scholarly journals Visualizing endogenous opioid receptors in living neurons using ligand-directed chemistry

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Seksiri Arttamangkul ◽  
Andrew Plazek ◽  
Emily J Platt ◽  
Haihong Jin ◽  
Thomas F Murray ◽  
...  
Keyword(s):  
Opioid Receptors ◽  
Physiological Role ◽  
Loss Of Function ◽  
Wild Type ◽  
Endogenous Opioid ◽  
Covalently Linked ◽  
Living Tissues ◽  
Living Neurons ◽  
Post Hoc

Identifying neurons that have functional opioid receptors is fundamental for the understanding of the cellular, synaptic and systems actions of opioids. Current techniques are limited to post hoc analyses of fixed tissues. Here we developed a fluorescent probe, naltrexamine-acylimidazole (NAI), to label opioid receptors based on a chemical approach termed ‘traceless affinity labeling’. In this approach, a high affinity antagonist naltrexamine is used as the guide molecule for a transferring reaction of acylimidazole at the receptor. This reaction generates a fluorescent dye covalently linked to the receptor while naltrexamine is liberated and leaves the binding site. The labeling induced by this reagent allowed visualization of opioid-sensitive neurons in rat and mouse brains without loss of function of the fluorescently labeled receptors. The ability to locate endogenous receptors in living tissues will aid considerably in establishing the distribution and physiological role of opioid receptors in the CNS of wild type animals.

scholarly journals Usp14 is required for spermatogenesis and ubiquitin stress responses in Drosophila melanogaster

2019 ◽  
Author(s):  
Levente Kovács ◽  
Ágota Nagy ◽  
Margit Pál ◽  
Peter Deák
Keyword(s):  
Male Sterility ◽  
Stress Responses ◽  
Expression Patterns ◽  
Loss Of Function ◽  
Wild Type ◽  
Cellular Processes ◽  
Protein Conjugates ◽  
Covalently Linked ◽  
Mutant Phenotypes

ABSTRACTDeubiquitinating (DUB) enzymes free covalently linked ubiquitins from ubiquitin-ubiquitin and ubiquitin-protein conjugates, and thereby maintain the equilibrium between free and conjugated ubiquitins and regulate ubiquitin-mediated cellular processes. The present genetic analyses of mutant phenotypes demonstrate that loss of Usp14 function results in male sterility, with defects in spermatid individualization and reduced testicular free monoubiquitin levels. These phenotypes were rescued by germline specific overexpression of wild type Usp14. Synergistic genetic interactions with Ubi-p63E and cycloheximide sensitivity suggest that ubiquitin shortage is a primary cause of male sterility. In addition, Usp14 is predominantly expressed in testes in Drosophila, and differential expression patterns may be causative of testis-specific loss of function Usp14 phenotypes. Collectively, these results suggest a major role of Usp14 in maintaining normal steady state free monoubiquitin levels during the later stages of Drosophila spermatogenesis.

scholarly journals The Constitutive Centromere Component CENP-50 Is Required for Recovery from Spindle Damage

2005 ◽  
Vol 25 (23) ◽  
pp. 10315-10328 ◽  
Author(s):  
Yukinori Minoshima ◽  
Tetsuya Hori ◽  
Masahiro Okada ◽  
Hiroshi Kimura ◽  
Tokuko Haraguchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Mitotic Checkpoint ◽  
Loss Of Function ◽  
Wild Type ◽  
Centromere Function ◽  
Dt40 Cells ◽  
Precise Role ◽  
Chicken Dt40 Cell

ABSTRACT We identified CENP-50 as a novel kinetochore component. We found that CENP-50 is a constitutive component of the centromere that colocalizes with CENP-A and CENP-H throughout the cell cycle in vertebrate cells. To determine the precise role of CENP-50, we examined its role in centromere function by generating a loss-of-function mutant in the chicken DT40 cell line. The CENP-50 knockout was not lethal; however, the growth rate of cells with this mutation was slower than that of wild-type cells. We observed that the time for CENP-50-deficient cells to complete mitosis was longer than that for wild-type cells. Centromeric localization of CENP-50 was abolished in both CENP-H- and CENP-I-deficient cells. Coimmunoprecipitation experiments revealed that CENP-50 interacted with the CENP-H/CENP-I complex in chicken DT40 cells. We also observed severe mitotic defects in CENP-50-deficient cells with apparent premature sister chromatid separation when the mitotic checkpoint was activated, indicating that CENP-50 is required for recovery from spindle damage.

