Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex

Protein kinase C (PKC) δ plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKCδ generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKCδ isoform named PKCδIX (Genebank Accession No. HQ840432). PKCδIX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKCδ. PKCδIX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKCδIX provided a possibility that this PKCδ isozyme functions as a novel dominant-negative form for PKCδ due to its lack of the ATP-binding domain that is required for the kinase activity of PKCδ. Indeed, overexpression of PKCδIX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKCδIX protein could competitively inhibit the kinase activity of PKCδ. We conclude that PKCδIX can function as a natural dominant-negative inhibitor of PKCδ in vivo.     

Effects of precipitation on photosynthesis and water potential in Andropogon gerardii and Schizachyrium scoparium in a southern mixed grass prairie

In grassland ecosystems, spatial and temporal variability in precipitation is a key driver of species distributions and population dynamics. We experimentally manipulated precipitation to understand the physiological basis for differences in responses of species to water availability in a southern mixed grass prairie. We focused on the performance of two dominant C₄ grasses, Andropogon gerardii Vitman and Schizachyrium scoparium (Michx.) Nash, in treatments that received ambient rainfall, half of ambient rainfall (“drought” treatment), or approximately double ambient rainfall (“irrigated” treatment). Water potentials of S. scoparium were lower than A. gerardii, suggesting superior ability to adjust to water deficit in S. scoparium. Additionally, drought reduced photosynthesis to a greater extent in A. gerardii compared to S. scoparium. Leaf-level photosynthesis rates were similar in ambient and irrigated treatments, but were significantly lower in the drought treatment. Although stomatal conductance was reduced by drought, this was not limiting for photosynthesis. Leaf δ¹³C values were decreased by drought, caused by an increase in C_i/C_a. Chlorophyll fluorescence measures indicated light-harvesting rates were highest in irrigated treatments, and were lower in ambient and drought treatments. Moreover, drought resulted in a greater proportion of absorbed photon energy being lost via thermal pathways. Reductions in photosynthesis came as a result of non-stomatal limitations in the C₄ cycle. Our results provide mechanistic support for the hypothesis that S. scoparium is more drought tolerant than A. gerardii.     

Part 2: Design, synthesis and evaluation of hydroxyproline-derived α2δ ligands

Conformational constraint has been used to design a potent series of α₂δ ligands derived from the readily available starting material (2S,4R)-hydroxy-l-proline. The ligands have improved physicochemistry and potency compared to their linear counterparts (described in our earlier publication) and the lead compound has been progressed to clinical development.     

Part 1: N-alkylated glycines as potent α2δ ligands

A new series of glycine-derived ligands of the α₂δ subunit of voltage gated calcium channels is described. Several novel compounds (7) based on (6) were prepared that possessed a potency <100 nM in the α₂δ binding assay.     

Phosphoinositide 3-kinase delta (PI3Kδ) in leukocyte signaling and function

PI3Kδ is a lipid kinase of the PI3K class IA family involved in early signaling events of leukocytes responding to a wide variety of stimuli. The leukocyte specificity of PI3Kδ is defined by its expression, whereas its signaling function is via the production of phosphoinositide 3,4,5-triphosphates at the proximity of activated receptors for recruiting other signaling molecules. The importance of PI3Kδ in B cell development and function is most apparent, and its role in other leukocyte cell types can be easily demonstrated as well. PI3Kδ participates in the development, activation and migration of T cells and NK cells. The role of PI3Kδ in myeloid cell activities, such as inflammation driven cell infiltration, neutrophil oxidative burst, immune complex mediated macrophage activation, as well as mast cell maturation and degranulation, has been well illustrated in various studies. As a result of the broad effects of PI3Kδ in leukocyte functions, the disruption of PI3Kδ expression or activity leads to decreased inflammatory and immune responses in vivo. The protective role of PI3Kδ inactivation in animal models of arthritis, asthma or obstructive respiratory diseases has been demonstrated. These findings suggest the potential efficacy achievable with PI3Kδ inhibitors in the treatment of autoimmune and respiratory diseases.     

Hiding in plain sight: Uncovering a new function of vitamin A in redox signaling

The protein kinase Cδ signalosome modulates the generation of acetyl-Coenzyme A from glycolytic sources. This module is composed of four interlinked components: PKCδ, the signal adapter p66Shc, cytochrome c, and vitamin A. It resides in the intermembrane space of mitochondria, and is at the center of a feedback loop that senses upstream the redox balance between oxidized and reduced cytochrome c as a measure of the workload of the respiratory chain, and transmits a forward signal to the pyruvate dehydrogenase complex to adjust the flux of fuel entering the tricarboxylic acid cycle. The novel role of vitamin A as co-activator and potential electron carrier, required for redox activation of PKCδ, is discussed. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.     

