Dissecting DISC regulation via pharmacological targeting of caspase-8/c-FLIPL heterodimer.

Pharmacological targeting via small molecule-based chemical probes has recently acquired an emerging importance as a valuable tool to delineate molecular mechanisms. Induction of apoptosis via CD95/Fas and TRAIL-R1/2 is triggered by the formation of the death-inducing signaling complex (DISC). Caspase-8 activation at the DISC is largely controlled by c-FLIP proteins. However molecular mechanisms of this control have just started to be uncovered. In this study we report the first-in-class chemical probe targeting c-FLIPL in the heterodimer caspase-8/c-FLIPL. This rationally designed small molecule was aimed to imitate the closed conformation of the caspase-8 L2' loop and thereby increase caspase-8 activity after initial processing of the heterodimer. In accordance with in silico predictions, this small molecule enhanced caspase-8 activity at the DISC, CD95L/TRAIL-induced caspase activation, and subsequent apoptosis. The generated computational model provided further evidence for the proposed effects of the small molecule on the heterodimer caspase-8/c-FLIPL. In particular, the model has demonstrated that boosting caspase-8 activity by the small molecule at the early time points after DISC assembly is crucial for promoting apoptosis induction. Taken together, our study allowed to target the heterodimer caspase-8/c-FLIPL and get new insights into molecular mechanisms of its activation.

A plausible mechanism for auxin patterning along the developing root.

BACKGROUND: In plant roots, auxin is critical for patterning and morphogenesis. It regulates cell elongation and division, the development and maintenance of root apical meristems, and other processes. In Arabidopsis, auxin distribution along the central root axis has several maxima: in the root tip, in the basal meristem and at the shoot/root junction. The distal maximum in the root tip maintains the stem cell niche. Proximal maxima may trigger lateral or adventitious root initiation. RESULTS: We propose a reflected flow mechanism for the formation of the auxin maximum in the root apical meristem. The mechanism is based on auxin's known activation and inhibition of expressed PIN family auxin carriers at low and high auxin levels, respectively. Simulations showed that these regulatory interactions are sufficient for self-organization of the auxin distribution pattern along the central root axis under varying conditions. The mathematical model was extended with rules for discontinuous cell dynamics so that cell divisions were also governed by auxin, and by another morphogen Division Factor which combines the actions of cytokinin and ethylene on cell division in the root. The positional information specified by the gradients of these two morphogens is able to explain root patterning along the central root axis. CONCLUSION: We present here a plausible mechanism for auxin patterning along the developing root, that may provide for self-organization of the distal auxin maximum when the reverse fountain has not yet been formed or has been disrupted. In addition, the proximal maxima are formed under the reflected flow mechanism in response to periods of increasing auxin flow from the growing shoot. These events may predetermine lateral root initiation in a rhyzotactic pattern. Another outcome of the reflected flow mechanism - the predominance of lateral or adventitious roots in different plant species - may be based on the different efficiencies with which auxin inhibits its own transport in different species, thereby distinguishing two main types of plant root architecture: taproot vs. fibrous.

Expression and molecular characterization of the Mycobacterium tuberculosis PII protein.

The signal transduction protein PII plays an important role in cellular nitrogen assimilation and regulation. The molecular characteristics of the Mycobacterium tuberculosis PII (Mtb PII) were investigated using biophysical experiments. The Mtb PII coding ORF Rv2919c was cloned and expressed in Escherichia coli. The binding characteristics of the purified protein with ATP and ADP were investigated using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Mtb PII binds to ATP strongly with K(d) in the range 1.93-6.44 microM. This binding strength was not significantly affected by the presence of 2-ketoglutarate even in molar concentrations of 66 (ITC) or 636 (SPR) fold excess of protein concentration. However, an additional enthalpy of 0.3 kcal/mol was released in presence of 2-ketoglutarate. Binding of Mtb PII to ADP was weaker by an order of magnitude. Binding of ATP and 2-ketoglutarate were analysed by docking studies on the Mtb PII crystal structure (PDB id 3BZQ). We observed that hydrogen bonds involving the gamma-phosphate of ATP contribute to enhanced binding of ATP compared with ADP. Glutaraldehyde crosslinking showed that Mtb PII exists in homotrimeric state which is consistent with other PII proteins. Phylogenetic analysis showed that Mtb PII consistently grouped with other actinobacterial PII proteins.

The complete mitochondrial genomes of the liver flukes Opisthorchis felineus and Clonorchis sinensis (Trematoda).