scholarly journals Bub1 mediates cell death in response to chromosome missegregation and acts to suppress spontaneous tumorigenesis

2007 ◽  
Vol 179 (2) ◽  
pp. 255-267 ◽  
Author(s):  
Karthik Jeganathan ◽  
Liviu Malureanu ◽  
Darren J. Baker ◽  
Susan C. Abraham ◽  
Jan M. van Deursen
Keyword(s):  
Cell Death ◽  
Chromosome Segregation ◽  
Physiological Role ◽  
Mitotic Checkpoint ◽  
Critical Threshold ◽  
Wild Type ◽  
Checkpoint Protein ◽  
Chromosome Missegregation ◽  
Mitotic Checkpoint Protein

The physiological role of the mitotic checkpoint protein Bub1 is unknown. To study this role, we generated a series of mutant mice with a gradient of reduced Bub1 expression using wild-type, hypomorphic, and knockout alleles. Bub1 hypomorphic mice are viable, fertile, and overtly normal despite weakened mitotic checkpoint activity and high percentages of aneuploid cells. Bub1 haploinsufficient mice, which have a milder reduction in Bub1 protein than Bub1 hypomorphic mice, also exhibit reduced checkpoint activity and increased aneuploidy, but to a lesser extent. Although cells from Bub1 hypomorphic and haploinsufficient mice have similar rates of chromosome missegregation, cell death after an aberrant separation decreases dramatically with declining Bub1 levels. Importantly, Bub1 hypomorphic mice are highly susceptible to spontaneous tumors, whereas Bub1 haploinsufficient mice are not. These findings demonstrate that loss of Bub1 below a critical threshold drives spontaneous tumorigenesis and suggest that in addition to ensuring proper chromosome segregation, Bub1 is important for mediating cell death when chromosomes missegregate.

The role of PCSK9 in infectious diseases.

2021 ◽  
Vol 28 ◽  
Author(s):  
Laura Magnasco ◽  
Chiara Sepulcri ◽  
Roberta Maria Antonello ◽  
Stefano Di Bella ◽  
Laura Labate ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Malaria Infection ◽  
Bacterial Infections ◽  
Viral Infections ◽  
Physiological Role ◽  
Protective Role ◽  
Loss Of Function ◽  
Pcsk9 Inhibition ◽  
Sepsis And Septic Shock

Background: In recent years, many aspects of the physiological role of PCSK9 have been elucidated, particularly regarding its role in lipid metabolism, cardiovascular risk, and its role in innate immunity. Increasing evidence is available about the involvement of PCSK9 in the pathogenesis of viral infections, mainly HCV, and the regulation of host response to bacterial infections, primarily sepsis and septic shock. Moreover, the action of PCSK9 has been investigated as a crucial step in the pathogenesis of malaria infection and disease severity. Objective: This paper aims to review the available published literature on the role of PCSK9 in a wide array of infectious diseases. Conclusion: Besides the ongoing investigation on PCSK9 inhibition among HIV-infected patients to treat HIV- and ART-related hyperlipidemia, preclinical studies indicate how PCSK9 is involved in reducing the replication of HCV. Interestingly, high plasmatic PCSK9 levels have been described in patients with sepsis. Moreover, a protective role of PCSK9 inhibition has also been proposed against dengue and SARS-CoV-2 viral infections. Finally, a loss of function in the PCSK9-encoding gene has been reported to reduce malaria infection mortality.

scholarly journals Similarities and differences between IL11 and IL11RA1 knockout mice for lung fibro-inflammation, fertility and craniosynostosis

2020 ◽  
Author(s):  
Benjamin Ng ◽  
Anissa A. Widjaja ◽  
Sivakumar Viswanathan ◽  
Jinrui Dong ◽  
Sonia P. Chothani ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Lung Fibroblasts ◽  
Loss Of Function ◽  
Wild Type ◽  
Reduced Body ◽  
Body Weights ◽  
Show Evidence ◽  
Similarities And Differences ◽  
The Impact

AbstractGenetic loss of function (LOF) in IL11RA infers IL11 signaling as important for fertility, fibrosis, inflammation and craniosynostosis. The impact of genetic LOF in IL11 has not been characterized. We generated IL11-knockout (Il11-/-) mice, which are born in normal Mendelian ratios, have normal hematological profiles and are protected from bleomycin-induced lung fibro-inflammation. Noticeably, baseline IL6 levels in the lungs of Il11-/- mice are lower than those of wild-type mice and are not induced by bleomycin damage, placing IL11 upstream of IL6. Lung fibroblasts from Il11-/- mice are resistant to pro-fibrotic stimulation and show evidence of reduced autocrine IL11 activity. Il11-/- female mice are infertile. Unlike Il11ra1-/- mice, Il11-/- mice do not have a craniosynostosis-like phenotype and exhibit mildly reduced body weights. These data highlight similarities and differences between LOF in IL11 or IL11RA while establishing further the role of IL11 signaling in fibrosis and stromal inflammation.

