Screening of cDNA Libraries with Oligonucleotides as Applied to Signal Transducing G Proteins,Receptors and Effectors

Abstract

Screening of cDNA libraries constructed in phage or plasmids with oligonucleotide probes has become one of the preferred cloning techniques with the least number of false positive failures. In this article we present our current protocols for designing the procedure to detect cDNA inserts and isolate them. We illustrate with primary screens for G protein subunits and membrane receptors.     

RGS7 Attenuates Signal Transduction Through the Gαq Family of Heterotrimeric G Proteins in Mammalian Cells

Abstract: The RGS proteins are a recently discovered family of G protein regulators that have been shown to act as GTPase-activating proteins (GAPs) on the G_αi and G_αq subfamilies of the heterotrimeric G proteins. Here, we demonstrate that RGS7 is a potent GAP in vitro on G_αi1 and G_αo heterotrimeric proteins and that RGS7 acts to down-regulate G_αq-mediated calcium mobilization in a whole-cell assay system using a transient expression protocol. This RGS protein and RGS4 are reported to be expressed predominantly in brain, and in situ hybridization studies have revealed similarities in the regional distribution of RGS and G_αq mRNA expression. Our findings provide further evidence to support a functional role for RGS4 and RGS7 in G_αq-mediated signaling in the CNS.     

Urea Impedes the Hydrophobic Collapse of Partially Unfolded Proteins

Proteins are denatured in aqueous urea solution. The nature of the molecular driving forces has received substantial attention in the past, whereas the question how urea acts at different phases of unfolding is not yet well understood at the atomic level. In particular, it is unclear whether urea actively attacks folded proteins or instead stabilizes unfolded conformations. Here we investigated the effect of urea at different phases of unfolding by molecular dynamics simulations, and the behavior of partially unfolded states in both aqueous urea solution and in pure water was compared. Whereas the partially unfolded protein in water exhibited hydrophobic collapses as primary refolding events, it remained stable or even underwent further unfolding steps in aqueous urea solution. Further, initial unfolding steps of the folded protein were found not to be triggered by urea, but instead, stabilized. The underlying mechanism of this stabilization is a favorable interaction of urea with transiently exposed, less-polar residues and the protein backbone, thereby impeding back-reactions. Taken together, these results suggest that, quite generally, urea-induced protein unfolding proceeds primarily not by active attack. Rather, thermal fluctuations toward the unfolded state are stabilized and the hydrophobic collapse of partially unfolded proteins toward the native state is impeded. As a result, the equilibrium is shifted toward the unfolded state.     

A Proteomic Investigation of Soluble Olfactory Proteins in Anopheles gambiae

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector.     

The Repertoire of Heterotrimeric G Proteins and RGS Proteins in Ciona intestinalis

Background

Heterotrimeric G proteins and regulators of G protein signaling (RGS) proteins are key downstream interacting partners in the G protein coupled receptor (GPCR) signaling pathway. The highly versatile GPCR transmembrane signaling system is a consequence of the coupling of a diverse set of receptors to downstream partners that include multiple subforms of G proteins and regulatory proteins including RGS proteins, among others. While the GPCR repertoire of Ciona intestinalis, representing the basal chordate is known, the repertoire of the heterotrimeric G proteins and RGS proteins is unknown.

Methodology/Principal Findings

In the present study, we performed an in-silico genome-wide search of C. intestinalis for its complement of G proteins and RGS proteins. The identification of several one-to-one orthologs of human G proteins at the levels of families, subfamilies and types and of homologs of the human RGS proteins suggests an evolutionarily conserved structure function relationship of the GPCR signaling mechanism in the chordates.

Conclusions

The C. intestinalis genome encodes a highly conserved, albeit, limited repertoire of the heterotrimeric G protein complexes with the size of subunit types comparable with that in lower eukaryotes.     

Solid-phase Chemistry: A Useful Tool to Discover Modulators of Protein Interactions

The Solid phase synthesis (SPS) concept, first developed for biopolymers, has spread in every field where organic synthesis is involved. While the potential of the solid-phase method was obvious in 1959 to its discoverer, Prof. R. B. Merrifield, it was unpredictable its dominance in peptide synthesis and especially in combinatorial chemistry, an area not yet conceived. SPS paved the way for solid-phase combinatorial approaches (extensively reviewed in (Choong, I. C. and Ellman, J. A.: 1996, Annu. Rep. Med. Chem. 31, 309–318; Obrecht, D. and Villalgordo, J. M.: 1998, Solid-supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries. Pergamon Press Ltd., Oxford, UK; Chabala, J. C.: 1995, Curr. Opin. Biotechnol. 6, 632–639; Kamal, A., Reddy, K. L., Devaiah, V., Shankaraiah, N., Reddy, D. R.: 2006, Mini Rev. Med. Chem. 6, 53–69; Whitehead, D. M., McKeown, S. C., Routledge, A.: 2005, Comb. Chem. HTS 8, 361–371; Nefzi, A., Ostresh, J. M., Houghten, R. A.: 1997, Chem. Rev. 97, 449–472; Gordon, E. M., Gallop, M. A., Patel, D. V.: 1996, Acc. Chem. Res. 29, 144–154)) as many laboratories and companies focused on the development of technologies and chemistry suitable to this new methodology. This resulted in the spectacular outburst of combinatorial chemistry, which profoundly changed the approach for new drug discovery. Combinatorial chemistry is currently considered a valid approach for a wide range of biomedical applications, such as, target validation and drug discovery.     

