The affinity of protein C for the thrombin.thrombomodulin complex is determined in a primary way by active site-dependent interactions

The interaction of thrombin (IIa) with thrombomodulin (TM) is essential for the efficient activation of protein C (PC). Interactions between PC and extended surfaces, likely contributed by TM within the IIa.TM complex, have been proposed to play a key role in PC activation. Initial velocities of PC activation at different concentrations of PC and TM could be accounted for by a model that did not require consideration of direct binding interactions between PC and TM. Reversible inhibitors directed toward the active site of IIa within the IIa.TM complex behaved as classic competitive inhibitors of both peptidyl substrate cleavage as well as PC activation. The ability of these small molecule inhibitors to block PC binding to the enzyme points to a principal role for active site-dependent substrate recognition in determining the affinity of IIa.TM for its protein substrate. Selective abrogation of active site docking by mutation of the P1 Arg in PC to Gln yielded an uncleavable derivative (PC(R15Q)). PC(R15Q) was a poor inhibitor (K(i) >or= 30 microm) of PC activation as well as peptidyl substrate cleavage by IIa.TM. Thus, inhibition by PC(R15Q) most likely results from its ability to weakly interfere with active site function rather than by blocking extended interactions with the enzyme complex. The data suggest a primary role for active site-dependent substrate recognition in driving the affinity of the IIa.TM complex for its protein substrate. Interactions between PC and extended surfaces contributed by IIa and/or TM within the IIa.TM complex likely contribute in a secondary or minor way to protein substrate affinity.     

Consensus based validation of membrane porins

Porins are outermembrane beta-barrel proteins. They have varied biological functionality ranging from phage receptors, immunogenicity, pathogenicity to apoptosis. However, only a small number has been structurally and functionally characterised. A validation mechanism and a database of porins would be useful for target selection in proteomics and structural genomics work. Here we report a validation mechanism developed for membrane porins. A database server for porins, PRNDS, has been created containing experimentally proven porins and likely putative porins. Each porin is validated and ranked using a weighted scoring system developed based on six, structure and sequence based criteria. The server also predicts possible porins.     

Construction and Characterization of an Escherichia coli Strain Genetically Engineered for Ni(II) Bioaccumulation

An Escherichia coli strain that accumulated Ni(II) was constructed by introducing the nixA gene (coding for a nickel transport system) from Helicobacter pylori into JM109 cells that expressed a glutathione S-transferase–pea metallothionein fusion protein. The resulting strain accumulated 15 μmol of Ni(II) per g (dry weight) from a 10 μM Ni(II) solution, four times the level taken up by JM109 cells. Ni(II) accumulation did not require an energy source, was inhibited by only 50% by 0.1 M NaCl, and occurred over the pH range from 3 to 9.     

Engineered Minichromosomes in Plants

Engineered minichromosomes provide the ability to target transgenes to a defined insertion position for predictable expression on an independent chromosome. This technology promises to provide a means to add many genes to a synthetic chromosome in sequential manner. An additional advantage is that the multiple transgenes will not be inserted into the normal chromosomes and thus will not exhibit linkage drag when converging the transgenes to different germplasm nor will they be mutagenic. Telomere truncation coupled with the introduction of site-specific recombination cassettes has proven to be an easy method to produce minichromosomes. Telomere truncation results from the transformation of plasmids carrying a block of telomere repeats at one end. Minichromosomes consisting of little more than a centromere have been produced for B chromosomes of maize. Such small chromosomes have been studied for their meiotic behavior, which differs from normal sized chromosomes in that homologue pairing is rare or nonexistent and sister chromatid cohesion fails at meiosis I. Potential modifications of the minichromosomes that can address these issues are discussed. Minichromosomes can be recovered from transformed plants that are polyploid or that carry an additional chromosome as the preferred target for truncation. Site-specific recombination has been demonstrated to operate on these terminally located sites. By introducing normal B chromosomes into lines with engineered mini-B chromosomes, the latter can be increased in copy number, which provides the potential to augment the expression of the introduced genes. Because the vast majority of plant species have the same telomere sequence, the truncating transgenes should be effective in most plants to generate engineered minichromosomes. Such chromosomes establish the means to add or subtract multiple transgenes, multigene complexes, or whole biochemical pathways to plants to change their properties for agronomic applications or to use plants as factories for the production of foreign proteins or metabolites.     

Vitexin prevents Aβ proteotoxicity in transgenic Caenorhabditis elegans model of Alzheimer's disease by modulating unfolded protein response

Alzheimer's disease (AD) accounts for an estimated 60% to 80% of all dementia cases. The present study is aimed at evaluating the neuroprotective efficacy of vitexin, an apigenin flavone glycoside using transgenic Caenorhabditis elegans strain (CL2006) of AD. The neuroprotective effect of vitexin was determined using physiological assays, quantitative polymerase chain reaction, and Western blotting. The results of survival and paralysis assay indicate that vitexin (200 μM) significantly extended the lifespan of the nematodes. Vitexin‐treated nematodes showed a significant reduction in the expression of Aβ, ace‐1, and ace‐2 genes when compared to control. Further, vitexin significantly upregulated the expression of acr‐8 and dnj‐14, and increased the lifespan of the nematodes. Vitexin was also found to modulate the unfolded protein response genes (hsp‐4, pek‐1, ire‐1, and xbp‐1) and suppress the expression of Aβ. Overall, the results show that vitexin acts as a neuroprotective agent and protects transgenic C. elegans strains from Aβ proteotoxicity.     

Human leg model predicts ankle muscle-tendon morphology, state, roles and energetics in walking

A common feature in biological neuromuscular systems is the redundancy in joint actuation. Understanding how these redundancies are resolved in typical joint movements has been a long-standing problem in biomechanics, neuroscience and prosthetics. Many empirical studies have uncovered neural, mechanical and energetic aspects of how humans resolve these degrees of freedom to actuate leg joints for common tasks like walking. However, a unifying theoretical framework that explains the many independent empirical observations and predicts individual muscle and tendon contributions to joint actuation is yet to be established. Here we develop a computational framework to address how the ankle joint actuation problem is resolved by the neuromuscular system in walking. Our framework is founded upon the proposal that a consideration of both neural control and leg muscle-tendon morphology is critical to obtain predictive, mechanistic insight into individual muscle and tendon contributions to joint actuation. We examine kinetic, kinematic and electromyographic data from healthy walking subjects to find that human leg muscle-tendon morphology and neural activations enable a metabolically optimal realization of biological ankle mechanics in walking. This optimal realization (a) corresponds to independent empirical observations of operation and performance of the soleus and gastrocnemius muscles, (b) gives rise to an efficient load-sharing amongst ankle muscle-tendon units and (c) causes soleus and gastrocnemius muscle fibers to take on distinct mechanical roles of force generation and power production at the end of stance phase in walking. The framework outlined here suggests that the dynamical interplay between leg structure and neural control may be key to the high walking economy of humans, and has implications as a means to obtain insight into empirically inaccessible features of individual muscle and tendons in biomechanical tasks.