pH-dependent Binding Engineering Reveals an FcRn Affinity Threshold That Governs IgG Recycling

The Fc domain of IgG has been the target of multiple mutational studies aimed at altering the pH-dependent IgG/FcRn interaction to modulate IgG pharmacokinetics. These studies have yielded antibody variants with disparate pharmacokinetic characteristics, ranging from extended in vivo half-life to those exhibiting extremely rapid clearance. To better understand pH-dependent binding parameters that govern these outcomes and limit FcRn-mediated half-life extension, we generated a panel of novel Fc variants with high affinity binding at acidic pH that vary in pH 7.4 affinities and assessed pharmacokinetic outcomes. Pharmacokinetic studies in human FcRn transgenic mice and cynomolgus monkeys showed that multiple variants with increased FcRn affinities at acidic pH exhibited extended serum half-lives relative to the parental IgG. Importantly, the results reveal an underappreciated affinity threshold of neutral pH binding that determines IgG recycling efficiency. Variants with pH 7.4 FcRn affinities below this threshold recycle efficiently and can exhibit increased serum persistence. Increasing neutral pH FcRn affinity beyond this threshold reduced serum persistence by offsetting the benefits of increased pH 6.0 binding. Ultra-high affinity binding to FcRn at both acidic and neutral pH leads to rapid serum clearance.     

Numerical Simulation of Dynamic Electro-Mechanical Response of Ionic Polymer-Metal Composites

Ionic Polymer-Metal Composites (IPMC) is an emerging class of Electro-Active Polymer (EAP) materials. IPMC has attractive features, such as high sensitivity and light weight, which are useful for developing novel designs in the fields of bionic actuators, artificial muscles and dynamic sensors. A Finite Element (FE) model was developed for simulating the dynamic electro-mechanical response of an IPMC structure under an external voltage input. A lumped Resistor-Capacitor (RC) model was used to describe the voltage-to-current relationship of a Nafion IPMC film for the computation of electric field intensity. Moreover, the viscoelastic property of the IPMC film was considered in the model and the non-uniform bending behavior was also taken into account. Based on the proposed model and the assumption that the thicknesses of the two electrodes are the same and uniform, the optimal coating thickness of the IPMC electrode was determined. It was demonstrated that the dynamic electro-mechanical response of the IPMC structure can be predicted by the proposed FE model, and the simulation results were in good agreement with the experimental findings.     

Cholesterol modulation of the expression of mitochondrial aconitase in human prostatic carcinoma cells

Mitochondrial aconitase (mACON) is the key enzyme for the citrate oxidation in the mitochondrial Krebs cycle. Cholesterol treatment (10 microg/ml of cholesterol and 1 microg/ml of 25-hydroxycholesterol) for 24 h stimulates mACON enzymatic activity in human prostatic carcinoma cells (PC-3) and hepatoma cells (HepG2). Mevastatin, a cholesterol synthesis antagonist, blocked the effect of cholesterol treatment on mACON. The cholesterol treatment stimulated mACON enzymatic activity, which enhanced the citrate utility but decreased intracellular ATP levels in PC-3 cells. The immunoblotting and transient gene expression assays demonstrated that cholesterol treatment enhances the gene expression of mACON. Mutation of the putative sterol response element (SRE) from GACGCCCCACT to GACGCCCATAT abolished the stimulating effects of cholesterol on the promoter activity of mACON gene. The results suggest that cholesterol treatment induces the mACON gene expression through the SRE signal transduction pathway. Our study demonstrated the deregulation of cholesterol on the citrate metabolism.     

The effects of phenytoin and its metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin on cellular glucose transport

Glucose is the principal fuel for brain metabolism and its movement across the blood-brain barrier depends on Glut1. Impaired glucose transport to the brain may have deleterious consequences. For example, Glut1 deficiency syndrome (Glut1DS) is the result of heterozygous loss of function Glut1 mutation leading to energy failure of the brain and subsequently, epileptic encephalopathy. To preserve the integrity of the energy supply to the brain in patients with compromised glucose transport function, consumption of compounds with glucose transport inhibiting properties should be avoided. Phenytoin is a widely used anticonvulsant that affects carbohydrate metabolism. In this study, the hypothesis that phenytoin and its metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) affect cellular glucose transport was tested. With a focus on Glut1, the effects of phenytoin and HPPH on cellular glucose transport were studied. Glucose uptake assay measuring the zero-trans influx of radioactive-labeled glucose analogues showed that phenytoin and HPPH did not exert immediate effects on erythrocyte Glut1 activity or glucose transport in Hs68 control fibroblasts, Glut1DS primary fibroblasts isolated from two patients, or in rat primary astrocytes. Prolonged exposure to the two compounds could stimulate glucose transport by up to 30-60% over the control level (p <0.05) in Hs68 and Glut1DS fibroblasts as well as in rat astrocytes. The stimulation of glucose transport by HPPH was dose-dependent and accompanied by an up-regulation of GLUT1 mRNA expression (p <0.05). In conclusion, phenytoin and HPPH do not compromise cellular glucose transport. Prolonged exposure to these compounds can modify carbohydrate homeostasis by up-regulating glucose transport in both normal and Glut1DS conditions in vitro.     

