Measuring the Binding Stoichiometry of HIV-1 Gag to Very-Low-Density Oligonucleotide Surfaces Using Surface Plasmon Resonance Spectroscopy

The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions, and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)(n) oligonucleotides using surface plasmon resonance (SPR) spectroscopy. We found a single NC protein is able to bind to more than one immobilized oligonucleotide, provided that the oligonucleotides are close enough together. As NC is believed to be the nucleic acid binding domain of Gag, we might expect Gag to show the same complex behavior. We wished to analyze the stoichiometry of Gag binding to oligonucleotides without this complication due to tertiary complex formation. We have therefore analyzed Gag binding to extremely low oligonucleotide density on SPR chips. Such low densities of oligonucleotides are difficult to accurately quantitate. We have determined by Fourier transform ion cyclotron (FTICR) mass spectrometry that four molecules of NC bind to d(TG)(10) (a 20-base oligonucleotide). We developed a method of calibrating low-density surfaces using NC calibration injections. Knowing the maximal response and the stoichiometry of binding, we can precisely determine the amount of oligonucleotide immobilized at these very-low-density surfaces (<1 Response Unit). Using this approach, we have measured the binding of Gag to d(TG)(10). Gag binds to a 20-mer with a stoichiometry of greater than 4. This suggests that once Gag is bound to the immobilized oligonucleotide, additional Gag molecules can bind to this complex.     

A systems biology approach to model neural stem cell regulation by notch, shh, wnt, and EGF signaling pathways

The Notch, Sonic Hedgehog (Shh), Wnt, and EGF pathways have long been known to influence cell fate specification in the developing nervous system. Here we attempted to evaluate the contemporary knowledge about neural stem cell differentiation promoted by various drug-based regulations through a systems biology approach. Our model showed the phenomenon of DAPT-mediated antagonism of Enhancer of split [E(spl)] genes and enhancement of Shh target genes by a SAG agonist that were effectively demonstrated computationally and were consistent with experimental studies. However, in the case of model simulation of Wnt and EGF pathways, the model network did not supply any concurrent results with experimental data despite the fact that drugs were added at the appropriate positions. This paves insight into the potential of crosstalks between pathways considered in our study. Therefore, we manually developed a map of signaling crosstalk, which included the species connected by representatives from Notch, Shh, Wnt, and EGF pathways and highlighted the regulation of a single target gene, Hes-1, based on drug-induced simulations. These simulations provided results that matched with experimental studies. Therefore, these signaling crosstalk models complement as a tool toward the discovery of novel regulatory processes involved in neural stem cell maintenance, proliferation, and differentiation during mammalian central nervous system development. To our knowledge, this is the first report of a simple crosstalk map that highlights the differential regulation of neural stem cell differentiation and underscores the flow of positive and negative regulatory signals modulated by drugs.     

Evaluation of macrocyclic Grb2 SH2 domain-binding peptide mimetics prepared by ring-closing metathesis of C-terminal allylglycines with an N-terminal beta-vinyl-substituted phosphotyrosyl mimetic

Preferential binding of ligands to Grb2 SH2 domains in beta-bend conformations has made peptide cyclization a logical means of effecting affinity enhancement. This is based on the concept that constraint of open-chain sequences to bend geometries may reduce entropy penalties of binding. The current study extends this approach by undertaking ring-closing metathesis (RCM) macrocyclization between i and i+3 residues through a process involving allylglycines and beta-vinyl-functionalized residues. Ring closure in this fashion results in minimal macrocyclic tetrapeptide mimetics. The predominant effects of such macrocyclization on Grb2 SH2 domain binding affinity were increases in rates of association (from 7- to 16-fold) relative to an open-chain congener, while decreases in dissociation rates were less pronounced (approximately 2-fold). The significant increases in association rates were consistent with pre-ordering of solution conformations to near those required for binding. Data from NMR experiments and molecular modeling simulations were used to interpret the binding results. An understanding of the conformational consequences of such i to i+3 ring closure may facilitate its application to other systems where bend geometries are desired.     

Tissue- and gene-specific recruitment of steroid receptor coactivator-3 by thyroid hormone receptor during development

Numerous coactivators that bind nuclear hormone receptors have been isolated and characterized in vitro. Relatively few studies have addressed the developmental roles of these cofactors in vivo. By using the total dependence of amphibian metamorphosis on thyroid hormone (T3) as a model, we have investigated the role of steroid receptor coactivator 3 (SRC3) in gene activation by thyroid hormone receptor (TR) in vivo. First, expression analysis showed that SRC3 was expressed in all tadpole organs analyzed. In addition, during natural as well as T3-induced metamorphosis, SRC3 was up-regulated in both the tail and intestine, two organs that undergo extensive transformations during metamorphosis and the focus of the current study. We then performed chromatin immunoprecipitation assays to investigate whether SRC3 is recruited to endogenous T3 target genes in vivo in developing tadpoles. Surprisingly, we found that SRC3 was recruited in a gene- and tissue-dependent manner to target genes by TR, both upon T3 treatment of premetamorphic tadpoles and during natural metamorphosis. In particular, in the tail, SRC3 was not recruited in a T3-dependent manner to the target TRbetaA promoter, suggesting either no recruitment or constitutive association. Finally, by using transgenic tadpoles expressing a dominant negative SRC3 (F-dnSRC3), we demonstrated that F-dnSRC3 was recruited in a T3-dependent manner in both the intestine and tail, blocking the recruitment of endogenous coactivators and histone acetylation. These results suggest that SRC3 is utilized in a gene- and tissue-specific manner by TR during development.     

Immunomodulation by 'Immuplus (AquaImmu)' in giant freshwater prawn, Macrobrachium rosenbergii (De Man)

ImmuPlus, a polyherbal commercial formulation was used to modulate the immune system of commercially important giant freshwater prawn M. rosenbergii. The prawns were fed with basal diet supplemented with ImmuPlus at 1g/kg feed for 4 weeks. Results showed that the phenoloxidase activity (PO), haemagglutination and lysozyme activities were significantly elevated in ImmuPlus-fed prawn up to 3 weeks of feeding and declined after 4 weeks of feeding. The total protein level in ImmuPlus-fed prawn raised up to 2nd week of feeding. Incorporation of ImmuPlus at the rate of 1g/kg feed in the diet of prawn for 3 weeks may be beneficial in raising the immune status of prawn.     

Augmented heme oxygenase-1 induces prostaglandin uptake via the prostaglandin transporter in micro-vascular endothelial cells

Previous studies show that expression of heme oxygenase-1 (HO-1) in endothelial cells results in decreased cyclooxygenase expression and prostaglandin (PG) levels through limiting heme availability. Regulation of PGs, important inflammatory mediators, may contribute to the anti-inflammatory potential of HO-1. Here we examine the effects of HO-1 expression on PG clearance via the prostaglandin transporter (PGT). Endothelial cells expressing human HO-1 via retroviral transfer exhibit approximately 7-fold higher levels of PGT RNA and equivalently elevated uptake of [(3)H]PGE(2). The pattern and extent of uptake and the substrate inhibitory constants of PGE(2), PGF(2alpha), and thromboxane B(2) are similar to those of cloned PGT. Treatment of cells with stannous chloride, an inducer of HO-1, results in increased expression of PGT while incubation of cells expressing human HO-1 with stannic mesophorphyrin, a substrate inhibitor of HO-1, decreases PG uptake. Therefore, PG clearance via PGT may contribute to the cellular regulation of PG levels by HO-1.