Cell-Free Membrane Expression into Hybrid Lipid/Polymer

SummarySpecific membrane glycoproteins (GP) expressed by the megakaryocyte-platelet system, including GPIa-lla, GPIb-V-IX, GPIIb-llla, and GPIV are involved in mediat-ing platelet adhesion to the subendothelial matrix. Among these glycoproteins, GPIIb-llla plays a pivotal role since platelet aggregation is exclusively mediated by this receptor and its interaction with soluble macromolecular proteins. Inherited defects of the GPIIb-llla or GPIb-V-IX receptor complexes are associated with bleeding disorders, known as Glanzmann's thrombasthenia, Bernard-Soulier syndrome, or platelet-type von Willebrand's disease, respectively. Using immuno-chemical and molecular biology techniques, rapid advances in our understanding of the molecular genetic basis of these disorders have been made during the last few years. Moreover, analyses of patients with congenital platelet membrane glycoprotein abnormalities have provided valuable insights into molecular mechanisms that are required for structural and functional integrity, normal biosynthesis of the glycoprotein complexes and coordinated membrane expression of their constituents. The present article reviews the current state of knowledge of the major membrane glycoproteins in health and disease. The spectrum of clinical bleeding manifestations and established diagnostic criteria for each of these dis-orders are summarized. In particular, the variety of molecular defects that have been identified so far and their genetic basis will be discussed.

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Faculty Opinions recommendation of Role of PDZK1 in membrane expression of renal brush border ion exchangers.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1027864.334836 ◽

2005 ◽

Author(s):

Mark Donowitz

Keyword(s):

Brush Border ◽

Ion Exchangers ◽

Membrane Expression ◽

Renal Brush Border

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Evaluation of E-Cadherin and β-Catenin Immunoreactivity for Determining Undifferentiated Cells in Anaplastic Thyroid Carcinoma

Pathobiology ◽

10.1159/000516263 ◽

2021 ◽

pp. 1-8

Author(s):

Risa Kanematsu ◽

Mitsuyoshi Hirokawa ◽

Aki Tanaka ◽

Ayana Suzuki ◽

Miyoko Higuchi ◽

...

Keyword(s):

Cell Membrane ◽

Thyroid Carcinoma ◽

Undifferentiated Carcinoma ◽

Anaplastic Thyroid Carcinoma ◽

Carcinoma Cells ◽

Membrane Expression ◽

Thyroid Carcinomas ◽

Nuclear Expression ◽

Thyroid Transcription Factor 1 ◽

E Cadherin

Introduction: An immunohistochemical study has occasionally been performed to diagnose anaplastic thyroid carcinoma (ATC). However, antibodies to confirm the undifferentiated nature of ATC have not yet been evaluated. The aim of this study was to evaluate E-cadherin and β-catenin expressions in immunoreactivity to determine undifferentiated carcinoma cells in the diagnosis of ATC. Methods: We immunohistochemically examined 29 ATCs, 30 poorly differentiated thyroid carcinomas (PDTCs), 22 well-differentiated thyroid carcinomas (WDTCs), and 3 squamous cell carcinomas. Antibodies for thyroid transcription factor-1 (TTF-1), paired-box gene 8 (PAX8), β-catenin, and E-cadherin were used. Results: All WDTCs tested positive for TTF-1, PAX8, and E-cadherin. The positive rates of TTF-1, PAX8, and E-cadherin were 93.3, 93.3, and 100%, respectively, in PDTCs and 17.2, 51.7, and 10.3%, respectively, in ATCs. WDTC expressed the lateral cell membrane staining for β-catenin and E-cadherin, whereas PDTC showed circumferential cell membranous expression (fishnet pattern). β-catenin cell membrane expression in ATCs is lost or discontinuous. Carcinoma cells with β-catenin nuclear expression without cell membranous expression were scattered in 72.4% of ATCs but were not observed in the other carcinomas. Conclusion: We propose 3 immunohistochemical findings to determine undifferentiated carcinoma cells in the diagnosis of ATC: (1) β-catenin nuclear expression with no or reduced cell membranous expression, (2) the loss or discontinuous pattern of E-cadherin expression, and (3) the loss of PAX8 nuclear expression.

