scholarly journals Identification of a cono-RFamide from the venom of Conus textile that targets ASIC3 and enhances muscle pain

2017 ◽  
Vol 114 (17) ◽  
pp. E3507-E3515 ◽  
Author(s):  
Catharina Reimers ◽  
Cheng-Han Lee ◽  
Hubert Kalbacher ◽  
Yuemin Tian ◽  
Chih-Hsien Hung ◽  
...  
Keyword(s):  
Ion Channels ◽  
Muscle Pain ◽  
Disulfide Bonds ◽  
High Specificity ◽  
Snail Species ◽  
Cone Snail ◽  
Neuronal Proteins ◽  
Pathological Pain ◽  
Electrophysiological Characterization

Acid-sensing ion channels (ASICs) are proton-gated Na+ channels that are expressed throughout the nervous system. ASICs have been implicated in several neuronal disorders, like ischemic stroke, neuronal inflammation, and pathological pain. Several toxins from venomous animals have been identified that target ASICs with high specificity and potency. These toxins are extremely useful in providing protein pharmacophores and to characterize function and structure of ASICs. Marine cone snails contain a high diversity of toxins in their venom such as conotoxins, which are short polypeptides stabilized by disulfide bonds, and conopeptides, which have no or only one disulfide bond. Whereas conotoxins selectively target specific neuronal proteins, mainly ion channels, the targets of conopeptides are less well known. Here, we perform an in vitro screen of venoms from 18 cone snail species to identify toxins targeting ASICs. We identified a small conopeptide of only four amino acids from the venom of Conus textile that strongly potentiated currents of ASIC3, which has a specific role in the pain pathway. This peptide, RPRFamide, belongs to the subgroup of cono-RFamides. Electrophysiological characterization of isolated dorsal root ganglion (DRG) neurons revealed that RPRFamide increases their excitability. Moreover, injection of the peptide into the gastrocnemius muscle strongly enhanced acid-induced muscle pain in mice that was abolished by genetic inactivation of ASIC3. In summary, we identified a conopeptide that targets the nociceptor-specific ion channel ASIC3.

scholarly journals Cardiac optogenetics

2013 ◽  
Vol 304 (9) ◽  
pp. H1179-H1191 ◽  
Author(s):  
Emilia Entcheva
Keyword(s):  
Ion Channels ◽  
Mammalian Cells ◽  
High Specificity ◽  
Emerging Technology ◽  
Inhibitory Response ◽  
Research Activity ◽  
Spatiotemporal Resolution ◽  
And Control ◽  
The Impact

Optogenetics is an emerging technology for optical interrogation and control of biological function with high specificity and high spatiotemporal resolution. Mammalian cells and tissues can be sensitized to respond to light by a relatively simple and well-tolerated genetic modification using microbial opsins (light-gated ion channels and pumps). These can achieve fast and specific excitatory or inhibitory response, offering distinct advantages over traditional pharmacological or electrical means of perturbation. Since the first demonstrations of utility in mammalian cells (neurons) in 2005, optogenetics has spurred immense research activity and has inspired numerous applications for dissection of neural circuitry and understanding of brain function in health and disease, applications ranging from in vitro to work in behaving animals. Only recently (since 2010), the field has extended to cardiac applications with less than a dozen publications to date. In consideration of the early phase of work on cardiac optogenetics and the impact of the technique in understanding another excitable tissue, the brain, this review is largely a perspective of possibilities in the heart. It covers the basic principles of operation of light-sensitive ion channels and pumps, the available tools and ongoing efforts in optimizing them, overview of neuroscience use, as well as cardiac-specific questions of implementation and ideas for best use of this emerging technology in the heart.

scholarly journals Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

2018 ◽  
Vol 150 (4) ◽  
pp. 637-646 ◽  
Author(s):  
Laura-Marie Winterstein ◽  
Kerri Kukovetz ◽  
Oliver Rauh ◽  
Daniel L. Turman ◽  
Christian Braun ◽  
...  
Keyword(s):  
Ion Channels ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Functional Characterization ◽  
In Vitro Translation ◽  
Target Membrane ◽  
Electrophysiological Characterization ◽  
Channel Properties