Calcium/calmodulin-dependent protein kinase II (CaMKII) localization acts in concert with substrate targeting to create spatial restriction for phosphorylation

Ca2+/calmodulin-dependent protein kinase II (CaMKII) acts in diverse cell types by phosphorylating proteins with key calcium-dependent functions such as synaptic plasticity, electrical excitability, and neurotransmitter synthesis. CaMKII displays calcium-dependent binding to proteins in vitro and translocation to synaptic sites after glutamatergic activity in neurons. We therefore hypothesized that subcellular targeting of CaMKII can direct its substrate specificity in an activity-dependent fashion. Here, we examined whether activity-dependent colocalization of CaMKII and its substrates could result in regulation of substrate phosphorylation in cells. We find that substrates localized at cellular membranes required CaMKII translocation to these compartments to achieve effective phosphorylation. Spatial barriers to phosphorylation could be overcome by translocation and anchoring to the substrate itself or to nearby target proteins within the membrane compartment. In contrast, phosphorylation of a cytoplasmic counterpart of the substrate does not require CaMKII translocation or stable protein-protein binding. Cytosolic phosphorylation is more permissive, exhibiting partial calcium-independence. Localization-dependent substrate specificity can also show more graded levels of regulation within signaling microdomains. We find that colocalization of translocated CaMKII and its substrate to lipid rafts in the plasma membrane can modulate the magnitude of phosphorylation. Thus, dynamic regulation of both substrate and kinase localization provides a powerful and nuanced way to regulate CaMKII signal specificity.     

Natural genetic variation caused by transposable elements in humans

Transposons and transposon-like repetitive elements collectively occupy 44% of the human genome sequence. In an effort to measure the levels of genetic variation that are caused by human transposons, we have developed a new method to broadly detect transposon insertion polymorphisms of all kinds in humans. We began by identifying 606,093 insertion and deletion (indel) polymorphisms in the genomes of diverse humans. We then screened these polymorphisms to detect indels that were caused by de novo transposon insertions. Our method was highly efficient and led to the identification of 605 nonredundant transposon insertion polymorphisms in 36 diverse humans. We estimate that this represents 25-35% of approximately 2075 common transposon polymorphisms in human populations. Because we identified all transposon insertion polymorphisms with a single method, we could evaluate the relative levels of variation that were caused by each transposon class. The average human in our study was estimated to harbor 1283 Alu insertion polymorphisms, 180 L1 polymorphisms, 56 SVA polymorphisms, and 17 polymorphisms related to other forms of mobilized DNA. Overall, our study provides significant steps toward (i) measuring the genetic variation that is caused by transposon insertions in humans and (ii) identifying the transposon copies that produce this variation.     

Dual action of epidermal growth factor: extracellular signal-stimulated nuclear–cytoplasmic export and coordinated translation of selected messenger RNA

We report the first example of a coordinated dual action of epidermal growth factor (EGF) in stimulating the nuclear–cytoplasmic export and translation of a select messenger RNA (mRNA). The effect of EGF is mediated by the RNA-binding protein Grb7 (growth factor receptor–bound protein 7), which serves as an adaptor for a specific mRNA–protein export complex and a translational regulator. Using the κ–opioid receptor (OR [KOR]) as a model, we demonstrate that EGF activates nuclear SHP-2 (Src homology region 2–containing tyrosine phosphatase), which dephosphorylates Grb7 in the nucleus. Hypophosphorylated Grb7 binds to the KOR mRNA and recruits the Hu antigen R–exportin-1 (CRM1) complex to form a nuclear–cytoplasmic export complex that exports KOR mRNA. EGF also activates focal adhesion kinase in the cytoplasm to rephosphorylate Grb7, releasing KOR mRNA for active translation. In summary, this study uncovers a coordinated, dual activity of EGF in facilitating nuclear export of a specific mRNA–protein complex as well as translational activation of the exported mRNA.     

Molecular systematics of Helicoma, Helicomyces and Helicosporium and their teleomorphs inferred from rDNA sequences

Three genera of asexual, helical-spored fungi, Helicoma, Helicomyces and Helicosporium traditionally have been differentiated by the morphology of their conidia and conidiophores. In this paper we assessed their phylogenetic relationships from ribosomal sequences from ITS, 5.8S and partial LSU regions using maximum parsimony, maximum likelihood and Bayesian analysis. Forty-five isolates from the three genera were closely related and were within the teleomorphic genus Tubeufia sensu Barr (Tubeufiaceae, Ascomycota). Most of the species could be placed in one of the seven clades that each received 78% or greater bootstrap support. However none of the anamorphic genera were monophyletic and all but one of the clades contained species from more than one genus. The 15 isolates of Helicoma were scattered through the phylogeny and appeared in five of the clades. None of the four sections within the genus were monophyletic, although species from Helicoma sect. helicoma were concentrated in Clade A. The Helicosporium species also appeared in five clades. The four Helicomyces species were distributed among three clades. Most of the clades supported by sequence data lacked unifying morphological characters. Traditional characters such as the thickness of the conidial filament and whether conidiophores were conspicuous or reduced proved to be poor predictors of phylogenetic relationships. However some combinations of characters including conidium colour and the presence of lateral, tooth-like conidiogenous cells did appear to be predictive of genetic relationships.