scholarly journals Structure and analysis of nanobody binding to the human ASIC1a ion channel

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Yangyu Wu ◽  
Zhuyuan Chen ◽  
Fred J Sigworth ◽  
Cecilia M Canessa
Keyword(s):  
Structural Studies ◽  
Coral Snake ◽  
Channel Activation ◽  
Isolation And Characterization ◽  
Cryo Electron Microscopy ◽  
Single Polypeptide ◽  
A Site ◽  
Snake Toxin

ASIC1a is a proton-gated sodium channel involved in modulation of pain, fear, addiction, and ischemia-induced neuronal injury. We report isolation and characterization of alpaca-derived nanobodies (Nbs) that specifically target human ASIC1a. Cryo-electron microscopy of the human ASIC1a channel at pH 7.4 in complex with one of these, Nb.C1, yielded a structure at 2.9 Å resolution. It is revealed that Nb.C1 binds to a site overlapping with that of the Texas coral snake toxin (MitTx1) and the black mamba venom Mambalgin-1; however, the Nb.C1-binding site does not overlap with that of the inhibitory tarantula toxin psalmotoxin-1 (PcTx1). Fusion of Nb.C1 with PcTx1 in a single polypeptide markedly enhances the potency of PcTx1, whereas competition of Nb.C1 and MitTx1 for binding reduces channel activation by the toxin. Thus, Nb.C1 is a molecular tool for biochemical and structural studies of hASIC1a; a potential antidote to the pain-inducing component of coral snake bite; and a candidate to potentiate PcTx1-mediated inhibition of hASIC1a in vivo for therapeutic applications.

scholarly journals Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes

2020 ◽  
Author(s):  
Tongqing Zhou ◽  
I-Ting Teng ◽  
Adam S. Olia ◽  
Gabriele Cerutti ◽  
Jason Gorman ◽  
...  
Keyword(s):  
Region Of Interest ◽  
Molecular Probes ◽  
Terminal Sequence ◽  
Protease Cleavage ◽  
Isolation And Characterization ◽  
Cryo Electron Microscopy ◽  
Specific Protease ◽  
A Site ◽  
Biotinylated Probes

SummaryBiotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.

scholarly journals Isolation and characterization of a gene (CBF2) specifying a protein component of the budding yeast kinetochore.

1993 ◽  
Vol 121 (3) ◽  
pp. 513-519 ◽  
Author(s):  
W Jiang ◽  
J Lechner ◽  
J Carbon
Keyword(s):  
Molecular Motors ◽  
Cell Biology ◽  
Budding Yeast ◽  
Isolation And Characterization ◽  
Sequence Homologies ◽  
Binding Factor ◽  
Microtubule Motor

We have cloned and determined the nucleotide sequence of the gene (CBF2) specifying the large (110 kD) subunit of the 240-kD multisubunit yeast centromere binding factor CBF3, which binds selectively in vitro to yeast centromere DNA and contains a minus end-directed microtubule motor activity. The deduced amino acid sequence of CBF2p shows no sequence homologies with known molecular motors, although a consensus nucleotide binding site is present. The CBF2 gene is essential for viability of yeast and is identical to NDC10, in which a conditional mutation leads to a defect in chromosome segregation (Goh, P.-Y., and J. V. Kilmartin, in this issue of The Journal of Cell Biology). The combined in vitro and in vivo evidence indicate that CBF2p is a key component of the budding yeast kinetochore.

scholarly journals Isolation and characterization of adrenocortical progenitors involved in the adaptation to stress

2018 ◽  
Vol 115 (51) ◽  
pp. 12997-13002 ◽  
Author(s):  
Charlotte Steenblock ◽  
Maria F. Rubin de Celis ◽  
Luis F. Delgadillo Silva ◽  
Verena Pawolski ◽  
Ana Brennand ◽  
...  
Keyword(s):  
Lineage Tracing ◽  
Differentiated Cells ◽  
Isolation And Characterization ◽  
Proliferation And Differentiation ◽  
Response To Stress ◽  
Steroidogenic Cells ◽  
High Degree

The adrenal gland is a master regulator of the human body during response to stress. This organ shows constant replacement of senescent cells by newly differentiated cells. A high degree of plasticity is critical to sustain homeostasis under different physiological demands. This is achieved in part through proliferation and differentiation of adult adrenal progenitors. Here, we report the isolation and characterization of a Nestin+ population of adrenocortical progenitors located under the adrenal capsule and scattered throughout the cortex. These cells are interconnected with progenitors in the medulla. In vivo lineage tracing revealed that, under basal conditions, this population is noncommitted and slowly migrates centripetally. Under stress, this migration is greatly enhanced, and the cells differentiate into steroidogenic cells. Nestin+ cells cultured in vitro also show multipotency, as they differentiate into mineralocorticoid and glucocorticoid-producing cells, which can be further influenced by the exposure to Angiotensin II, adrenocorticotropic hormone, and the agonist of luteinizing hormone-releasing hormone, triptorelin. Taken together, Nestin+ cells in the adult adrenal cortex exhibit the features of adrenocortical progenitor cells. Our study provides evidence for a role of Nestin+ cells in organ homeostasis and emphasizes their role under stress. This cell population might be a potential source of cell replacement for the treatment of adrenal insufficiency.

scholarly journals Pharmaceutical properties of 'sucupira' (Pterodon spp.)