Recent studies have shown that membrane proteins can be efficiently synthesized in vitro before spontaneously inserting into soluble nanoscale lipid bilayers called nanodiscs (NDs). In this paper, we present experimental details that allow a combination of in vitro translation of ion channels into commercially available NDs followed by their direct reconstitution from these nanobilayers into standard bilayer setups for electrophysiological characterization. We present data showing that two model K+ channels, Kcv and KcsA, as well as a recently discovered dual-topology F− channel, Fluc, can be reliably reconstituted from different types of NDs into bilayers without contamination from the in vitro translation cocktail. The functional properties of Kcv and KcsA were characterized electrophysiologically and exhibited sensitivity to the lipid composition of the target DPhPC bilayer, suggesting that the channel proteins were fully exposed to the target membrane and were no longer surrounded by the lipid/protein scaffold. The single-channel properties of the three tested channels are compatible with studies from recordings of the same proteins in other expression systems. Altogether, the data show that synthesis of ion channels into NDs and their subsequent reconstitution into conventional bilayers provide a fast and reliable method for functional analysis of ion channels.

A Physiologic Regulator of Collagen-Induced Platelet Aggregation: Inhibition by Clq

1981 ◽  
Vol 45 (02) ◽  
pp. 110-115 ◽  
Author(s):  
György Csákó ◽  
Eva A Suba
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Platelet Reactivity ◽  
High Specificity ◽  
Turbidimetric Method ◽  
Specific Manner ◽  
Aggregation Inhibition ◽  
Different Tissues

SummaryPlatelet aggregations were studied by a turbidimetric method in citrated human platelet-rich plasmas (PRP) in vitro. Human Clq inhibited the aggregations caused by collagens derived from different tissues and species. Clq was needed by weight in comparable quantities to collagen for neutralizing the aggregating effect. The dependence of the inhibitory reaction on the preincubation of platelets with Clq and the differences in the occurrence of aggregating substances in supernatants of PRP triggered with collagen in the presence or absence of Clq, confirmed that Clq exerts its effect by preventing fixation of collagen to platelets. In addition, the high specificity of the inhibitory action of Clq for collagen-induced platelet aggregation was demonstrated by results obtained for testing a variety of aggregating agents in combination with Clq and/or collagen.Since normal concentrations of Clq in the blood are in the range of inhibitory doses of Clq for collagen-induced platelet aggregations in vitro and upon activation of complement Clq is known to dissociate from Cl, it is proposed that Clq may participate in a highly specific manner in regulating platelet reactivity to collagen in vivo.

Enzyme Promiscuous Activity: How to Define it and its Evolutionary Aspects

2020 ◽  
Vol 27 (5) ◽  
pp. 400-410
Author(s):  
Valentina De Luca ◽  
Luigi Mandrich
Keyword(s):  
Gene Evolution ◽  
Sequence Divergence ◽  
High Specificity ◽  
Industrial Applications ◽  
Point Of View ◽  
Enzyme Promiscuity ◽  
Primary Activity ◽  
Starting Point ◽  
Main Activity

: Enzymes are among the most studied biological molecules because better understanding enzymes structure and activity will shed more light on their biological processes and regulation; from a biotechnological point of view there are many examples of enzymes used with the aim to obtain new products and/or to make industrial processes less invasive towards the environment. Enzymes are known for their high specificity in the recognition of a substrate but considering the particular features of an increasing number of enzymes this is not completely true, in fact, many enzymes are active on different substrates: this ability is called enzyme promiscuity. Usually, promiscuous activities have significantly lower kinetic parameters than to that of primary activity, but they have a crucial role in gene evolution. It is accepted that gene duplication followed by sequence divergence is considered a key evolutionary mechanism to generate new enzyme functions. In this way, promiscuous activities are the starting point to increase a secondary activity in the main activity and then get a new enzyme. The primary activity can be lost or reduced to a promiscuous activity. In this review we describe the differences between substrate and enzyme promiscuity, and its rule in gene evolution. From a practical point of view the knowledge of promiscuity can facilitate the in vitro progress of proteins engineering, both for biomedical and industrial applications. In particular, we report cases regarding esterases, phosphotriesterases and cytochrome P450.