Abstract 361: Creg1 Interacts With Sec8 to Promote Cell-cell Adhesion During Cardiomyogenesis

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Jie Liu ◽  
Yanmei Qi ◽  
Shu-Chan Hsu ◽  
Siavash Saadat ◽  
Saum Rahimi ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Es Cells ◽  
Embryonic Stem ◽  
Intercellular Junctions ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Wild Type ◽  
Adhesion Sites ◽  
Cell Cell

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a 24 kD glycoprotein essential for early embryonic development. Our immunofluorescence studies revealed that CREG1 is highly expressed at myocyte junctions in both embryonic and adult hearts. To explore it role in cardiomyogenesis, we employed gain- and loss-of-function analyses demonstrating that CREG1 is required for the differentiation of mouse embryonic stem (ES) cell into cohesive myocardium-like structures. Chimeric cultures of wild-type and CREG1 knockout ES cells expressing cardiac-specific reporters showed that the cardiomyogenic effect of CREG1 is cell autonomous. Furthermore, we identified a novel interaction between CREG1 and Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Mutations of the amino acid residues D141 and P142 to alanine in CREG1 abolished its binding to Sec8. To address the role of the CREG1-Sec8 interaction in cardiomyogenesis, we rescued CREG1 knockout ES cells with wild-type and Sec8-binding mutant CREG1 and showed that CREG1 binding to Sec8 promotes cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8 and N-cadherin all localize at cell-cell adhesion sites. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. Finally, shRNA-mediated knockdown of Sec8 leads to cardiomyogenic defects similar to CREG1 knockout. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis.

scholarly journals Deletion of the rpoZ gene, encoding the ω subunit of RNA polymerase, results in pleiotropic surface-related phenotypes in Mycobacterium smegmatis

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1741-1750 ◽  
Author(s):  
Renjith Mathew ◽  
Raju Mukherjee ◽  
Radhakrishnan Balachandar ◽  
Dipankar Chatterji
Keyword(s):  
Rna Polymerase ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Physiological Role ◽  
Wild Type ◽  
Gene Encoding ◽  
Biofilm Growth ◽  
Mutant Bacterium ◽  
Sliding Motility

The ω subunit, the smallest subunit of bacterial RNA polymerase, is known to be involved in maintaining the conformation of the β′ subunit and aiding its recruitment to the rest of the core enzyme assembly in Escherichia coli. It has recently been shown in Mycobacterium smegmatis, by creating a deletion mutation of the rpoZ gene encoding ω, that the physiological role of the ω subunit also includes providing physical protection to β′. Interestingly, the mutant had altered colony morphology. This paper demonstrates that the mutant mycobacterium has pleiotropic phenotypes including reduced sliding motility and defective biofilm formation. Analysis of the spatial arrangement of biofilms by electron microscopy suggests that the altered phenotype of the mutant arises from a deficiency in generation of extracellular matrix. Complementation of the mutant strain with a copy of the wild-type rpoZ gene integrated in the bacterial chromosome restored both sliding motility and biofilm formation to the wild-type state, unequivocally proving the role of ω in the characteristics observed for the mutant bacterium. Analysis of the cell wall composition demonstrated that the mutant bacterium had an identical glycopeptidolipid profile to the wild-type, but failed to synthesize the short-chain mycolic acids characteristic of biofilm growth in M. smegmatis.

scholarly journals Loss of Wwox Causes Defective Development of Cerebral Cortex with Hypomyelination in a Rat Model of Lethal Dwarfism with Epilepsy

2019 ◽  
Vol 20 (14) ◽  
pp. 3596 ◽  
Author(s):  
Yuki Tochigi ◽  
Yutaka Takamatsu ◽  
Jun Nakane ◽  
Rika Nakai ◽  
Kentaro Katayama ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Rat Model ◽  
Physiological Role ◽  
Early Death ◽  
Neurite Growth ◽  
Loss Of Function ◽  
Age Related ◽  
Putative Tumor Suppressor ◽  
Severe Reduction