2010 ◽  
Vol 46 (4) ◽  
pp. 607-616 ◽  
Author(s):  
Daiane Hansen ◽  
Mitsue Haraguchi ◽  
Antonio Alonso
Keyword(s):  
Biological Effects ◽  
Chemical Compounds ◽  
Experimental Models ◽  
Popular Medicine ◽  
Isolation And Characterization ◽  
Pharmaceutical Properties ◽  
The Relationship

The plant of the genus Pterodon (Fabaceae, Leguminosae), commonly known as 'sucupira' or 'faveira', are disseminated throughout the central region of Brazil and has frequently been used in popular medicine for its anti-rheumatic, analgesic, and anti-inflammatory properties. In recent years, interest in these plants has increased considerably. The biological effects of different phytoextracts and pure metabolites have been investigated in several experimental models in vivo and in vitro. The literature describes flavonoids, triterpene and steroids, while one paper presented studies with proteins isolated from the genus. This review provides an overview of phytochemical and pharmacological research in Pterodon, showing the main chemical compounds studied to date, and focusing on the relationship between these molecules and their biological activity. Furthermore, this study paves the way for more in-depth investigation, isolation and characterization of the molecules of this plant genus.

scholarly journals Isolation and characterization of the dopa decarboxylase gene of Drosophila melanogaster

1981 ◽  
Vol 1 (6) ◽  
pp. 475-485
Author(s):  
J Hirsh ◽  
N Davidson
Keyword(s):  
Drosophila Melanogaster ◽  
Ribonucleic Acid ◽  
Deoxyribonucleic Acid ◽  
Isolation Procedure ◽  
Chromosome Band ◽  
Dopa Decarboxylase ◽  
Developmental Profile ◽  
Isolation And Characterization

We have isolated chromosomal deoxyribonucleic acid clones containing the Drosophila dopa decarboxylase gene. We describe an isolation procedure which can be applied to other nonabundantly expressed Drosophila genes. The dopa decarboxylase gene lies within or very near polytene chromosome band 37C1-2. The gene is interrupted by at least one intron, and the primary mode of regulation is pretranslational. At least two additional sequences hybridized by in vivo ribonucleic acid-derived probes are found within a 35-kilobase region surrounding the gene. The developmental profile of ribonucleic acid transcribed from one of these regions differs from that of the dopa decarboxylase transcript.

scholarly journals Ex Vivo Isolation and Characterization of Cd4+Cd25+ T Cells with Regulatory Properties from Human Blood

2001 ◽  
Vol 193 (11) ◽  
pp. 1303-1310 ◽  
Author(s):  
Detlef Dieckmann ◽  
Heidi Plottner ◽  
Susanne Berchtold ◽  
Thomas Berger ◽  
Gerold Schuler
Keyword(s):  
T Cells ◽  
Cytotoxic T Lymphocyte ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Regulatory Cells ◽  
Suppressor T Cells ◽  
Isolation And Characterization ◽  
Regulatory Properties

It has been known for years that rodents harbor a unique population of CD4+CD25+ “professional” regulatory/suppressor T cells that is crucial for the prevention of spontaneous autoimmune diseases. Here we demonstrate that CD4+CD25+CD45RO+ T cells (mean 6% of CD4+ T cells) are present in the blood of adult healthy volunteers. In contrast to previous reports, these CD4+CD25+ T cells do not constitute conventional memory cells but rather regulatory cells exhibiting properties identical to their rodent counterparts. Cytotoxic T lymphocyte–associated antigen (CTLA)-4 (CD152), for example, which is essential for the in vivo suppressive activity of CD4+CD25+ T cells, was constitutively expressed, and remained strongly upregulated after stimulation. The cells were nonproliferative to stimulation via their T cell receptor for antigen, but the anergic state was partially reversed by interleukin (IL)-2 and IL-15. Upon stimulation with allogeneic (but not syngeneic) mature dendritic cells or platebound anti-CD3 plus anti-CD28 the CD4+CD25+ T cells released IL-10, and in coculture experiments suppressed the activation and proliferation of CD4+ and CD8+ T cells. Suppression proved IL-10 independent, yet contact dependent as in the mouse. The identification of regulatory CD4+CD25+ T cells has important implications for the study of tolerance in man, notably in the context of autoimmunity, transplantation, and cancer.

