A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.     

Ribosomes Lacking Protein S20 Are Defective in mRNA Binding and Subunit Association

The functional significance of ribosomal proteins is still relatively unclear. Here, we examined the role of small subunit protein S20 in translation using both in vivo and in vitro techniques. By means of lambda red recombineering, the rpsT gene, encoding S20, was removed from the chromosome of Salmonella enterica var. Typhimurium LT2 to produce a ΔS20 strain that grew markedly slower than the wild type while maintaining a wild-type rate of peptide elongation. Removal of S20 conferred a significant reduction in growth rate that was eliminated upon expression of the rpsT gene on a high-copy-number plasmid. The in vitro phenotype of mutant ribosomes was investigated using a translation system composed of highly active, purified components from Escherichia coli. Deletion of S20 conferred two types of initiation defects to the 30S subunit: (i) a significant reduction in the rate of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in association with the 50S subunit. Both of these impairments were partially relieved by an extended incubation time with mRNA, fMet-tRNA^fMet, and initiation factors, indicating that absence of S20 disturbs the structural integrity of 30S subunits. Considering the topographical location of S20 in complete 30S subunits, the molecular mechanism by which it affects mRNA binding and subunit docking is not entirely obvious. We speculate that its interaction with helix 44 of the 16S ribosomal RNA is crucial for optimal ribosome function.     

Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 Å crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G–ribosome complex.     

The physiological effects of deleting the mouse SLC30A8 gene encoding zinc transporter-8 are influenced by gender and genetic background

Objective

The SLC30A8 gene encodes the islet-specific transporter ZnT-8, which is hypothesized to provide zinc for insulin-crystal formation. A polymorphic variant in SLC30A8 is associated with altered susceptibility to type 2 diabetes. Several groups have examined the effect of global Slc30a8 gene deletion but the results have been highly variable, perhaps due to the mixed 129SvEv/C57BL/6J genetic background of the mice studied. We therefore sought to remove the conflicting effect of 129SvEv-specific modifier genes.

Methods

The impact of Slc30a8 deletion was examined in the context of the pure C57BL/6J genetic background.

Results

Male C57BL/6J Slc30a8 knockout (KO) mice had normal fasting insulin levels and no change in glucose-stimulated insulin secretion (GSIS) from isolated islets in marked contrast to the ∼50% and ∼35% decrease, respectively, in both parameters observed in male mixed genetic background Slc30a8 KO mice. This observation suggests that 129SvEv-specific modifier genes modulate the impact of Slc30a8 deletion. In contrast, female C57BL/6J Slc30a8 KO mice had reduced (∼20%) fasting insulin levels, though this was not associated with a change in fasting blood glucose (FBG), or GSIS from isolated islets. This observation indicates that gender also modulates the impact of Slc30a8 deletion, though the physiological explanation as to why impaired insulin secretion is not accompanied by elevated FBG is unclear. Neither male nor female C57BL/6J Slc30a8 KO mice showed impaired glucose tolerance.

Conclusions

Our data suggest that, despite a marked reduction in islet zinc content, the absence of ZnT-8 does not have a substantial impact on mouse physiology.     

Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM

The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNA^Phe, Trp-tRNA^Trp, or Leu-tRNA^LeuI. The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same ‘loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon–anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection.     

Inhibition of DNA replication fork progression and mutagenic potential of 1, N6-ethenoadenine and 8-oxoguanine in human cell extracts