Prospective Application of Aptamer-based Assays and Therapeutics in Bloodstream Infections

2020 ◽  
Vol 20 (10) ◽  
pp. 831-840
Author(s):  
Weibin Li
Keyword(s):  
Pathogenic Bacteria ◽  
Genetic Diagnosis ◽  
Bloodstream Infections ◽  
High Specificity ◽  
Peptide Sequence ◽  
High Morbidity ◽  
Specificity And Sensitivity ◽  
Prospective Application ◽  
Small Molecule Therapeutics

Sepsis is still a severe health problem worldwide with high morbidity and mortality. Blood bacterial culture remains the gold standard for the detection of pathogenic bacteria in bloodstream infections, but it is time-consuming, and both the sophisticated equipment and well-trained personnel are required. Immunoassays and genetic diagnosis are expensive and limited to specificity and sensitivity. Aptamers are single-stranded deoxyribonucleic acid (ssDNA) and ribonucleic acid (RNA) oligonucleotide or peptide sequence generated in vitro based on the binding affinity of aptamer-target by a process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). By taking several advantages over monoclonal antibodies and other conventional small-molecule therapeutics, such as high specificity and affinity, negligible batch-to-batch variation, flexible modification and production, thermal stability, low immunogenicity and lack of toxicity, aptamers are presently becoming promising novel diagnostic and therapeutic agents. This review describes the prospective application of aptamerbased laboratory diagnostic assays and therapeutics for pathogenic bacteria and toxins in bloodstream infections.

Acid-Sensing Ion Channels

2020 ◽  
Author(s):  
Stefan Gründer
Keyword(s):  
Nervous System ◽  
Ion Channels ◽  
Excitatory Synapses ◽  
Detailed Characterization ◽  
High Affinity ◽  
Acid Sensing Ion Channels ◽  
Long Time ◽  
Pathological Pain

Acid-sensing ion channels (ASICs) are proton-gated Na+ channels. Being almost ubiquitously present in neurons of the vertebrate nervous system, their precise function remained obscure for a long time. Various animal toxins that bind to ASICs with high affinity and specificity have been tremendously helpful in uncovering the role of ASICs. We now know that they contribute to synaptic transmission at excitatory synapses as well as to sensing metabolic acidosis and nociception. Moreover, detailed characterization of mouse models uncovered an unanticipated role of ASICs in disorders of the nervous system like stroke, multiple sclerosis, and pathological pain. This review provides an overview on the expression, structure, and pharmacology of ASICs plus a summary of what is known and what is still unknown about their physiological functions and their roles in diseases.

Standardization of in vitro nervous tissue culture for honeybee: A high specificity toxicological approach

2020 ◽  
Vol 189 ◽  
pp. 110040 ◽  
Author(s):  
Patricia Azevedo ◽  
Nicole Pavan Butolo ◽  
Luciano Delmondes de Alencar ◽  
Hellen Maria Soares-Lima ◽  
Victor Ribeiro Sales ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Nervous Tissue ◽  
High Specificity ◽  
Nervous Tissue Culture

scholarly journals Calcium Carbonate Nanoparticles—Toxicity and Effect of In Ovo Inoculation on Chicken Embryo Development, Broiler Performance and Bone Status

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 932
Author(s):  
Arkadiusz Matuszewski ◽  
Monika Łukasiewicz ◽  
Jan Niemiec ◽  
Maciej Kamaszewski ◽  
Sławomir Jaworski ◽  
...  
Keyword(s):  
Calcium Carbonate ◽  
Embryo Development ◽  
Bone Quality ◽  
Chicken Embryo ◽  
Broiler Chickens ◽  
Selection Procedure ◽  
High Specificity ◽  
Control Group ◽  
In Ovo

