequester Ca²⁺. One of the mechanisms responsible for this ability is believed to be the rapid mode (RaM) of Ca²⁺ uptake. Despite the existence of many models of mitochondrial Ca²⁺ dynamics, very few consider RaM as a potential mechanism that regulates mitochondrial Ca²⁺ dynamics. To fill this gap, a novel mathematical model of the RaM mechanism is developed herein. The model is able to simulate the available experimental data of rapid Ca²⁺ uptake in isolated mitochondria from both chicken heart and rat liver tissues with good fidelity. The mechanism is based on Ca²⁺ binding to an external trigger site(s) and initiating a brief transient of high Ca²⁺ conductivity. It then quickly switches to an inhibited, zero-conductive state until the external Ca²⁺ level is dropped below a critical value (∼100–150 nM). RaM''s Ca²⁺- and time-dependent properties make it a unique Ca²⁺ transporter that may be an important means by which mitochondria take up Ca²⁺ in situ and help enable mitochondria to decode cytosolic Ca²⁺ signals. Integrating the developed RaM model into existing models of mitochondrial Ca²⁺ dynamics will help elucidate the physiological role that this unique mechanism plays in mitochondrial Ca²⁺-homeostasis and bioenergetics.     

Late immature mortality is the major influence on reproductive success of African black beetle, Heteronychus arator (Fabricius) (Coleoptera: Scarabaeidae), in a Mediterranean-climate region of Australia

In field and laboratory studies, mortality of African black beetle, Heteronychus arator, in the winter-rainfall, Mediterranean-type climate region of south-western Australia was higher in the late immature stages during summer than in the early immature stages that occur during spring, a contrast to summer-rainfall climatic regions. Greatest mortality occurred around the pupal stage in contrasting soil types, despite drying differences in summer and supplementary watering in some plots. Sampling of natural populations confirmed experimental results that mortality in late immature stages is the major factor limiting H. arator populations under a Mediterranean-type climate. Inter-generation increase in H. arator abundance was uncommon, explaining the consistent abundance typically observed between years in south-western Australia. Random dispersal of newly emerged adults in autumn was inferred to restore uniformity in adult abundance between areas of varying favourability for immature survival.     

Shedding as a mechanism of down-modulation of CD14 on stimulated human monocytes

CD14, expressed on the surface of monocytes as a phospholipid-linked protein, is a receptor for serum LPS binding protein/LPS complex. It was specifically down-modulated after stimulation of monocytes by physiologic activating/differentiating agents such as bacterial LPS and IFN-gamma, by the pharmacologic agents PMA and calcium ionophore A23187, and by anti-CD14 antibodies. The down-modulation was almost totally blocked at 4 degrees C or at pH 4.5 and markedly inhibited by the protease inhibitors diisopropylfluorophosphate and PMSF. A soluble labeled CD14 was isolated from culture supernatant of surface iodinated monocytes after their activation, indicating that CD14 is shed from the cell surface rather than internalized. The size of the soluble CD14 shed from the monocytes in vitro was smaller than that of either the membrane-bound form or a soluble CD14 cleaved from the cell surface by phosphatidyl inositol-specific phospholipase C, but identical to the size of one of the two major soluble CD14 forms normally found in human serum. These data suggest that CD14 shedding induced by monocyte stimulation may play an important role in the regulation of surface CD14 expression.