The complete mitochondrial genomes of the parasitic trematodes Opisthorchis felineus and Clonorchis sinensis (family Opisthorchiidae) were fully sequenced in order to develop markers for DNA diagnostics of the liver flukes infection, molecular ecology, population and phylogenetic studies. The complete sequences of mitochondrial genomes of these species comprise 14,277 and 13,875bp, respectively, and are thus the shortest trematode mitochondrial genomes sequenced to date. The gene content and arrangement are identical to that of Fasciola hepatica. ATG and GTG are used as the start-codons and TAG and TAA are used as the stop-codons. The stop-codon TAG of the C. sinensis nad1 gene overlap by 1nt with the downstream tRNA-Asn gene. Alternative structures for the Ser(UCN) tRNAs were found for both species. The noncoding control regions are separated into two parts by the tRNA-Gly gene and contain neither tandem repeats, which are characteristic for trematode control regions, nor secondary structures. In conclusion, the complete mitochondrial DNA sequences of O. felineus and C. sinensis will serve as a resource for comparative mitochondrial genomics and systematic studies of parasitic trematodes.

A novel nuclear marker, Pm-int9, for phylogenetic studies of Opisthorchis felineus, Opisthorchis viverrini, and Clonorchis sinensis (Opisthorchiidae, Trematoda).

Opisthorchis felineus, O. viverrini, and Clonorchis sinensis, the trematodes of the family Opisthorchiidae, are important human parasites. Two previous studies (Kang et al. Parasitol Int 57:191-197, 2008; Katokhin et al. Dokl Biochem Biophys 421:214-217, 2008) have provided evidence using ribosomal and mitochondrial sequences that O. viverrini, O. felineus, and C. sinensis are closely related. We developed a novel nuclear marker, Pm-int9, which included the ninth intron of the paramyosin gene and flanking exon sequences. Samples of O. felineus from four localities of West Siberia, C. sinensis from the Russian Far East, and O. viverrini from Thailand were genotyped by Pm-int9. Little variation was detected in exon sequences, however, intron sequences turned out to be more variable than ribosomal internal transcribed spacers. We can conclude that Pm-int9 is valuable for interspecific variation studies. Phylogenetic analysis based on Pm-int9 revealed that O. viverrini and C. sinensis were closer to each other than either of them to O. felineus, supporting the opinion that C. sinensis should be considered the sister species of Opisthorchis spp.

Application of bioinformatics resources for genosensor design.

Two novel databases, GenSensor and ConSensor, have been developed. GenSensor accumulates information on the sensitivities of the prokaryotic genes to external stimuli and may facilitate designing of novel genosensors; ConSensor contains data about the structure and efficiency of the available genosensor plasmid constructs. Using these databases, candidate genes for the design of novel multiple functional genosensors were searched, and the Escherichia coli dps gene was chosen as the candidate. The genetic construct derived from its promoter was developed and tested for its sensitivity to various stress agents: hydrogen peroxide (oxidative stress), phenol (protein and membrane damaging), and mitomycin C (DNA damaging). This genosensor was found to be sensitive to all stress conditions applied confirming its ability to serve as multi-functional genosensor. The GenSensor and ConSensor databases are available at http://wwwmgs.bionet.nsc.ru/mgs/dbases/gensensor/index.html.

Mathematical model for suppression of subgenomic hepatitis C virus RNA replication in cell culture.

A mathematical model for suppression of the hepatitis C virus RNA replicon replication in Huh-7 cell culture in the presence of potential drugs was built. There was a good agreement between the experimental and theoretical kinetic data for the decrease in the level of viral RNA in the cell in the presence of the competitive HCV NS3 protease inhibitor. Using the model, we verified the estimates for the efficiency of the effect of potential drugs on replication of viral RNA and viral protein processing. It was demonstrated that the tested drugs are most efficient at the replication step of viral RNA. The efficiency of the combined action of real and putative inhibitors target on the host and viral proteins was also studied. It was found that the action of the inhibitor at low concentrations on the host factors considerably enhances the suppressive effect on viral RNA replication in the presence of even the low affine NS3 protease inhibitors. The developed mathematical model may serve as a tool for the evaluation of the efficiency of potential drugs on the HCV genome.

A cellular automaton to model the development of primary shoot meristems of Arabidopsis thaliana.

Development of organisms is a very complex process in which a lot of gene networks of different cell types are integrated. Development of a cellular automaton (Ermentrout and Edelshtein-Keshet, J Theor Biol 160:97-133, 1993) that models the morphodynamics of different cell types is the first step in understanding and analysis of the regulatory mechanisms underlying the functioning of developmental gene networks. A model of a cellular automaton has been developed, which simulates the embryonic development of shoot meristem in Arabidopsis thaliana. The model adequately describes the basic stages in development of this organ in wild and mutant types.