Roles of surface residues of intracellular domains of heag potassium channels

Ether-a-go-go potassium channels have large intracellular regions containing ‘Per-Ant-Sim’ (PAS) and cyclic nucleotide binding (cNBD) domains at the N- and C-termini, respectively. In heag1 and heag2 channels, recent studies have suggested that the N- and C-terminal domains interact, and affect activation properties. Here, we have studied the effect of mutations of residues on the surfaces of PAS and cNBD domains. For this, we introduced alanine and lysine mutations in heag1 channels, and recorded currents by two-electrode voltage clamp. In both the PAS domain and the cNBD domain, contiguous areas of conserved residues on the surfaces of these domains were found which affected the activation kinetics of the channel. Next, we investigated possible effects of mutations on domain interactions of PAS and cNBD proteins in heag2 by co-expressing these domain proteins followed by analysis with native gels and western blotting. We found oligomeric association between these domains. Mutations F30A and A609K (on the surfaces of the PAS and cNBD domains, respectively) affected oligomeric compositions of these domains when proteins for PAS and cNBD domains were expressed together. Taken together, the data suggest that the PAS and cNBD domains form interacting oligomers that have roles in channel function.     

Parallel mapping and simultaneous sequencing reveals deletions in BCAN and FAM83H associated with discrete inherited disorders in a domestic dog breed

The domestic dog (Canis familiaris) segregates more naturally-occurring diseases and phenotypic variation than any other species and has become established as an unparalled model with which to study the genetics of inherited traits. We used a genome-wide association study (GWAS) and targeted resequencing of DNA from just five dogs to simultaneously map and identify mutations for two distinct inherited disorders that both affect a single breed, the Cavalier King Charles Spaniel. We investigated episodic falling (EF), a paroxysmal exertion-induced dyskinesia, alongside the phenotypically distinct condition congenital keratoconjunctivitis sicca and ichthyosiform dermatosis (CKCSID), commonly known as dry eye curly coat syndrome. EF is characterised by episodes of exercise-induced muscular hypertonicity and abnormal posturing, usually occurring after exercise or periods of excitement. CKCSID is a congenital disorder that manifests as a rough coat present at birth, with keratoconjunctivitis sicca apparent on eyelid opening at 10–14 days, followed by hyperkeratinisation of footpads and distortion of nails that develops over the next few months. We undertook a GWAS with 31 EF cases, 23 CKCSID cases, and a common set of 38 controls and identified statistically associated signals for EF and CKCSID on chromosome 7 (P_raw 1.9×10⁻¹⁴; P_genome = 1.0×10⁻⁵) and chromosome 13 (P_raw 1.2×10⁻¹⁷; P_genome = 1.0×10⁻⁵), respectively. We resequenced both the EF and CKCSID disease-associated regions in just five dogs and identified a 15,724 bp deletion spanning three exons of BCAN associated with EF and a single base-pair exonic deletion in FAM83H associated with CKCSID. Neither BCAN or FAM83H have been associated with equivalent disease phenotypes in any other species, thus demonstrating the ability to use the domestic dog to study the genetic basis of more than one disease simultaneously in a single breed and to identify multiple novel candidate genes in parallel.

Authors Summary

The Cavalier King Charles Spaniel (CKCS) is popular as a companion breed of dog in many countries worldwide. However, in common with other breeds, it is documented to suffer from a high frequency of inherited disorders, which are largely the result of routine breeding practices. The homogeneous population structure of individual breeds is advantageous for mapping inherited conditions, and we sought to utilise this by mapping two disorders, episodic falling (EF) and congenital keratoconjunctivitis sicca and ichthyosiform dermatosis (CKCSID), using a genome-wide association study approach comprising a set of cases for each condition and a single set of common controls. Independent disease-associated regions were identified for EF and CKCSID, both containing approximately 100 genes. In the absence of any provocative candidate genes, we resequenced both entire regions simultaneously using two cases for each disease and one clinically unaffected control. A 15.7 kb deletion in the BCAN gene was associated with EF and a 1 bp deletion in FAM83H was associated with CKCSID. Neither gene has been associated with similar conditions previously. This investigation highlights how multiple disease-associated mutations can be simultaneously identified in the dog with a minimal set of individuals.     