Emerging role of NF-κB signaling in the induction of senescence-associated secretory phenotype (SASP)

The major hallmark of cellular senescence is an irreversible cell cycle arrest and thus it is a potent tumor suppressor mechanism. Genotoxic insults, e.g. oxidative stress, are important inducers of the senescent phenotype which is characterized by an accumulation of senescence-associated heterochromatic foci (SAHF) and DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS). Interestingly, senescent cells secrete pro-inflammatory factors and thus the condition has been called the senescence-associated secretory phenotype (SASP). Emerging data has revealed that NF-κB signaling is the major signaling pathway which stimulates the appearance of SASP. It is known that DNA damage provokes NF-κB signaling via a variety of signaling complexes containing NEMO protein, an NF-κB essential modifier, as well as via the activation of signaling pathways of p38MAPK and RIG-1, retinoic acid inducible gene-1. Genomic instability evoked by cellular stress triggers epigenetic changes, e.g. release of HMGB1 proteins which are also potent enhancers of inflammatory responses. Moreover, environmental stress and chronic inflammation can stimulate p38MAPK and ceramide signaling and induce cellular senescence with pro-inflammatory responses. On the other hand, two cyclin-dependent kinase inhibitors, p16INK4a and p14ARF, are effective inhibitors of NF-κB signaling. We will review in detail the signaling pathways which activate NF-κB signaling and trigger SASP in senescent cells.     

Energetics and forces of the Dionaea muscipula trap closing

The Venus flytrap is the most famous carnivorous plant. The electrical stimulus between a midrib and a lobe closes the Venus flytrap upper leaf in 0.3 s without mechanical stimulation of trigger hairs. Here we present results for direct measurements of the closing force of the trap of Dionaea muscipula Ellis after mechanical or electrical stimulation of the trap using the piezoelectric thin film or Fuji Prescale indicating sensor film. The closing force was 0.14 N and the corresponding pressure between rims of two lobes was 38 kPa. We evaluated theoretically using the Hydroelastic Curvature Model and compared with experimental data velocity, acceleration and kinetic energy from the time dependencies of distance between rims of lobes during the trap closing. The Charge Stimulation Method was used for trap electrostimulation between the midrib and lobes. From the dependence of voltage between two Ag/AgCl electrodes in the midrib and one of the lobes, we estimated electrical charge, current, resistance, electrical energy and electrical power dependencies on time during electrostimulation of the trap.     

Blind assessment of vertebrate taxonomic diversity across spatial scales by clustering environmental DNA metabarcoding sequences

Human activities impact all ecosystems on Earth, which urges scientists to better understand biodiversity changes across temporal and spatial scales. Environmental DNA (eDNA) metabarcoding is a promising non-invasive method to assess species composition in a wide range of ecosystems. Yet, this method requires the completeness of a reference database, i.e. a list of DNA sequences attached to each species of the regional pool, which is rarely met. As an alternative, molecular operational taxonomic units (MOTUs) can be extracted as clusters of sequences. However, the extent to which the diversity of MOTUs can predict the diversity of species across spatial scales is unknown. Here, we used 196 samples along the Rhone river (France) for which the reference database is complete to assess whether a blind eDNA approach can reliably predict the ground-truth number of species at different spatial scales. Using the 12S rDNA teleo primer, we curated and clustered 60 million sequences into MOTUs using a new assembled bioinformatic pipeline. We show that stringent quality filters were necessary to remove artefact noise, notably MOTUs present in a single PCR replicate, which represented 55% of MOTUs (103). Post-clustering cleaning also removed 19 additional erroneous MOTUs and only discarded one truly present species. We then show that the diversity of retained fish MOTUs accurately predicted the local (α, r = 0.98) and regional (γ) ground-truth species diversity (67 MOTUs versus 63 species), but also the species dissimilarity between samples (β-diversity, r = 0.98). This work paves the way towards extending the use of eDNA metabarcoding in community ecology and biogeography despite major gaps in genetic reference databases.     

Design,synthesis, and biological activity of new endomorphin analogs with multi-site modifications

Endomorphin (EM)-1 and EM-2 are the most effective endogenous analgesics with efficient separation of analgesia from the risk of adverse effects. Poor metabolic stability and ineffective analgesia after peripheral administration were detrimental for the use of EMs as novel clinical analgesics. Therefore, here, we aimed to establish new EM analogs via introducing different bifunctional d-amino acids at position 2 of [(2-furyl)Map⁴]EMs. The combination of [(2-furyl)Map⁴]EMs with D-Arg² or D-Cit² yielded analogs with enhanced binding affinity to the μ-opioid receptor (MOR) and increased stability against enzymatic degradation (t_1/2 > 300 min). However, the agonistic activities of these analogs toward MOR were slightly reduced. Similar to morphine, peripheral administration of the analog [D-Cit², (2-furyl)Map⁴]EM-1 (10) significantly inhibited the pain behavior of mice in multiple pain models. In addition, this EM-1 analog was associated with reduced tolerance, less effect on gastrointestinal mobility, and no significant motor impairment. Compared to natural EMs, the EM analogs synthesized herein had enhanced metabolic stability, bioavailability, and analgesic properties.