WW domain-containing oxidoreductase (Wwox) is a putative tumor suppressor. Several germline mutations of Wwox have been associated with infant neurological disorders characterized by epilepsy, growth retardation, and early death. Less is known, however, about the pathological link between Wwox mutations and these disorders or the physiological role of Wwox in brain development. In this study, we examined age-related expression and histological localization of Wwox in forebrains as well as the effects of loss of function mutations in the Wwox gene in the immature cortex of a rat model of lethal dwarfism with epilepsy (lde/lde). Immunostaining revealed that Wwox is expressed in neurons, astrocytes, and oligodendrocytes. lde/lde cortices were characterized by a reduction in neurite growth without a reduced number of neurons, severe reduction in myelination with a reduced number of mature oligodendrocytes, and a reduction in cell populations of astrocytes and microglia. These results indicate that Wwox is essential for normal development of neurons and glial cells in the cerebral cortex.

scholarly journals MtBZR1 Plays an Important Role in Nodule Development in Medicago truncatula

2019 ◽  
Vol 20 (12) ◽  
pp. 2941
Author(s):  
Can Cui ◽  
Hongfeng Wang ◽  
Limei Hong ◽  
Yiteng Xu ◽  
Yang Zhao ◽  
...  
Keyword(s):  
Medicago Truncatula ◽  
Sinorhizobium Meliloti ◽  
Normal Growth ◽  
Dry Mass ◽  
Growth Conditions ◽  
Nodule Development ◽  
Functional Study ◽  
Loss Of Function ◽  
Wild Type

Brassinosteroid (BR) is an essential hormone in plant growth and development. The BR signaling pathway was extensively studied, in which BRASSINAZOLE RESISTANT 1 (BZR1) functions as a key regulator. Here, we carried out a functional study of the homolog of BZR1 in Medicago truncatula R108, whose expression was induced in nodules upon Sinorhizobium meliloti 1021 inoculation. We identified a loss-of-function mutant mtbzr1-1 and generated 35S:MtBZR1 transgenic lines for further analysis at the genetic level. Both the mutant and the overexpression lines of MtBZR1 showed no obvious phenotypic changes under normal growth conditions. After S. meliloti 1021 inoculation, however, the shoot and root dry mass was reduced in mtbzr1-1 compared with the wild type, caused by partially impaired nodule development. The transcriptomic analysis identified 1319 differentially expressed genes in mtbzr1-1 compared with wild type, many of which are involved in nodule development and secondary metabolite biosynthesis. Our results demonstrate the role of MtBZR1 in nodule development in M. truncatula, shedding light on the potential role of BR in legume–rhizobium symbiosis.

scholarly journals Caenorhabditits elegans LRK-1 and PINK-1 Act Antagonistically in Stress Response and Neurite Outgrowth

2009 ◽  
Vol 284 (24) ◽  
pp. 16482-16491 ◽  
Author(s):  
Julia Sämann ◽  
Jan Hegermann ◽  
Erika von Gromoff ◽  
Stefan Eimer ◽  
Ralf Baumeister ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Response ◽  
Parkinson Disease ◽  
Neurite Outgrowth ◽  
Physiological Role ◽  
Model Organisms ◽  
Loss Of Function ◽  
C Elegans ◽  
Genes Encoding

Mutations in two genes encoding the putative kinases LRRK2 and PINK1 have been associated with inherited variants of Parkinson disease. The physiological role of both proteins is not known at present, but studies in model organisms have linked their mutants to distinct aspects of mitochondrial dysfunction, increased vulnerability to oxidative and endoplasmic reticulum stress, and intracellular protein sorting. Here, we show that a mutation in the Caenorhabditits elegans homologue of the PTEN-induced kinase pink-1 gene resulted in reduced mitochondrial cristae length and increased paraquat sensitivity of the nematode. Moreover, the mutants also displayed defects in axonal outgrowth of a pair of canal-associated neurons. We demonstrate that in the absence of lrk-1, the C. elegans homologue of human LRRK2, all phenotypic aspects of pink-1 loss-of-function mutants were suppressed. Conversely, the hypersensitivity of lrk-1 mutant animals to the endoplasmic reticulum stressor tunicamycin was reduced in a pink-1 mutant background. These results provide the first evidence of an antagonistic role of PINK-1 and LRK-1. Due to the similarity of the C. elegans proteins to human LRRK2 and PINK1, we suggest a common role of both factors in cellular functions including stress response and regulation of neurite outgrowth. This study might help to link pink-1/PINK1 and lrk-1/LRRK2 function to the pathological processes resulting from Parkinson disease-related mutants in both genes, the first manifestations of which are cytoskeletal defects in affected neurons.

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