Comparative mutagenesis of 1,N⁶-ethenoadenine (εA) and 8-oxoguanine (8-oxoG), two endogenous DNA lesions that are also formed by exogenous DNA damaging agents, have been evaluated in HeLa and xeroderma pigmentosum variant (XPV) cell extracts. Two-dimensional gel electrophoresis of the duplex M13mp2SV vector containing these lesions established that there was significant inhibition of replication fork movement past εA, whereas 8-oxoG caused only minor stalling of fork progression. In extracts of HeLa cells, εA was weakly mutagenic inducing all three base substitutions in approximately equal frequency, whereas 8-oxoG was 10-fold more mutagenic inducing primarily G→T transversions. These data suggest that 8-oxoG is a miscoding lesion that presents a minimal, if any, block to DNA replication in human cells. We hypothesized that bypass of εA proceeded principally by an error-free mechanism in which the undamaged strand was used as a template, since this lesion strongly blocked fork progression. To examine this, we determined the sequence of replication products derived from templates in which a G was placed across from the εA. Consistent with our hypothesis, 93% of the progeny were derived from replication of the undamaged strand. When translesion synthesis occurred, εA→T mutations increased 3-fold in products derived from the mismatched εA: G construct compared with those derived from the εA: T construct. More efficient repair of εA in the εA: T construct may have been responsible for lower mutation frequency. Primer extension studies with purified pol η have shown that this polymerase is highly error-prone when bypassing εA. To examine if pol η is the primary mutagenic translesion polymerase in human cells, we determined the lesion bypass characteristics of extracts derived from XPV cells, which lack this polymerase. The εA: T construct induced εA→G and εA→C mutant frequencies that were approximately the same as those observed using the HeLa extracts. However, εA→T events were increased 5-fold relative to HeLa extracts. These data support a model in which pol η-mediated translesion synthesis past this adduct is error-free in the context of semiconservative replication in the presence of fidelity factors such as PCNA.     

Dimensional analysis for modeling oxygen transfer in rotating biological contactor

In rotating biological contactors (RBC), the oxygen mass transfer coefficient (KLa) is often inadequately predicted by the available models. Hence, dimensional analysis based empirical models were developed for predicting KLa and the component of KLa due to turbulence (KLat) using data available in the literature. The overall oxygen transfer number (OTN) and its component due to turbulence (OTNt) were defined as dimensionless groups based on KLa and KLat, respectively. They were expressed as a function of dimensionless groups, formed using disc diameter (D), area of discs (Ad), rotational speed (omega) and cross-sectional area of the tank (At). OTN was also a function of thickness of the water film on the disc (delta) and working volume of the reactor (V). Decrease in number of discs and decrease in (Ad/At) resulted in decreasing OTN but increasing OTNt. Both OTNt and OTN increased with increase in omega. The proposed empirical models will facilitate scale-up of RBCs.     

Insight to structural subsite recognition in plant thiol protease-inhibitor complexes : Understanding the basis of differential inhibition and the role of water

Background  

This work represents an extensive MD simulation / water-dynamics studies on a series of complexes of inhibitors (leupeptin, E-64, E-64-C, ZPACK) and plant cysteine proteases (actinidin, caricain, chymopapain, calotropin DI) of papain family to understand the various interactions, water binding mode, factors influencing it and the structural basis of differential inhibition.     

The folding of dimeric cytoplasmic malate dehydrogenase. Equilibrium and kinetic studies.

Porcine heart cytoplasmic malate dehydrogenase (s-MDH) is a dimeric protein (2 x 35 kDa). We have studied equilibrium unfolding and refolding of s-MDH using activity assay, fluorescence, far-UV and near-UV circular dichroism (CD) spectroscopy, hydrophobic probe-1-anilino-8-napthalene sulfonic acid binding, dynamic light scattering, and chromatographic (HPLC) techniques. The unfolding and refolding transitions are reversible and show the presence of two equilibrium intermediate states. The first one is a compact monomer (MC) formed immediately after subunit dissociation and the second one is an expanded monomer (ME), which is little less compact than the native monomer and has most of the characteristic features of a 'molten globule' state. The equilibrium transition is fitted in the model: 2U <--> 2M(E) <--> 2M(C) <--> D. The time course of kinetics of self- refolding of s-MDH revealed two parallel folding pathways [Rudolph, R., Fuchs, I. & Jaenicke, R. (1986) Biochemistry 25, 1662-1669]. The major pathway (70%) is 2U-->2M*-->2M-->D, the rate limiting step being the isomerization of the monomers (K1 = 1.7 x 10(-3) s(-1)). The minor pathway (30%) involves an association step leading to the incorrectly folding dimers, prior to the very slow D*-->D folding step. In this study, we have characterized the folding-assembly pathway of dimeric s-MDH. Our kinetic and equilibrium experiments indicate that the folding of s-MDH involves the formation of two folding intermediates. However, whether the equilibrium intermediates are equivalent to the kinetic ones is beyond the scope of this study.     

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.