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Lentiviral CRISPR-guided RNA library screening identified Adam17 as an upstream negative regulator of Procr in mammary epithelium

BMC Biotechnology ◽

10.1186/s12896-021-00703-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Ting Wu ◽

Yinghua Wang ◽

Tianxiong Xiao ◽

Yirui Ai ◽

Jinsong Li ◽

...

Keyword(s):

Breast Cancer ◽

Membrane Proteins ◽

Epithelial Cells ◽

Adult Stem Cells ◽

Vascular System ◽

Negative Regulator ◽

Breast Cancer Cell Lines ◽

Screening System ◽

Membrane Expression ◽

Sorting Technique

Abstract Background Protein C receptor (Procr) has recently been shown to mark resident adult stem cells in the mammary gland, vascular system, and pancreatic islets. More so, high Procr expression was also detected and used as indicator for subsets of triple-negative breast cancers (TNBCs). Previous study has revealed Procr as a target of Wnt/β-catenin signaling; however, direct upstream regulatory mechanism of Procr remains unknown. To comprehend the molecular role of Procr during physiology and pathology, elucidating the upstream effectors of Procr is necessary. Here, we provide a system for screening negative regulators of Procr, which could be adapted for broad molecular analysis on membrane proteins. Results We established a screening system which combines CRISPR-Cas9 guided gene disruption with fluorescence activated cell sorting technique (FACS). CommaDβ (murine epithelial cells line) was used for the initial Procr upstream effector screening using lentiviral CRISPR-gRNA library. Shortlisted genes were further validated through individual lentiviral gRNA infection followed by Procr expression evaluation. Adam17 was identified as a specific negative inhibitor of Procr expression. In addition, MDA-MB-231 cells and Hs578T cells (human breast cancer cell lines) were used to verify the conserved regulation of ADAM17 over PROCR expression. Conclusion We established an efficient CRISPR-Cas9/FACS screening system, which identifies the regulators of membrane proteins. Through this system, we identified Adam17 as the negative regulator of Procr membrane expression both in mammary epithelial cells and breast cancer cells.

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Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01988-6 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Andreia Peixoto ◽

Dylan Ferreira ◽

Rita Azevedo ◽

Rui Freitas ◽

Elisabete Fernandes ◽

...

Keyword(s):

Bladder Cancer ◽

Cell Surface ◽

Protein Identification ◽

Glucose Deprivation ◽

Invasive Bladder Cancer ◽

Nutrient Deprivation ◽

Molecular Heterogeneity ◽

Cell Models ◽

Membrane Expression ◽

Bladder Tumours

Abstract Background Muscle invasive bladder cancer (MIBC) remains amongst the deadliest genitourinary malignancies due to treatment failure and extensive molecular heterogeneity, delaying effective targeted therapeutics. Hypoxia and nutrient deprivation, oversialylation and O-glycans shortening are salient features of aggressive tumours, creating cell surface glycoproteome fingerprints with theranostics potential. Methods A glycomics guided glycoproteomics workflow was employed to identify potentially targetable biomarkers using invasive bladder cancer cell models. The 5637 and T24 cells O-glycome was characterized by mass spectrometry (MS), and the obtained information was used to guide glycoproteomics experiments, combining sialidase, lectin affinity and bottom-up protein identification by nanoLC-ESI-MS/MS. Data was curated by a bioinformatics approach developed in-house, sorting clinically relevant molecular signatures based on Human Protein Atlas insights. Top-ranked targets and glycoforms were validated in cell models, bladder tumours and metastases by MS and immunoassays. Cells grown under hypoxia and glucose deprivation disclosed the contribution of tumour microenvironment to the expression of relevant biomarkers. Cancer-specificity was validated in healthy tissues by immunohistochemistry and MS in 20 types of tissues/cells of different individuals. Results Sialylated T (ST) antigens were found to be the most abundant glycans in cell lines and over 900 glycoproteins were identified potentially carrying these glycans. HOMER3, typically a cytosolic protein, emerged as a top-ranked targetable glycoprotein at the cell surface carrying short-chain O-glycans. Plasma membrane HOMER3 was observed in more aggressive primary tumours and distant metastases, being an independent predictor of worst prognosis. This phenotype was triggered by nutrient deprivation and concomitant to increased cellular invasion. T24 HOMER3 knockdown significantly decreased proliferation and, to some extent, invasion in normoxia and hypoxia; whereas HOMER3 knock-in increased its membrane expression, which was more pronounced under glucose deprivation. HOMER3 overexpression was associated with increased cell proliferation in normoxia and potentiated invasion under hypoxia. Finally, the mapping of HOMER3-glycosites by EThcD-MS/MS in bladder tumours revealed potentially targetable domains not detected in healthy tissues. Conclusion HOMER3-glycoforms allow the identification of patients’ subsets facing worst prognosis, holding potential to address more aggressive hypoxic cells with limited off-target effects. The molecular rationale for identifying novel bladder cancer molecular targets has been established. Graphical abstract

