scholarly journals A Reinterpretation of Evidence for the Endothelial Glycocalyx Filtration Structure

2021 ◽  
Vol 9 ◽  
Author(s):  
Kenton P. Arkill
Keyword(s):  
Core Protein ◽  
Endothelial Glycocalyx ◽  
Actin Binding ◽  
Surface Coat ◽  
Fixed Tissue ◽  
Transmission Electron ◽  
Square Packing ◽  
New Interpretation ◽  
Fiber Thickness

The endothelial glycocalyx (eGlx) is thought to be the primary macromolecular filter for fluid flux out of the vasculature. This filter maintains the higher protein concentration within the vessel lumen relative to the tissue. Whilst the arguments for the eGlx being the size filter are convincing the structural evidence has been limited to specialized stains of perfusion fixed tissue, which are further processed for resin embedding for transmission electron microscopy. The staining and processing of the delicate pore structure has left many researchers struggling to interpret the observed surface coat. Previous work has alluded to a 19.5 nm spacing between fibers; however, whilst repeatable it does not give an eGlx pore size consistent with known glycosaminoglycan (GAG) molecular structure due to the required fiber thickness of >10 nm. Here a new interpretation is proposed based on the likelihood that the electron micrographs of are often of collapsed eGlx. The 19.5 nm spacing measured may therefore be the core protein of the proteoglycans (PGs) with the GAGs wrapped up around them rather than in an expanded in vivo state. The concept is explored to determine that this is indeed consistent with experimental measurements of permeability if the syndecans are predominately dimerized. Further an alteration of core protein lattice from hexagonal packing to square packing dramatically changes the permeability which could be facilitated via known mechanisms such as transient actin binding.

Effect of brefeldin A (BFA) on the unique surface coat of pulmonary intravascular macrophages (PIMs) of sheep: Ultrastructural and cytochemical study

1993 ◽  
Vol 51 ◽  
pp. 18-19
Author(s):  
Baljit Singh
Keyword(s):  
Brefeldin A ◽  
Ultrastructural Feature ◽  
Surface Coat ◽  
Anterograde Transport ◽  
Cytochemical Study ◽  
Active Involvement ◽  
Electron Lucent ◽  
Pulmonary Intravascular Macrophages ◽  
Unique Surface

The PIM of sheep, calf, goat and horse has a characteristic ultrastructural feature in the form of a unique, heparin sensitive, globular surface coat present around the plasma membrane with an intervening electron lucent space of 32-40 nm. We previously showed the active involvement of this surface coat in the phagocytosis of tracer material like monastral blue and cationized ferritin. The surface coat is capable of reconstitution in vivo following disruption with heparin. The present study was aimed to investigate whether PIM is the source of surface coat or not. In the recent years the BFA has been extensively used to understand the secretory pathways in the cells because of its ability to cause a rapid and reversible block to the anterograde transport of proteins from the endoplasmic reticulum to the Golgi.Sheep (n=6) were weighed, their plasma volume was calculated indirectly and based on which a sufficient single intravenous dose of BFA was given so as to reach a concentration of 4-5 microgram/ml of plasma.

Pharmacokinetic Evaluation of 99mTc-radiolabeled Solid Lipid Nanoparticles and Chitosan Coated Solid Lipid Nanoparticles

2020 ◽  
Vol 20 (13) ◽  
pp. 1044-1052
Author(s):  
Nasrin Abbasi Gharibkandi ◽  
Sajjad Molavipordanjani ◽  
Jafar Akbari ◽  
Seyed Jalal Hosseinimehr
Keyword(s):  
Solid Lipid Nanoparticles ◽  
Lipid Nanoparticles ◽  
High Resistivity ◽  
Scanning Calorimetry ◽  
Liver Uptake ◽  
Solid Lipid ◽  
Transmission Electron ◽  
Main Portion ◽  
Pharmacokinetic Behavior