Confocal microscopy imaging of NR2B-containing NMDA receptors based on fluorescent ifenprodil-like conjugates

We describe the synthesis and pharmacological characterization of a first generation of ifenprodil conjugates 4-7 as fluorescent probes for the confocal microscopy imaging of the NR2B-containing NMDA receptor. The fluorescein conjugate 6 displayed a moderate affinity for NMDAR but a high selectivity for the NR2B subunit and its NTD. Fluorescence imaging of DS-red labeled cortical neurons showed an exact colocalization of the probe 6 with small protrusions along the dendrites related to a specific binding on NR2B-containing NMDARs.     

Does the Clock Make the Poison? Circadian Variation in Response to Pesticides

Background

Circadian clocks govern daily physiological and molecular rhythms, and putative rhythms in expression of xenobiotic metabolizing (XM) genes have been described in both insects and mammals. Such rhythms could have important consequences for outcomes of chemical exposures at different times of day. To determine whether reported XM gene expression rhythms result in functional rhythms, we examined daily profiles of enzyme activity and dose responses to the pesticides propoxur, deltamethrin, fipronil, and malathion.

Methodology/Principal Findings

Published microarray expression data were examined for temporal patterns. Male Drosophila were collected for ethoxycoumarin-O-deethylase (ECOD), esterase, glutathione-S-transferase (GST), and, and uridine 5′-diphosphoglucosyltransferase (UGT) enzyme activity assays, or subjected to dose-response tests at four hour intervals throughout the day in both light/dark and constant light conditions. Peak expression of several XM genes cluster in late afternoon. Significant diurnal variation was observed in ECOD and UGT enzyme activity, however, no significant daily variation was observed in esterase or GST activity. Daily profiles of susceptibility to lethality after acute exposure to propoxur and fipronil showed significantly increased resistance in midday, while susceptibility to deltamethrin and malathion varied little. In constant light, which interferes with clock function, the daily variation in susceptibility to propoxur and in ECOD and UGT enzyme activity was depressed.

Conclusions/Significance

Expression and activities of specific XM enzymes fluctuate during the day, and for specific insecticides, the concentration resulting in 50% mortality varies significantly during the day. Time of day of chemical exposure should be an important consideration in experimental design, use of pesticides, and human risk assessment.     

Synthesis and biological evaluation of halogenated naphthyridone carboxamides as potential ligands for in vivo imaging studies of substance P receptors

With the aim of developing new radioligands for in vivo studies of substance P receptors using positron emission tomography or single photon emission computed tomography, 2- and 3-halo naphthyridone-6-carboxamide derivatives were synthesized. Their affinities toward the target receptors were evaluated on CHO cells and compared to the unsubstituted analogue EP 00652218 (IC(50) = 100 nM +/- 20). The IC(50) value was not altered in the case of 2-chloro compound 1 (IC(50) = 100 nM +/- 15) and only slightly reduced for the 2-fluoro and -iodo analogues 6 and 8 (IC(50) = 500 nM +/- 80). A drastic reduction in binding (IC(50) > 1000 nM) was observed for the halogenated compounds 2-5, 7, and 9.     

Citrate synthase from Thermus aquaticus: a thermostable bacterial enzyme with a five-membered inter-subunit ionic network

A bacterial thermostable citrate synthase has been analyzed to investigate the structural basis of its thermostability, and to compare such features with those previously identified in archaeal citrate synthases. The gene encoding the citrate synthase from Thermus aquaticus was identified from a gene library by screening with a PCR fragment amplified from genomic DNA using a primer based on the determined N-terminal amino acid sequence and a citrate synthase consensus primer. Apart from high sequence similarities with citrate synthase sequences within the Thermus/ Deinococcus group, the analyzed enzyme has highest similarities with the enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The recombinant enzyme is a dimer with high specific activity. Compared to its thermoactivity (T(opt)at 80 degrees C), the thermal stability of the enzyme is high, as judged from its T(m) (101 degrees C), and from irreversible thermal inactivation assays. Molecular modeling of the structure revealed an inter-subunit ion-pair network, comparable in size to the network found in the citrate synthase from P. furiosus; these networks are discussed in relation to the high thermal stability of these bacterial and archaeal enzymes.     