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The Dual Role of the GABAA Receptor in Peripheral Inflammation and Neuroinflammation: A Study in Hyperammonemic Rats

International Journal of Molecular Sciences ◽

10.3390/ijms22136772 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6772

Author(s):

Michele Malaguarnera ◽

Tiziano Balzano ◽

Mari Carmen Castro ◽

Marta Llansola ◽

Vicente Felipo

Keyword(s):

Gabaa Receptors ◽

Motor Impairment ◽

Minimal Hepatic Encephalopathy ◽

Peripheral Inflammation ◽

Membrane Expression ◽

Gabaergic Neurotransmission ◽

Gaba Transporters ◽

Suitable Target ◽

Gabaa Antagonist

Cognitive and motor impairment in minimal hepatic encephalopathy (MHE) are mediated by neuroinflammation, which is induced by hyperammonemia and peripheral inflammation. GABAergic neurotransmission in the cerebellum is altered in rats with chronic hyperammonemia. The mechanisms by which hyperammonemia induces neuroinflammation remain unknown. We hypothesized that GABAA receptors can modulate cerebellar neuroinflammation. The GABAA antagonist bicuculline was administrated daily (i.p.) for four weeks in control and hyperammonemic rats. Its effects on peripheral inflammation and on neuroinflammation as well as glutamate and GABA neurotransmission in the cerebellum were assessed. In hyperammonemic rats, bicuculline decreases IL-6 and TNFα and increases IL-10 in the plasma, reduces astrocyte activation, induces the microglia M2 phenotype, and reduces IL-1β and TNFα in the cerebellum. However, in control rats, bicuculline increases IL-6 and decreases IL-10 plasma levels and induces microglial activation. Bicuculline restores the membrane expression of some glutamate and GABA transporters restoring the extracellular levels of GABA in hyperammonemic rats. Blocking GABAA receptors improves peripheral inflammation and cerebellar neuroinflammation, restoring neurotransmission in hyperammonemic rats, whereas it induces inflammation and neuroinflammation in controls. This suggests a complex interaction between GABAergic and immune systems. The modulation of GABAA receptors could be a suitable target for improving neuroinflammation in MHE.

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Claudin-19 mediates the effects of NO on the paracellular pathway in thick ascending limbs

AJP Renal Physiology ◽

10.1152/ajprenal.00065.2019 ◽

2019 ◽

Vol 317 (2) ◽

pp. F411-F418

Author(s):

Casandra M. Monzon ◽

Jeffrey L. Garvin

Keyword(s):