Background: Solid Lipid Nanoparticles (SLNs) possess unique in vivo features such as high resistivity, bioavailability, and habitation at the target site. Coating nanoparticles with polymers such as chitosan greatly affects their pharmacokinetic behavior, stability, tissue uptake, and controlled drug delivery. The aim of this study was to prepare and evaluate the biodistribution of 99mTc-labeled SLNs and chitosan modified SLNs in mice. Methods: 99mTc-oxine was prepared and utilized to radiolabel pre-papered SLNs or chitosan coated SLNs. After purification of radiolabeled SLNs (99mTc-SLNs) and radiolabeled chitosan-coated SLNs (99mTc-Chi-SLNs) using Amicon filter, they were injected into BALB/c mice to evaluate their biodistribution patterns. In addition, nanoparticles were characterized using Transmission Electron Microscopy (TEM), Fourier-transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), X-ray Powder Diffraction (XRD) and Dynamic Light Scattering (DLS). Results: 99mTc-oxine with high radiochemical purity (RCP~100%) and stability (RCP > 97% at 24 h) was used to provide 99mTc-SLNs and 99mTc-Chi-SLNs with high initial RCP (100%). TEM image and DLS data suggest 99mTc- SLNs susceptibility to aggregation. To that end, the main portion of 99mTc-SLNs radioactivity accumulates in the liver and intestines, while 99mTc-Chi-SLNs sequesters in the liver, intestines and kidneys. The blood radioactivity of 99mTc-Chi-SLNs was higher than that of 99mTc-SLNs by 7.5, 3.17 and 3.5 folds at 1, 4 and 8 h post-injection. 99mTc- Chi-SLNs uptake in the kidneys in comparison with 99mTc-SLNs was higher by 37.48, 5.84 and 11 folds at 1, 4 and 8h. Conclusion: The chitosan layer on the surface of 99mTc-Chi-SLNs reduces lipophilicity in comparison with 99mTc- SLNs. Therefore, 99mTc-Chi-SLNs are less susceptible to aggregation, which leads to their lower liver uptake and higher kidney uptake and blood concentration.

scholarly journals Mice with cleavage-resistant N-cadherin exhibit synapse anomaly in the hippocampus and outperformance in spatial learning tasks

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
M. Asada-Utsugi ◽  
K. Uemura ◽  
M. Kubota ◽  
Y. Noda ◽  
Y. Tashiro ◽  
...  
Keyword(s):  
Memory Function ◽  
Three Dimensional ◽  
Excitatory Synapses ◽  
Missense Mutations ◽  
Glutamatergic Synapses ◽  
Learning Tasks ◽  
Transmission Electron ◽  
Nmdar Activation ◽  
And Function

AbstractN-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 s than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.

scholarly journals Protectin conjugates in tissue regeneration 1 restores lipopolysaccharide-induced pulmonary endothelial glycocalyx loss via ALX/SIRT1/NF-kappa B axis

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xin-Yang Wang ◽  
Xin-Yu Li ◽  
Cheng-Hua Wu ◽  
Yu Hao ◽  
Pan-Han Fu ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Inflammatory Cytokines ◽  
Tissue Regeneration ◽  
Umbilical Vein ◽  
Endothelial Glycocalyx ◽  
Lipid Mediator ◽  
Protective Effects ◽  
Pulmonary Vascular Permeability

Abstract Background Endothelial glycocalyx loss is integral to increased pulmonary vascular permeability in sepsis-related acute lung injury. Protectin conjugates in tissue regeneration 1 (PCTR1) is a novel macrophage-derived lipid mediator exhibiting potential anti-inflammatory and pro-resolving benefits. Methods PCTR1 was administrated intraperitoneally with 100 ng/mouse after lipopolysaccharide (LPS) challenged. Survival rate and lung function were used to evaluate the protective effects of PCTR1. Lung inflammation response was observed by morphology and inflammatory cytokines level. Endothelial glycocalyx and its related key enzymes were measured by immunofluorescence, ELISA, and Western blot. Afterward, related-pathways inhibitors were used to identify the mechanism of endothelial glycocalyx response to PCTR1 in mice and human umbilical vein endothelial cells (HUVECs) after LPS administration. Results In vivo, we show that PCTR1 protects mice against lipopolysaccharide (LPS)-induced sepsis, as shown by enhanced the survival and pulmonary function, decreased the inflammatory response in lungs and peripheral levels of inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1β. Moreover, PCTR1 restored lung vascular glycocalyx and reduced serum heparin sulphate (HS), syndecan-1 (SDC-1), and hyaluronic acid (HA) levels. Furthermore, we found that PCTR1 downregulated heparanase (HPA) expression to inhibit glycocalyx degradation and upregulated exostosin-1 (EXT-1) protein expression to promote glycocalyx reconstitution. Besides, we observed that BAY11-7082 blocked glycocalyx loss induced by LPS in vivo and in vitro, and BOC-2 (ALX antagonist) or EX527 (SIRT1 inhibitor) abolished the restoration of HS in response to PCTR1. Conclusion PCTR1 protects endothelial glycocalyx via ALX receptor by regulating SIRT1/NF-κB pathway, suggesting PCTR1 may be a significant therapeutic target for sepsis-related acute lung injury.