Cathodoluminescence Microscopy: A Useful Tool for Assessing Incremental Chemical Variation in Otoliths

Otoliths taken from fish from Eden Lake, Manitoba show yellow–green and red cathodoluminescence of varying intensity that corresponds to their annular structure. Proton-induced X-ray emission analysis shows manganese (Mn) concentrations of between 2 and 205ppm, zinc (Zn) concentrations between 2 and 290ppm and strontium (Sr) concentrations up to 1500ppm in the otoliths. The distribution of luminescence correlates with the distribution of Mn. The Mn, Zn and Sr are likely derived from the monzonitic rocks surrounding the lake. Variations in the distribution of cathodoluminescence may be a useful tool for evaluating changes in environmental chemistry and fish life histories.     

Influenza A Virus Encoding Secreted Gaussia Luciferase as Useful Tool to Analyze Viral Replication and Its Inhibition by Antiviral Compounds and Cellular Proteins

Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.     

正常与患白内障大鼠晶状体中脲溶性蛋白质性质的研究

本文研究了正常及三种类型白内障大鼠晶状体中脲溶性蛋白质的含量及性质的变化,发现在每种类型白内障晶状体中,水溶性蛋白质均减少,水不溶性蛋白质则都相对增加。经SephadexG-200柱层析及SDS聚丙烯酰胺凝胶电泳发现,晶状体中脲溶性蛋白质主要是由二硫键交联而成的高分子聚合物。经巯基乙醇还原后,绝大部分高分子聚合物可分解成低分子量蛋白质,其分子量与水溶性的γ晶体蛋白相同。这提示晶状体中脲溶性蛋白质的主要成分很可能是以二硫键交联而成的γ晶体蛋白聚合物。此结果与本实验室所得白内障晶状体水溶性蛋白质的变化相吻合。     

QHELIX: A Computational Tool for the Improved Measurement of Inter-Helical Angles in Proteins

Knowledge about the assembled structures of the secondary elements in proteins is essential to understanding protein folding and functionality. In particular, the analysis of helix geometry is required to study helix packing with the rest of the protein and formation of super secondary structures, such as, coiled coils and helix bundles, formed by packing of two or more helices. Here we present an improved computational method, QHELIX, for the calculation of the orientation angles between helices. Since a large number of helices are known to be in curved shapes, an appropriate definition of helical axes is a prerequisite for calculating the orientation angle between helices. The present method provides a quantitative measure on the irregularity of helical shape, resulting in discriminating irregular-shaped helices from helices with an ideal geometry in a large-scale analysis of helix geometry. It is also capable of straightforwardly assigning the direction of orientation angles in a consistent way. These improvements will find applications in finding a new insight on the assembly of protein secondary structure. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular Identification of Melanised Non-Sporulating Moulds: A Useful Tool for Studying the Epidemiology of Phaeohyphomycosis

Subcutaneous infections caused by melanised fungi have been increasingly reported among transplant patients, and these infections have the potential for blood and visceral dissemination. Some moulds, such as Mycelia sterilia, cannot grow and sporulate on different media, making their identification impossible by conventional methods. The fast and accurate identification of melanised fungi at the species level is important because species may have tropism to different organs and different susceptibilities to antifungal agents. Molecular tools have been reported to be helpful for the species identification of non-sporulating moulds. Our goal was to identify the species of M. sterilia isolates obtained from clinical samples of transplant patients using sequences of ITS and the D1/D2 regions of rDNA. Clinical samples were obtained from eight kidney transplant recipients who developed subcutaneous fungal infections. The diagnosis was confirmed by histopathology and conventional culture. Histopathology showed septated, melanised hyphae, and the cultures identified non-sporulating fungi. Therefore, the DNA from the M. sterilia isolates was subjected to PCR amplification and sequencing of the ITS and D1/D2 regions. Genus/species identification was obtained by comparison with gene banks. We obtained the following identifications: Alternaria sp. (2), Cochliobolus lunatus/Curvularia lunata (2), Cochliobolus hawaiiensis/Bipolaris hawaiiensis (1), Ochroconis sp. (1), Medicocopsis romeroi/Pyrenochaeta romeroi (1) and Nigrograna mackinnonii/Pyrenochaeta mackinnonii (1).     

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.     