Plasma Membrane ◽

Control Period ◽

Paracellular Pathway ◽

Membrane Expression ◽

No Donor ◽

Charge Selectivity ◽

Plasma Membrane Expression ◽

Cgmp Signaling ◽

Blocking Antibodies ◽

Dilution Potential

Claudins are a family of tight junction proteins that provide size and charge selectivity to solutes traversing the paracellular space. Thick ascending limbs (TALs) express numerous claudins, including claudin-19. Nitric oxide (NO), via cGMP, reduces dilution potentials in perfused TALs, a measure of paracellular permeability, but the role of claudin-19 is unknown. We hypothesized that claudin-19 mediates the effects of NO/cGMP on the paracellular pathway in TALs via increases in plasma membrane expression of this protein. We measured the effect of the NO donor spermine NONOate (SPM) on dilution potentials with and without blocking antibodies and plasma membrane expression of claudin-19. During the control period, the dilution potential was −18.2 ± 1.8 mV. After treatment with 200 μmol/l SPM, it was −14.7 ± 2.0 mV ( P < 0.04). In the presence of claudin-19 antibody, the dilution potential was −12.7 ± 2.1 mV. After SPM, it was −12.9 ± 2.4 mV, not significantly different. Claudin-19 antibody alone had no effect on dilution potentials. In the presence of Tamm-Horsfall protein antibody, SPM reduced the dilution potential from −9.7 ± 1.0 to −6.3 ± 1.1 mV ( P < 0.006). Dibutyryl-cGMP (500 µmol/l) reduced the dilution potential from −19.6 ± 2.6 to −17.2 ± 2.3 mV ( P < 0.002). Dibutyryl-cGMP increased expression of claudin-19 in the plasma membrane from 29.9 ± 3.8% to 65.9 ± 10.1% of total ( P < 0.011) but did not change total expression. We conclude that claudin-19 mediates the effects of the NO/cGMP signaling cascade on the paracellular pathway.

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Flow cytometric analysis of megakaryocytes from patients with abnormal platelet counts

Blood ◽

10.1182/blood.v74.2.594.594 ◽

1989 ◽

Vol 74 (2) ◽

pp. 594-601 ◽

Cited By ~ 48

Author(s):

A Tomer ◽

P Friese ◽

R Conklin ◽

W Bales ◽

L Archer ◽

...

Keyword(s):

Cell Size ◽

Flow Cytometric Analysis ◽

Distribution Analysis ◽

Membrane Expression ◽

Platelet Destruction ◽

Platelet Production ◽

Flow Cytometric ◽

High Ploidy ◽

The Right ◽

Primary Thrombocytosis

Abstract Megakaryocytes (MKs) from 40 patients with quantitative platelet disorders and 19 normal volunteers were analyzed by flow cytometry for size, fine cell internal structure and granularity, membrane expression of the glycoprotein (GP) IIb/IIIa complex, and for ploidy distribution. Analysis was performed on unfractionated minimally manipulated marrows obtained from routine bone marrow aspirates. MKs were labeled with a fluorescent lineage-specific monoclonal antibody to the GPIIb/IIIa complex followed by DNA staining with propidium iodide. Eight hundred to 3,000 MKs were analyzed in each sample. The modal ploidy distribution in normals was 16N, comprising about half of the megakaryocytic population, with 22.6% of the cells less than or equal to 8N and 22.0% greater than or equal to 32N. Twelve thrombocytopenic patients with decreased marrow MKs on biopsy (mean platelet count [MPC] 44,600/microliters) showed an increase in low ploidy cells with 53.2% less than or equal to 8N (P less than .01); cell size was reduced in three patients when compared to normal cells of identical ploidy (P less than .05). Eight thrombocytopenic patients with enhanced platelet destruction (with normal or increased MKs on biopsy and shortened platelet survival; MPC 41,400/microliters) showed an increased proportion of high ploidy cells greater than or equal to 32N to 39.2% (P less than .01). Increased cell size and granularity were found in four of these patients (P less than .05). Six patients with thrombocytopenia secondary to multiple mechanisms affecting both platelet production and destruction (MPC 66,700/microliters) showed no shift in ploidy. Four patients with primary thrombocytosis (two with thrombocythemia and two with polycythemia vera; MPC 822,500/microliters) showed a marked shift toward high ploidy cells with 42.3% greater than or equal to 32N and 7.6% greater than or equal to 64N cells (P less than .01). The shift was accompanied by a marked increase in cell size and granularity in the patients with thrombocythemia. Ten patients with thrombocytosis secondary to chronic blood loss, malignant or inflammatory disorders (MPC 714,000/microliters), showed variable distributions with four patients exhibiting a shift in ploidy to the right similar to that found in the patients with increased platelet destruction. Based upon the present data, flow cytometric ploidy distribution may be diagnostically useful in thrombocytopenic patients by discriminating between disorders of platelet production and destruction.

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