Discovery of renin inhibitors containing a simple aspartate binding moiety that imparts reduced P450 inhibition

Discovery of potent renin inhibitors which contain a simplified alkylamino Asp-binding group and exhibit improved selectivity for renin over Cyp3A4 is described. Structure-function results in this series are rationalized based on analysis of selected compounds bound to renin, and the contribution of each molecular feature leading to the reduced P450 inhibition is quantified.     

Development of Lactococcus lactis encoding fluorescent proteins,GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.     

The Ultimate Basis of the Caching Preferences of Rodents, and the Oak-Dispersal Syndrome: Tannins, Insects, and Seed Germination

We have shown that eastern gray squirrels and other animalsconsistently prefer to store intact acorns from the red oakgroup rather than those from the white oak group. We hypothesizedthat the ultimate advantage to this behavior comes from thedormancy of red oak acorns. Acorns of the white oak group germinateearly in the autumn; thus, we hypothesize that avoiding germinationis the primary selective advantage to the preference for cachingred oak acorns. Here, we test two alternative hypotheses toexplain the benefits of this caching preference: 1) storingred oak acorns allows the high tannin concentrations in redoak acorns to decline (making them more palatable), and 2) storingred oak acorns minimizes losses to insects, presuming they areless infested with insects. We also test the effect of germinationschedule on squirrel caching preferences directly, by presentingthem with dormant red oak acorns, and red oak acorns about togerminate. We find no evidence that tannin concentrations inred oak acorns decline, although tannin levels did decline inour white oak acorns. We found high losses to insect infestationsin one white oak species, but a second white oak species lostvery little mass to insects. Finally, we found that germinationschedule directly affects squirrel caching preferences: redoak acorns that are near germination are treated like whiteoak acorns. We conclude that the primary advantage to the preferencefor caching red oak acorns is that they are less perishable,due to their dormancy. We discuss the effects of this preferenceon the dispersal of red and white oak acorns, and the subsequenteffects of differential dispersal on the ecology and evolutionof oaks.     

Genetic architecture modulates diet-induced hepatic mRNA and miRNA expression profiles in Diversity Outbred mice

Genetic approaches in model organisms have consistently demonstrated that molecular traits such as gene expression are under genetic regulation, similar to clinical traits. The resulting expression quantitative trait loci (eQTL) have revolutionized our understanding of genetic regulation and identified numerous candidate genes for clinically relevant traits. More recently, these analyses have been extended to other molecular traits such as protein abundance, metabolite levels, and miRNA expression. Here, we performed global hepatic eQTL and microRNA expression quantitative trait loci (mirQTL) analysis in a population of Diversity Outbred mice fed two different diets. We identified several key features of eQTL and mirQTL, namely differences in the mode of genetic regulation (cis or trans) between mRNA and miRNA. Approximately 50% of mirQTL are regulated by a trans-acting factor, compared to ∼25% of eQTL. We note differences in the heritability of mRNA and miRNA expression and variance explained by each eQTL or mirQTL. In general, cis-acting variants affecting mRNA or miRNA expression explain more phenotypic variance than trans-acting variants. Finally, we investigated the effect of diet on the genetic architecture of eQTL and mirQTL, highlighting the critical effects of environment on both eQTL and mirQTL. Overall, these data underscore the complex genetic regulation of two well-characterized RNA classes (mRNA and miRNA) that have critical roles in the regulation of clinical traits and disease susceptibility     

Diverse Heterologous Primary Infections Radically Alter Immunodominance Hierarchies and Clinical Outcomes Following H7N9 Influenza Challenge in Mice

The recent emergence of a novel H7N9 influenza A virus (IAV) causing severe human infections in China raises concerns about a possible pandemic. The lack of pre-existing neutralizing antibodies in the broader population highlights the potential protective role of IAV-specific CD8+ cytotoxic T lymphocyte (CTL) memory specific for epitopes conserved between H7N9 and previously encountered IAVs. In the present study, the heterosubtypic immunity generated by prior H9N2 or H1N1 infections significantly, but variably, reduced morbidity and mortality, pulmonary virus load and time to clearance in mice challenged with the H7N9 virus. In all cases, the recall of established CTL memory was characterized by earlier, greater airway infiltration of effectors targeting the conserved or cross-reactive H7N9 IAV peptides; though, depending on the priming IAV, each case was accompanied by distinct CTL epitope immunodominance hierarchies for the prominent K^bPB1₇₀₃, D^bPA₂₂₄, and D^bNP₃₆₆ epitopes. While the presence of conserved, variable, or cross-reactive epitopes between the priming H9N2 and H1N1 and the challenge H7N9 IAVs clearly influenced any change in the immunodominance hierarchy, the changing patterns were not tied solely to epitope conservation. Furthermore, the total size of the IAV-specific memory CTL pool after priming was a better predictor of favorable outcomes than the extent of epitope conservation or secondary CTL expansion. Modifying the size of the memory CTL pool significantly altered its subsequent protective efficacy on disease severity or virus clearance, confirming the important role of heterologous priming. These findings establish that both the protective efficacy of heterosubtypic immunity and CTL immunodominance hierarchies are reflective of the immunological history of the host, a finding that has implications for understanding human CTL responses and the rational design of CTL-mediated vaccines.     