scholarly journals Qualitative ultrastructural analysis of the submandibular salivary glands after administration of khat: in vivo study

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Gamilah Al-Qadhi ◽  
Rabab Mubarak
Keyword(s):  
Salivary Glands ◽  
Arabian Peninsula ◽  
Submandibular Salivary Gland ◽  
Catha Edulis ◽  
Ductal Cells ◽  
Cytoplasmic Vacuoles ◽  
Transmission Electron ◽  
Salivary Gland Cells ◽  
Submandibular Salivary Glands

Abstract Objective Khat (Catha edulis Forssk) plant has been widely chewed for its psychostimulatory effects in the African and Arabian Peninsula, particularly in Yemen. Considering the khat leaves are gradually chewed without swallowing, while its active constituents are extracted into saliva, studying the effect of khat on salivary glands is necessary. This work is an extension of the previously published work that studied the effect of khat extract on the rats' submandibular salivary glands in terms of histological and immunohistochemical evaluations. The current research note aimed to better understand this effect on the ultrastructure of submandibular salivary gland cells by using transmission electron microscope. Results Oral administration of khat extract produced degenerative changes in the secretory and ductal cells of rats' submandibular salivary glands. These changes involved irregular boundaries of variable sized-nuclei, dilated RER, cytoplasmic vacuoles as well as swollen and degenerated mitochondria.

scholarly journals Specific heparan sulfate modifications stabilize the synaptic organizer MADD-4/Punctin at C. elegans neuromuscular junctions

Genetics ◽  
2021 ◽  
Author(s):  
Mélissa Cizeron ◽  
Laure Granger ◽  
Hannes E BÜlow ◽  
Jean-Louis Bessereau
Keyword(s):  
Heparan Sulfate ◽  
Matrix Protein ◽  
Neuromuscular Junctions ◽  
Core Protein ◽  
Extracellular Matrix Protein ◽  
Inhibitory Synapses ◽  
Single Chain ◽  
C Elegans ◽  
Sugar Chains

Abstract Heparan sulfate proteoglycans contribute to the structural organization of various neurochemical synapses. Depending on the system, their role involves either the core protein or the glycosaminoglycan chains. These linear sugar chains are extensively modified by heparan sulfate modification enzymes, resulting in highly diverse molecules. Specific modifications of glycosaminoglycan chains may thus contribute to a sugar code involved in synapse specificity. Caenorhabditis elegans is particularly useful to address this question because of the low level of genomic redundancy of these enzymes, as opposed to mammals. Here, we systematically mutated the genes encoding heparan sulfate modification enzymes in C. elegans and analyzed their impact on excitatory and inhibitory neuromuscular junctions. Using single chain antibodies that recognize different heparan sulfate modification patterns, we show in vivo that these two heparan sulfate epitopes are carried by the SDN-1 core protein, the unique C. elegans syndecan orthologue, at neuromuscular junctions. Intriguingly, these antibodies differentially bind to excitatory and inhibitory synapses, implying unique heparan sulfate modification patterns at different neuromuscular junctions. Moreover, while most enzymes are individually dispensable for proper organization of neuromuscular junctions, we show that 3-O-sulfation of SDN-1 is required to maintain wild-type levels of the extracellular matrix protein MADD-4/Punctin, a central synaptic organizer that defines the identity of excitatory and inhibitory synaptic domains at the plasma membrane of muscle cells.