Lysis and Lysogenization of Groups A, C, and G Streptococci by a Transducing Bacteriophage Induced from a Group G Streptococcus

A temperate bacteriophage, designated GT-234, was isolated from a group G Streptococcus after ultraviolet irradiation. After several single-plaque passages in a group G indicator strain, this phage formed plaques in 3 of 14 group A strains, in 3 of 15 group C strains, and in 4 of 13 group G strains-but not in some representatives of several other serogroups. After propagation in each of the sensitive strains, the progeny from each was shown to be the same phage by (i) adsorption and plaque formation in each of the other groups, (ii) lysogenization in each of the other groups, (iii) high titers on infection of each serogroup, regardless of the group of propagating strain, and (iv) neutralization of infection in each of the other groups by antiserum against the phage propagated in group G. Phage GT-234 is serologically related to virulent group A phage A25, from which it is morphologically indistinguishable. Like A25, it is a transducing phage. Other studies showed that A25, as well as a group A temperate transducing phage (AT-298), could also infect strains of group C and G. These results indicate a need for reassessment of group specificity and phage receptors among streptococci of groups A, C, and G and raise possibilities for intergroup transduction.     

一种同时纯化钙谓神经磷酸酶和钙调素的有效方法

本文报导了一种能同时纯化钙调神经磷酸酶和钙调素的有效方法。牛脑粗提液经DE-52纤维素层析分段洗脱:0.5mol/L NaCl缓冲液洗脱峰经phenyl-sepharose亲和柱和G75 sephadex制得电泳纯钙调素。0.18mol/L KCl缓冲液洗脱峰经Affigel-Blue层析,硫酸铵盐析,钙调素亲和层析,G-200 Sephadex凝胶过滤制得电泳纯钙调神经磷酸酶。     

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent^1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions^3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling^3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation^7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading^9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas^{7, 8,13}, and can be used to study coupling between other retinal neurons, as described here.     

A Transmural Gradient in the Cardiac Na/K Pump Generates a Transmural Gradient in Na/Ca Exchange

We previously demonstrated a transmural gradient in Na/K pump current (I _P) and [Na⁺] _i , with the highest maximum I _P and lowest [Na⁺] _i in epicardium. The present study examines the relationship between the transmural gradient in I _P and Na/Ca exchange (NCX). Myocytes were isolated from canine left ventricle. Whole-cell patch clamp was used to measure current generated by NCX (I _NCX) and inward background calcium current (I _ibCa), defined as inward current through Ca²⁺ channels less outward current through Ca²⁺-ATPase. When resting myocytes from endocardium (Endo), midmyocardium (Mid) or epicardium (Epi) were studied in the same conditions, I _NCX was the same and I _ibCa was zero. Moreover, Western blots were consistent with NCX protein being uniform across the wall. However, the gradient in [Na⁺] _i , with I _ibCa = 0, should create a gradient in [Ca²⁺] _i . To test this hypothesis, we measured resting [Ca²⁺] _i using two methods, based on either transport or the Ca²⁺-sensitive dye Fura2. Both methods demonstrated a significant transmural gradient in resting [Ca²⁺] _i , with Endo > Mid > Epi. This gradient was eliminated by exposing Epi to sufficient ouabain to partially inhibit Na/K pumps, thus increasing [Na⁺] _i to values similar to those in Endo. These data support the existence of a transmural gradient for Ca²⁺ removal by NCX. This gradient is not due to differences in expression of NCX; rather, it is generated by a transmural gradient in [Na⁺] _i , which is due to a transmural gradient in plasma membrane expression of the Na/K pump.     

CarSPred: A Computational Tool for Predicting Carbonylation Sites of Human Proteins

Protein carbonylation is one of the most pervasive oxidative stress-induced post-translational modifications (PTMs), which plays a significant role in the etiology and progression of several human diseases. It has been regarded as a biomarker of oxidative stress due to its relatively early formation and stability compared with other oxidative PTMs. Only a subset of proteins is prone to carbonylation and most carbonyl groups are formed from lysine (K), arginine (R), threonine (T) and proline (P) residues. Recent advancements in analysis of the PTM by mass spectrometry provided new insights into the mechanisms of protein carbonylation, such as protein susceptibility and exact modification sites. However, the experimental approaches to identifying carbonylation sites are costly, time-consuming and capable of processing a limited number of proteins, and there is no bioinformatics method or tool devoted to predicting carbonylation sites of human proteins so far. In the paper, a computational method is proposed to identify carbonylation sites of human proteins. The method extracted four kinds of features and combined the minimum Redundancy Maximum Relevance (mRMR) feature selection criterion with weighted support vector machine (WSVM) to achieve total accuracies of 85.72%, 85.95%, 83.92% and 85.72% for K, R, T and P carbonylation site predictions respectively using 10-fold cross-validation. The final optimal feature sets were analysed, the position-specific composition and hydrophobicity environment of flanking residues of modification sites were discussed. In addition, a software tool named CarSPred has been developed to facilitate the application of the method. Datasets and the software involved in the paper are available at https://sourceforge.net/projects/hqlstudio/files/CarSPred-1.0/.