N-terminal myristoylation regulates calcium-induced conformational changes in neuronal calcium sensor-1

Neuronal calcium sensor-1 (NCS-1), a Ca(2+)-binding protein, plays an important role in the modulation of neurotransmitter release and phosphatidylinositol signaling pathway. It is known that the physiological activity of NCS-1 is governed by its myristoylation. Here, we present the role of myristoylation of NSC-1 in governing Ca(2+) binding and Ca(2+)-induced conformational changes in NCS-1 as compared with the role in the nonmyristoylated protein. The (45)Ca binding and isothermal titration calorimetric data show that myristoylation increases the degree of cooperativity; thus, the myristoylated NCS-1 binds Ca(2+) more strongly (with three Ca(2+) binding sites) than the non-myristoylated one (with two Ca(2+) binding sites). Both forms of protein show different conformational features in far-UV CD when titrated with Ca(2+). Large conformational changes were seen in the near-UV CD with more changes in the case of nonmyristoylated protein than the myristoylated one. Although the changes in the far-UV CD upon Ca(2+) binding were not seen in E120Q mutant (disabling EF-hand 3), the near-UV CD changes in conformation also were not influenced by this mutation. The difference in the binding affinity of myristoylated and non-myristoylated proteins to Ca(2+) also was reflected by Trp fluorescence. Collisional quenching by iodide showed more inaccessibility of the fluorophore in the myristoylated protein. Mg(2+)-induced changes in near-UV CD are different from Ca(2+)-induced changes, indicating ion selectivity. 8-Anilino-1-naphthalene sulfonic acid binding data showed solvation of the myristoyl group in the presence of Ca(2+), which could be attributed to the myristoyl-dependent conformational changes in NCS-1. These results suggest that myristoylation influences the protein conformation and Ca(2+) binding, which might be crucial for its physiological functions.     

CRD-BP/IMP1 expression characterizes cord blood CD34+ stem cells and affects c-myc and IGF-II expression in MCF-7 cancer cells

The coding region determinant-binding protein/insulin-like growth factor II mRNA-binding protein (CRD-BP/IMP1) is an RNA-binding protein specifically recognizing c-myc, leader 3' IGF-II and tau mRNAs, and the H19 RNA. CRD-BP/IMP1 is predominantly expressed in embryonal tissues but is de novo activated and/or overexpressed in various human neoplasias. To address the question of whether CRD-BP/IMP1 expression characterizes certain cell types displaying distinct proliferation and/or differentiation properties (i.e. stem cells), we isolated cell subpopulations from human bone marrow, mobilized peripheral blood, and cord blood, all sources known to contain stem cells, and monitored for its expression. CRD-BP/IMP1 was detected only in cord blood-derived CD34(+) stem cells and not in any other cell type of either adult or cord blood origin. Adult BM CD34(+) cells cultured in the presence of 5'-azacytidine expressed de novo CRD-BP/IMP1, suggesting that epigenetic modifications may be responsible for its silencing in adult non-expressing cells. Furthermore, by applying the short interfering RNA methodology in MCF-7 cells, we observed, subsequent to knocking down CRD-BP/IMP1, decreased c-myc expression, increased IGF-II mRNA levels, and reduced cell proliferation rates. These data 1) suggest a normal role for CRD-BP/IMP1 in pluripotent stem cells with high renewal capacity, like the CB CD34(+) cells, 2) indicate that altered methylation may directly or indirectly affect its expression in adult cells, 3) imply that its de novo activation in cancer cells may affect the expression of c-Myc and insulin-like growth factor II, and 4) indicate that the inhibition of CRD-BP/IMP1 expression might affect cancer cell proliferation.     

Identification and classification of host cell proteins during biopharmaceutical process development

As significant improvements in volumetric antibody productivity have been achieved by advances in upstream processing over the last decade, and harvest material has become progressively more difficult to recover with these intensified upstream operations, the segregation of upstream and downstream processing has remained largely unchanged. By integrating upstream and downstream process development, product purification issues are given consideration during the optimization of upstream operating conditions, which mitigates the need for extensive and expensive clearance strategies downstream. To investigate the impact of cell culture duration on critical quality attributes, CHO-expressed IgG1 was cultivated in two 2 L bioreactors with samples taken on days 8, 10, 13, 15, and 17. The material was centrifuged, filtered and protein A purified on a 1 ml HiTrap column. Host cell protein (HCP) identification by mass spectrometry (MS) was applied to this system to provide insights into cellular behavior and HCP carryover during protein A purification. It was shown that as cultivation progressed from day 8 to 17 and antibody titer increased, product quality declined due to an increase in post-protein A HCPs (from 72 to 475 peptides detected by MS) and a decrease in product monomer percentage (from 98% to 95.5%). Additionally, the MS data revealed an increase in the abundance of several classes of post-protein A HCPs (e.g., stress response proteins and indicators of cell age), particularly on days 15 and 17 of culture, which were associated with significant increases in total overall HCP levels. This provides new insight into the specific types of HCPs that are retained during mAb purification and may be used to aid process development strategies.