scholarly journals Cell-associated, Heparin-like Molecules Modulate the Ability of LDL to Regulate PCSK9 Uptake

2018 ◽  
Author(s):  
Adri M. Galvan ◽  
John S. Chorba
Keyword(s):  
Ldl Receptor ◽  
Sulfate Proteoglycan ◽  
Biochemical Basis ◽  
A Cell ◽  
Epidermal Growth ◽  
Ldl Binding ◽  
Truncated Variants ◽  
The Relationship ◽  
Predominant Effect

AbstractProprotein convertase subtilisin/kexin type 9 (PCSK9) targets the LDL receptor (LDLR) for degradation, increasing plasma LDL and, consequently, cardiovascular risk. Uptake of secreted PCSK9 is required for its predominant effect on the LDLR. LDL itself inhibits this uptake, though the mechanism by which it does so remains unclear. In this study, we investigated the relationship between LDL, the PCSK9:LDLR interaction, and PCSK9 uptake. We show that LDL inhibits binding of PCSK9 to the epidermal growth factor precursor homology domain A (EGF-A) domain of the LDLR in vitro more impressively than it inhibits PCSK9 uptake in cells. Furthermore, a cell-based factor responsive to heparin-targeting treatments can explain this difference, consistent with its identity as a cell surface heparan sulfate proteoglycan (HSPG), a known co-receptor for PCSK9. Furthermore, we show that the entire PCSK9 prodomain, but not truncated variants, rescues PCSK9 uptake in the presence of LDL, suggesting that PCSK9:LDL binding requires the entire prodomain. Additionally, we show that the gain-of-function (GOF) PCSK9 variant S127R has increased affinity for heparin-like molecules such as HSPGs, potentially explaining the biochemical basis for its phenotype. Overall, our findings suggest a model where PCSK9, LDL, and HSPGs all interact to regulate PCSK9 uptake into the hepatocyte.

scholarly journals SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO. III

1933 ◽  
Vol 16 (4) ◽  
pp. 571-577 ◽  
Author(s):  
J. M. Nelson ◽  
B. G. Wilkes
Keyword(s):  
Physiological Activity ◽  
Water Concentration ◽  
Invertase Activity ◽  
Yeast Cells ◽  
Identical Manner ◽  
A Cell ◽  
Relationship Of ◽  
The Relationship

1. The relationship of sucrose and water concentration to invertase activity in vivo and in vitro has been studied under the same environmental conditions. 2. The sucroclastic activity of S. cerevisiae cells and of invertase solutions prepared from them reacts to changes in sucrose and water concentration in an identical manner. 3. The invertase contained in living yeast cells is just as freely exposed to the conditions of sucrose and water concentrations of the suspending medium as it would be if it were contained in a cell-free solution. Weight is added to the previous suggestion (2) that yeast invertase exerts its physiological activity in a region quite close to the surface of the cell.

Scanning Studies of Cell Surfaces

1973 ◽  
Vol 31 ◽  
pp. 608-609
Author(s):  
Virginia Fonte ◽  
Nancy Weller ◽  
Keith R. Porter
Keyword(s):  
Cell Cycle ◽  
Structural Organization ◽  
Cell Surfaces ◽  
Detailed Characterization ◽  
A Cell ◽  
Relationship Of ◽  
The Relationship ◽  
Scanning Electron

The surfaces of a cell in its topography and anti-genicity expresses subtle variations in the effective genome, as well as the physiology and structural organization of the underlying cytoplasm. Understanding the relationship of these various factors to the surface depends in part on obtaining a detailed characterization of the topography of cells and how this topography changes with phases in the cell cycle, with transformation to malignancy and with the cell's response to such physiologically active agents as cyclic AMP.We have therefore explored the usefulness of the scanning electron microscope in investigations of the cell's topography. Cells grown under favourable in vitro conditions have been fixed in glutaraldehyde, dehydrated in acetone and dried by the critical point method of Anderson.

The role of yan in mediating the choice between cell division and differentiation

Development ◽  
1995 ◽  
Vol 121 (12) ◽  
pp. 3947-3958 ◽  
Author(s):  
R. Rogge ◽  
P.J. Green ◽  
J. Urano ◽  
S. Horn-Saban ◽  
M. Mlodzik ◽  
...  
Keyword(s):  
Cell Division ◽  
Growth Factor Receptor ◽  
Developmental Context ◽  
Eye Disc ◽  
A Cell ◽  
Epidermal Growth ◽  
Notch Pathways ◽  
Relationship Of ◽  
The Relationship

An allele of the yan locus was isolated as an enhancer of the Ellipse mutation of the Drosophila epidermal growth factor receptor (Egfr) gene. This yan allele is an embryonic lethal and also fails to complement the lethality of anterior open (aop) mutations. Phenotypic and complementation analysis revealed that aop is allelic to yan and genetically the lethal alleles act as null mutations for the yan gene. Analysis of the lethal alleles in the embryo and in mitotic clones showed that loss of yan function causes cells to overproliferate in the dorsal neuroectoderm of the embryo and in the developing eye disc. Our studies suggest that the role of yan is defined by the developmental context of the cells in which it functions. An important role of this gene is in allowing a cell to choose between cell division and differentiation. The relationship of the Egfr and Notch pathways to this developmental role of yan is discussed.

scholarly journals A rapid decrease in epidermal growth factor-binding capacity accompanies the terminal differentiation of mouse myoblasts in vitro.

