Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury: efficacy and mechanism

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P<0.05; n=4 pigs). PostC induced by four cycles of 30-s reperfusion/reocclusion at the onset of reperfusion after 4 h ischemia reduced muscle infarction from 44+/-2 to 22+/-2% at 48 h reperfusion. This infarct protective effect of PostC was mimicked by intravenous injection of the mPTP opening inhibitor cyclosporin A or NIM-811 (10 mg/kg) at 5 min before the end of 4 h ischemia and was abolished by intravenous injection of the mPTP opener atractyloside (10 mg/kg) at 5 min before PostC (P<0.05; n=4-5 pigs). PostC or intravenous cyclosporin A injection at 5 min before reperfusion caused a decrease in muscle myeloperoxidase activity and mitochondrial free Ca2+ concentration and an increase in muscle ATP content after 4 h ischemia and 2 h reperfusion compared with the time-matched controls. These effects of PostC were abolished by intravenous injection of atractyloside at 5 min before PostC (P<0.05; n=6 pigs). These observations support our hypothesis that PostC is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mPTP.     

An essential role for the proximal but not the distal cytoplasmic tail of glycoprotein M in murid herpesvirus 4 infection

Murid herpesvirus-4 (MuHV-4) provides a tractable model with which to define common, conserved features of gamma-herpesvirus biology. The multi-membrane spanning glycoprotein M (gM) is one of only 4 glycoproteins that are essential for MuHV-4 lytic replication. gM binds to gN and is thought to function mainly secondary envelopment and virion egress, for which several predicted trafficking motifs in its C-terminal cytoplasmic tail could be important. We tested the contribution of the gM cytoplasmic tail to MuHV-4 lytic replication by making recombinant viruses with varying C-terminal deletions. Removing an acidic cluster and a distal YXXPhi motif altered the capsid distribution somewhat in infected cells but had little effect on virus replication, either in vitro or in vivo. In contrast, removing a proximal YXXPhi motif as well completely prevented productive replication. gM was still expressed, but unlike its longer forms showed only limited colocalization with co-transfected gN, and in the context of whole virus appeared to support gN expression less well. We conclude that some elements of the gM cytoplasmic tail are dispensible for MuHV-4 replication, but the tail as a whole is not.     

Protein kinase A RII-like (R2D2) proteins exhibit differential localization and AKAP interaction

A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an amphipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process.     

Different thermodynamic binding mechanisms and peptide fine specificities associated with a panel of structurally similar high-affinity T cell receptors

To understand the mechanisms that govern T cell receptor (TCR)-peptide MHC (pMHC) binding and the role that different regions of the TCR play in affinity and antigen specificity, we have studied the TCR from T cell clone 2C. High-affinity mutants of the 2C TCR that bind QL9-L(d) as a strong agonist were generated previously by site-directed mutagenesis of complementarity determining regions (CDRs) 1beta, 2alpha, 3alpha, or 3beta. We performed isothermal titration calorimetry to assess whether they use similar thermodynamic mechanisms to achieve high affinity for QL9-L(d). Four of the five TCRs examined bound to QL9-L(d) in an enthalpically driven, entropically unfavorable manner. In contrast, the high-affinity CDR1beta mutant resembled the wild-type 2C TCR interaction, with favorable entropy. To assess fine specificity, we measured the binding and kinetics of these mutants for both QL9-L(d) and a single amino acid peptide variant of QL9, called QL9-Y5-L(d). While 2C and most of the mutants had equal or higher affinity for the Y5 variant than for QL9, mutant CDR1beta exhibited 8-fold lower affinity for Y5 compared to QL9. To examine possible structural correlates of the thermodynamic and fine specificity signatures of the TCRs, the structure of unliganded QL9-L(d) was solved and compared to structures of the 2C TCR/QL9-L(d) complex and three high-affinity TCR/QL9-L(d) complexes. Our findings show that the QL9-L(d) complex does not undergo major conformational changes upon binding. Thus, subtle changes in individual CDRs account for the diverse thermodynamic and kinetic binding mechanisms and for the different peptide fine specificities.     