The use of intensive selection procedure in modern broiler chicken lines has led to the development of several skeletal disorders in broiler chickens. Therefore, current research is focused on methods to improve the bone quality in birds. In ovo technology, using nanoparticles with a high specificity to bones, is a potential approach. The present study aimed to evaluate the effect of in ovo inoculation (IOI) of calcium carbonate nanoparticles (CCN) on chicken embryo development, health status, bone characteristics, and on broiler production results and bone quality. After assessing in vitro cell viability, the IOI procedure was performed with an injection of 500 μg/mL CCN. The control group was not inoculated with CCN. Hatchability, weight, and selected bone and serum parameters were measured in embryos. Part of hatchlings were reared under standard conditions until 42 days, and production results, meat quality, and bone quality of broilers were determined. CCN did not show cytotoxicity to cells and chicken embryo and positively influenced bone parameters of the embryos and of broilers later (calcification) without negatively affecting the production results. Thus, the IOI of CCN could modify the molecular responses at the stage of embryogenesis, resulting in better mineralization, and could provide a sustained effect, thereby improving bone quality in adult birds.

scholarly journals A molecularly defined skin test reagent for the diagnosis of bovine tuberculosis compatible with vaccination against Johne’s Disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sonya Middleton ◽  
Sabine Steinbach ◽  
Michael Coad ◽  
Kevina McGill ◽  
Colm Brady ◽  
...  
Keyword(s):  
Skin Test ◽  
Recombinant Proteins ◽  
Bovine Tuberculosis ◽  
Synthetic Peptides ◽  
Blood Test ◽  
High Specificity ◽  
Active Components ◽  
Interferon Gamma Release Assays ◽  
Skin Responses

AbstractTuberculin Purified Protein Derivatives (PPDs) exhibit multiple limitations: they are crude extracts from mycobacterial cultures with largely unknown active components; their production depends on culture of mycobacteria requiring expensive BCL3 production facilities; and their potency depends on the technically demanding guinea pig assay. To overcome these limitations, we developed a molecularly defined tuberculin (MDT) by adding further antigens to our prototype reagent composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test, DST). In vitro screening using PBMC from infected and uninfected cattle shortlisted four antigens from a literature-based list of 18 to formulate the MDT. These four antigens plus the previously identified Rv3020c protein, produced as recombinant proteins or overlapping synthetic peptides, were formulated together with the three DST antigens into the MDT to test cattle experimentally and naturally infected with M. bovis, uninfected cattle and MAP vaccinated calves. We demonstrated significant increases in MDT-induced skin responses compared to DST in infected animals, whilst maintaining high specificity in unvaccinated or MAP vaccinated calves. Further, MDT can also be applied in in vitro blood-based interferon-gamma release assays. Thus, MDT promises to be a robust diagnostic skin and blood test reagent overcoming some of the limitations of PPDs and warrants full validation.

scholarly journals Emerging Roles for Ion Channels in Ovarian Cancer: Pathomechanisms and Pharmacological Treatment

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 668
Author(s):  
Concetta Altamura ◽  
Maria Raffaella Greco ◽  
Maria Rosaria Carratù ◽  
Rosa Angela Cardone ◽  
Jean-François Desaphy
Keyword(s):  
Ovarian Cancer ◽  
Ion Channels ◽  
Transient Receptor Potential ◽  
Critical Role ◽  
Gynecologic Cancer ◽  
Receptor Potential ◽  
Transient Receptor Potential Channels ◽  
Platinum Resistance

Ovarian cancer (OC) is the deadliest gynecologic cancer, due to late diagnosis, development of platinum resistance, and inadequate alternative therapy. It has been demonstrated that membrane ion channels play important roles in cancer processes, including cell proliferation, apoptosis, motility, and invasion. Here, we review the contribution of ion channels in the development and progression of OC, evaluating their potential in clinical management. Increased expression of voltage-gated and epithelial sodium channels has been detected in OC cells and tissues and shown to be involved in cancer proliferation and invasion. Potassium and calcium channels have been found to play a critical role in the control of cell cycle and in the resistance to apoptosis, promoting tumor growth and recurrence. Overexpression of chloride and transient receptor potential channels was found both in vitro and in vivo, supporting their contribution to OC. Furthermore, ion channels have been shown to influence the sensitivity of OC cells to neoplastic drugs, suggesting a critical role in chemotherapy resistance. The study of ion channels expression and function in OC can improve our understanding of pathophysiology and pave the way for identifying ion channels as potential targets for tumor diagnosis and treatment.

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