An epitope of rice threonine- and hydroxyproline-rich glycoprotein is common to cell wall and hydrophobic plasma-membrane glycoproteins

A monoclonal antibody, LM1, has been derived that has a high affinity for an epitope of hydroxyproline-rich glycoproteins (HRGPs). In suspension-cultured rice (Oryza sativa L.) cells the epitope is carried by three major proteins with different biochemical properties. The most abundant is the 95-kDa extracellular rice extensin, a threonine- and hydroxyproline-rich glycoprotein (THRGP) occurring in the cell wall and secreted into the medium. This THRGP can be selectively oxidatively cross-linked in the presence of hydrogen peroxide and an endogenous peroxidase with the result that it does not enter a protein gel. A second polypeptide with the LM1 epitope (180 kDa), also occurring in the suspension-cultured cells and medium, is not oxidatively cross-linked. Three further polypeptides (52, 65 and 110 kDa) with the characteristics of hydrophobic proteins of the plasma-membrane also carry the LM1 epitope as determined by immuno-blotting of detergent/aqueous partitions of a plasma-membrane preparation and immuno-fluorescence studies with rice protoplasts. At the rice root apex the LM1 epitope is carried by four glycoproteins and is developmentally regulated. The major locations of the epitope are at the surface of cells associated with the developing protoxylem and metaxylem in the stele, the longitudinal radial walls of epidermal cells and a sheath-like structure at the surface of the root apex.Abbreviations AGP arabinogalactan protein - ELISA enzyme-linked immunosorbent assay - HRGP hydroxyproline-rich glycoprotein - THRGP threonine- and hydroxyproline-rich glycoprotein This work was supported by The Leverhulme Trust. We also acknowledge support from The Royal Society and thank Prof. L.A. Staehelin for the carrot extensin, N. Stacey for the rice cell culture and Dr. J. Keen for protein sequencing.     

Radiographic Evaluation of Appendicular Skeletal Maturation in the Common Marmoset (Callithrix jacchus)

The thoracic appendicular skeletal development of five common marmosets was monitored radiographically at weekly intervals from 1 day to 94 weeks of age and the times of appearance and fusion of 47 ossification centers were recorded. A range and average age for the appearance and fusion of each ossification center were calculated and compared to data available for the rhesus monkey and man.     

Mediating molecular recognition by methionine oxidation: conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin

The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca(2+)-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145 leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, (1)H(alpha), (13)C(alpha), and (13)CO chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using (13)C-filtered, (13)C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.     

Regulation of Hermissenda K+ Channels by Cytoplasmic and Membrane-Associated C-Kinase

Pharmacologic activation of endogenous protein kinase C (PKC) together with elevation of the intracellular Ca2+ level was previously shown to cause reduction of two voltage-dependent K+ currents (IA and ICa2+-K+) across the soma membrane of the type B photoreceptor within the eye of the mollusc Hermissenda crassicornis. Similar effects were also found to persist for days after acquisition of a classically conditioned response. Also, the state of phosphorylation of a low-molecular-weight protein was changed only within the eyes of conditioned Hermissenda. To examine the role of PKC in causing K+ current changes as well as changes of phosphorylation during conditioning (and possibly other physiologic contexts), we studied here the effects of endogenous PKC activation and exogenous PKC injection on phosphorylation and K+ channel function. Several phosphoproteins (20, 25, 56, and 165 kilodaltons) showed differences in phosphorylation in response to PKC activators applied to intact nervous systems or to isolated eyes. Specific differences were observed for membrane and cytosolic fractions in response to both the phorbol ester 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA) or exogenous PKC in the presence of Ca2+ and phosphatidylserine/diacylglycerol. Type B cells pretreated with DPBA responded to PKC injection with a persistent reduction of K+ currents. In the absence of DPBA, PKC injection also caused K+ current reduction only following Ca2+ loading conditions. However, the direct effect of PKC injection in the absence of DPBA was only to increase ICa2+-K+. According to a proposed model, the amplitude of the K+ currents would depend on the steady-state balance of effects mediated by PKC within the cytoplasm and membrane-associated PKC. The model further specifies that the effects on K+ currents of cytoplasmic PKC require an intervening proteolytic step. Such a model predicts that increasing the concentration of cytoplasmic protease, e.g., with trypsin, will increase K+ currents, whereas blocking endogenous protease, e.g., with leupeptin, will decrease K+ currents. These effects should be opposed by preexposure of the cells to DPBA. Furthermore, prior injection of leupeptin should block or reverse the effects of subsequent injection of PKC into the type B cell. All of these predictions were confirmed by results reported here. Taken together, the results of this and previous studies suggest that PKC regulation of membrane excitability critically depends on its cellular locus. The implications of such function for long-term physiologic transformations are discussed.