scholarly journals Cemp1-p3 Peptide Promotes the Transformation of Octacalcium Phosphate into Hydroxyapatite Crystals

Crystals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1131
Author(s):  
Maricela Santana ◽  
Gonzalo Montoya ◽  
Raúl Herrera ◽  
Lía Hoz ◽  
Enrique Romo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Octacalcium Phosphate ◽  
Sequence Motifs ◽  
Simple Method ◽  
Transmission Electron ◽  
Precursor Phase ◽  
Mineralized Tissues ◽  
Potent Growth

Dental cementum contains unique molecules that regulate the mineralization process in vitro and in vivo, such as cementum protein 1 (CEMP1). This protein possesses amino acid sequence motifs like the human recombinant CEMP1 with biological activity. This novel cementum protein 1-derived peptide (CEMP1-p3, from the CEMP1’s N-terminal domain: (QPLPKGCAAVKAEVGIPAPH), consists of 20 amino acids. Hydroxyapatite (HA) crystals could be obtained through the combination of the amorphous precursor phase and macromolecules such as proteins and peptides. We used a simple method to synthesize peptide/hydroxyapatite nanocomposites using OCP and CEMP1-p3. The characterization of the crystals through scanning electron microscopy (SEM), powder X-ray diffraction (XRD), high--resolution transmission electron microscopy (HRTEM), and Raman spectroscopy revealed that CEMP1-p3 transformed OCP into hydroxyapatite (HA) under constant ionic strength and in a buffered solution. CEMP1-p3 binds and highly adsorbs to OCP and is a potent growth stimulator of OCP crystals. CEMP1-p3 fosters the transformation of OCP into HA crystals with crystalline planes (300) and (004) that correspond to the cell of hexagonal HA. Octacalcium phosphate crystals treated with CEMP1-p3 grown in simulated physiological buffer acquired hexagonal arrangement corresponding to HA. These findings provide new insights into the potential application of CEMP1-p3 on possible biomimetic approaches to generate materials for the repair and regeneration of mineralized tissues, or restorative materials in the orthopedic field.

scholarly journals Villin Severing Activity Enhances Actin-based Motility In Vivo

2007 ◽  
Vol 18 (3) ◽  
pp. 827-838 ◽  
Author(s):  
Céline Revenu ◽  
Matthieu Courtois ◽  
Alphée Michelot ◽  
Cécile Sykes ◽  
Daniel Louvard ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Capping Protein ◽  
Function Strategy ◽  
In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

scholarly journals Ezrin is an Actin Binding Protein That Regulates Sertoli Cell and Spermatid Adhesion During Spermatogenesis

Endocrinology ◽  
2014 ◽  
Vol 155 (10) ◽  
pp. 3981-3995 ◽  
Author(s):  
N. Ece Gungor-Ordueri ◽  
Elizabeth I. Tang ◽  
Ciler Celik-Ozenci ◽  
C. Yan Cheng
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Adherens Junction ◽  
Actin Binding ◽  
Permeability Barrier ◽  
Tight Junction Permeability ◽  
Residual Bodies ◽  
Cell Interface

Abstract During spermatogenesis, the transport of spermatids and the release of sperms at spermiation and the remodeling of the blood-testis barrier (BTB) in the seminiferous epithelium of rat testes require rapid reorganization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction at the spermatid-Sertoli cell interface called apical ectoplasmic specialization (ES) in the adluminal compartment and the Sertoli cell-cell interface known as basal ES at the BTB. Ezrin is expressed at the basal ES/BTB in all stages, except from late VIII to IX, of the epithelial cycle. Its knockdown by RNA interference (RNAi) in vitro perturbs the Sertoli cell tight junction-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (eg, N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede spermatid transport, causing defects in spermiation in which spermatids were embedded deep inside the epithelium, and associated with a loss of spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilament organization at the ES in rat testes.

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