Isolation and characterization of dominant mutations resistant to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae

1981 ◽  
Vol 1 (2) ◽  
pp. 83-93
Author(s):  
K Matsumoto ◽  
A Toh-e ◽  
Y Oshima
Keyword(s):  
Partial Resistance ◽  
Carbon Catabolite Repression ◽  
Glucose Repression ◽  
Isocitrate Lyase ◽  
Isolation And Characterization ◽  
Enhanced Resistance ◽  
Leloir Pathway ◽  
Resistant Mutants ◽  
A Site

Seven dominant mutations showing greatly enhanced resistance to the glucose repression of galactokinase synthesis have been isolated from GAL81 mutants, which have the constitutive phenotype but are still strongly repressible by glucose for the synthesis of the Leloir enzymes. These glucose-resistant mutants were due to semidominant mutations at either of two loci, GAL82 and GAL83. Both loci are unlinked to the GAL81- gal4, gal80, or gal7 X gal10 X gal1 locus or to each other. The GAL83 locus was mapped on chromosome V at a site between arg9 and cho1. The GAL82 and GAL83 mutations produced partial resistance of galactokinase to glucose repression only when one or both of these mutations were combined with a GAL81 or a gal80 mutation. The GAL82 and GAL83 mutations are probably specific for expression of the Leloir pathway and related enzymes, because they do not affect the synthesis of alpha-D-glucosidase, invertase, or isocitrate lyase.

Isolation and Characterization of Human Platelet β-Thromboglobulin

1975 ◽  
Author(s):  
D. S. Pepper ◽  
S. Moore ◽  
J. D. Cash
Keyword(s):  
Molecular Weight ◽  
Plasma Proteins ◽  
Human Platelet ◽  
Weight Fraction ◽  
Human Platelets ◽  
Low Molecular Weight ◽  
Purification Procedure ◽  
Isolation And Characterization

The thrombin released products from washed human platelets were separated by filtration on 4% agarose in 0.15 M NaCl. The high molecular weight PF4 complex was dissociated and re-chromatographed in 0.75 M NaCl. The low molecular weight fraction, including β thromboglobulin and a low MW anti-heparin was freed of plasminogen anti-activator by dissociation and chromatography in pH 3.5 pyridine acetic acid. The anti-activator was irreversibly denatured and albumin was removed in the void volume of the column. A more suitable purification procedure for recovery of all activities was affinity chromatography on heparin-agarose. The anti-activator was excluded and could be obtained free of plasma proteins by Sephadex G-200 chromatography. The βTG eluted at 0.3 M NaCl and the low MW anti-heparin at 1.5 M NaCl. The pure βTG (MW 36,000) was injected into rabbits and the resulting antiserum used to produce a radioimmunoassay for the release reaction in vivo.

scholarly journals Functional and Genomic Characterization of Ligilactobacillus salivarius TUCO-L2 Isolated From Lama glama Milk: A Promising Immunobiotic Strain to Combat Infections

2020 ◽  
Vol 11 ◽  
Author(s):  
Sandra Quilodrán-Vega ◽  
Leonardo Albarracin ◽  
Flavia Mansilla ◽  
Lorena Arce ◽  
Binghui Zhou ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Functional Characterization ◽  
Comparative Genomic ◽  
Cytokines And Chemokines ◽  
Isolation And Characterization ◽  
Intestinal Pathogens ◽  
Lama Glama

Potential probiotic or immunobiotic effects of lactic acid bacteria (LAB) isolated from the milk of the South American camelid llama (Lama glama) have not been reported in published studies. The aim of the present work was to isolate beneficial LAB from llama milk that can be used as potential probiotics active against bacterial pathogens. LAB strains were isolated from llama milk samples. In vitro functional characterization of the strains was performed by evaluating the resistance against gastrointestinal conditions and inhibition of the pathogen growth. Additionally, the adhesive and immunomodulatory properties of the strains were assessed. The functional studies were complemented with a comparative genomic evaluation and in vivo studies in mice. Ligilactobacillus salivarius TUCO-L2 showed enhanced probiotic/immunobiotic potential compared to that of other tested strains. The TUCO-L2 strain was resistant to pH and high bile salt concentrations and demonstrated antimicrobial activity against Gram-negative intestinal pathogens and adhesion to mucins and epithelial cells. L. salivarius TUCO-L2 modulated the innate immune response triggered by Toll-like receptor (TLR)-4 activation in intestinal epithelial cells. This effect involved differential regulation of the expression of inflammatory cytokines and chemokines mediated by the modulation of the negative regulators of the TLR signaling pathway. Moreover, the TUCO-L2 strain enhanced the resistance of mice to Salmonella infection. This is the first report on the isolation and characterization of a potential probiotic/immunobiotic strain from llama milk. The in vitro, in vivo, and in silico investigation performed in this study reveals several research directions that are needed to characterize the TUCO-L2 strain in detail to position this strain as a probiotic or immunobiotic that can be used against infections in humans or animals, including llama.

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