Wagner and Dollo: a stochastic duet by composing two parsimonious solos

New contributions toward generalizing evolutionary models expand greatly our ability to analyze complex evolutionary characters and advance phylogeny reconstruction. In this article, we extend the binary stochastic Dollo model to allow for multi-state characters. In doing so, we align previously incompatible Wagner and Dollo parsimony principles under a common probabilistic framework by embedding arbitrary continuous-time Markov chains into the binary stochastic Dollo model. This approach enables us to analyze character traits that exhibit both Dollo and Wagner characteristics throughout their evolutionary histories. Utilizing Bayesian inference, we apply our novel model to analyze intron conservation patterns and the evolution of alternatively spliced exons. The generalized framework we develop demonstrates potential in distinguishing between phylogenetic hypotheses and providing robust estimates of evolutionary rates. Moreover, for the two applications analyzed here, our framework is the first to provide an adequate stochastic process for the data. We discuss possible extensions to the framework from both theoretical and applied perspectives.     

Towards a general theory of neural computation based on prediction by single neurons

Although there has been tremendous progress in understanding the mechanics of the nervous system, there has not been a general theory of its computational function. Here I present a theory that relates the established biophysical properties of single generic neurons to principles of Bayesian probability theory, reinforcement learning and efficient coding. I suggest that this theory addresses the general computational problem facing the nervous system. Each neuron is proposed to mirror the function of the whole system in learning to predict aspects of the world related to future reward. According to the model, a typical neuron receives current information about the state of the world from a subset of its excitatory synaptic inputs, and prior information from its other inputs. Prior information would be contributed by synaptic inputs representing distinct regions of space, and by different types of non-synaptic, voltage-regulated channels representing distinct periods of the past. The neuron's membrane voltage is proposed to signal the difference between current and prior information ("prediction error" or "surprise"). A neuron would apply a Hebbian plasticity rule to select those excitatory inputs that are the most closely correlated with reward but are the least predictable, since unpredictable inputs provide the neuron with the most "new" information about future reward. To minimize the error in its predictions and to respond only when excitation is "new and surprising," the neuron selects amongst its prior information sources through an anti-Hebbian rule. The unique inputs of a mature neuron would therefore result from learning about spatial and temporal patterns in its local environment, and by extension, the external world. Thus the theory describes how the structure of the mature nervous system could reflect the structure of the external world, and how the complexity and intelligence of the system might develop from a population of undifferentiated neurons, each implementing similar learning algorithms.     

PTP1B inhibitors: synthesis and evaluation of difluoro-methylenephosphonate bioisosteres on a sulfonamide scaffold

We have synthesized and evaluated a series of triaryl sulfonamide-based PTP1B inhibitors in which a difluoro-methylenephosphonate group of a potent lead has been replaced by potential bioisosteric replacements. Several mono- or di-charged compounds (8a, 8b, and 15a) were shown exhibit inhibitory activity in the low micromolar range, demonstrating the feasibility of using this approach in identifying non-phosphonate pTyr mimetics in a small molecular scaffold. These results also provide a useful indication of the relative effectiveness of these pTyr mimetics.     

The effects of temperature and nutrients on the growth and dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms

In temperate latitudes, toxic cyanobacteria blooms often occur in eutrophied ecosystems during warm months. Many common bloom-forming cyanobacteria have toxic and non-toxic strains which co-occur and are visually indistinguishable but can be quantified molecularly. Toxic Microcystis cells possess a suite of microcystin synthesis genes (mcyA–mcyJ), while non-toxic strains do not. For this study, we assessed the temporal dynamics of toxic and non-toxic strains of Microcystis by quantifying the microcystin synthetase gene (mcyD) and the small subunit ribosomal RNA gene, 16S (an indicator of total Microcystis), from samples collected from four lakes across the Northeast US over a two-year period. Nutrient concentrations and water quality were measured and experiments were conducted which examined the effects of elevated levels of temperatures (+4 °C), nitrogen, and phosphorus on the growth rates of toxic and non-toxic strains of Microcystis. During the study, toxic Microcystis cells comprised between 12% and 100% of the total Microcystis population in Lake Ronkonkoma, NY, and between 0.01% and 6% in three other systems. In all lakes, molecular quantification of toxic (mcyD-possessing) Microcystis was a better predictor of in situ microcystin levels than total cyanobacteria, total Microcystis, chlorophyll a, or other factors, being significantly correlated with the toxin in every lake studied. Experimentally enhanced temperatures yielded significantly increased growth rates of toxic Microcystis in 83% of experiments conducted, but did so for non-toxic Microcystis in only 33% of experiments, suggesting that elevated temperatures yield more toxic Microcystis cells and/or cells with more mcyD copies per cell, with either scenario potentially yielding more toxic blooms. Furthermore, concurrent increases in temperature and P concentrations yielded the highest growth rates of toxic Microcystis cells in most experiments suggesting that future eutrophication and climatic warming may additively promote the growth of toxic, rather than non-toxic, populations of Microcystis, leading to blooms with higher microcystin content.