1984 ◽  
Vol 98 (2) ◽  
pp. 739-747 ◽  
Author(s):  
R W Lim ◽  
S D Hauschka
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Capacity ◽  
Growth Factor Receptor ◽  
Terminal Differentiation ◽  
Growth Stimulation ◽  
Cycle Arrest ◽  
A Cell ◽  
Epidermal Growth

Specific mitogens stimulate the proliferation and repress the differentiation of mouse myoblasts (MM14). When mitogens are depleted, MM14 cells cease proliferation, commit to terminal differentiation, and become refractory to growth stimulation. The behavior of mitogen receptors during the transition from a proliferative to a permanently postmitotic state was examined using the epidermal growth factor receptor (EGFR) as a model system. Whereas proliferating myoblasts bound substantial amounts of EGF, their binding capacity declined rapidly upon exposure to low-mitogen medium. The decline became irreversible when a cell differentiated. Within 24 h, less than 5% of the original EGF binding capacity remained. Since the ability to internalize and degrade bound EGF was unaffected, the change presumably reflected a decrease in EGFR availability. Several observations indicated that loss of EGFR following mitogen removal is related to differentiation rather than the result of starvation or cell-cycle arrest. First, the decline is correlated with the absence of a single mitogen (fibroblast growth factor) and is independent of serum concentrations. Second, myoblasts that are either cycling through G1 or arrested at G0, but prevented from differentiating, all bind large amounts of EGF. These findings suggest that specific reduction in mitogen receptors could be part of a mechanism whereby terminally differentiating cells become refractory to mitogenic stimulation.

scholarly journals A transient amphipathic helix in the prodomain of PCSK9 facilitates binding to low-density lipoprotein particles

2020 ◽  
Vol 295 (8) ◽  
pp. 2285-2298
Author(s):  
Samantha K. Sarkar ◽  
Alexander C. Y. Foo ◽  
Angela Matyas ◽  
Ikhuosho Asikhia ◽  
Tanja Kosenko ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Loss Of Function ◽  
Ldl Particles ◽  
Ldl Binding ◽  
Α Helix

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein (LDL) receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30–40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding in vitro requires a disordered N-terminal region in PCSK9's prodomain. Here, we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N terminus adopt helical structure in a membrane-mimetic environment. This effect was greatly enhanced by an R46L substitution representing an atheroprotective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested that the transient α-helix aligns multiple polar residues to interact with positively charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, and F515L) inhibited LDL binding, which was completely abolished in the case of the R496W variant. These findings shed light on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, the initial identification of FH-associated mutations that diminish PCSK9's ability to bind LDL reported here supports the notion that PCSK9-LDL association in the circulation inhibits PCSK9 activity.

scholarly journals Novel Porous Forsterite Ceramics Biocompatibility and Bioactivity Evaluation

2020 ◽  
Vol 71 (2) ◽  
pp. 343-351
Author(s):  
Maria Gorea ◽  
Marieta-Adriana Naghiu ◽  
Alexandra Avram ◽  
Ioan Petean ◽  
Aurora Mocanu ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Sol Gel ◽  
Bone Substitutes ◽  
Poly Vinyl Alcohol ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
A Cell ◽  
The Relationship ◽  
Binding Component

This study is aimed to evaluate the biocompatibility and bioactivity of some new porous forsterite ceramics (FCs) produced from high-purity nano forsterite powder, synthesized by an original sol-gel method, which was subjected to pressing into pellets, by using a poly vinyl alcohol solution as a binding component. Then, the raw pellets were sintered at 1200 �C, 1300 �C, 1400 �C and 1450 �C. The obtained four forsterite ceramics, FC-1200, FC-1300, FC-1400 and FC-1450, were fully characterized by density, porosity and shrinkage measurements. The forsterite ceramics exhibited excellent biocompatibility determined by an in vitro cell viability assay, such as MTT test. Furthermore, the in vitro bioactivity test was performed by immersing the forsterite ceramics into simulated body fluid (SBF) and examining the hydroxyapatite (HAP) formation on forsterite ceramics, as evidenced by XRD, FTIR, SEM with EDX. Moreover, the relationship between porous structure and bioactivity of forsterite ceramics in SBF as well as the performance of FC in a cell culture was evaluated. The findings strongly recommend these forsterite ceramics for biomedical applications, as potential bone substitutes.

scholarly journals Design of a GAK/EGFR inhibitor set to interrogate the relationship of EGFR and GAK in chordoma

2018 ◽  
Author(s):  
Christopher R. M. Asquith ◽  
Kaleb M. Naegeli ◽  
Michael P. East ◽  
Tuomo Laitinen ◽  
Tammy M. Havener ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor Receptor ◽  
Egfr Inhibitor ◽  
Target Engagement ◽  
Cell Target ◽  
Epidermal Growth ◽  
Cyclin G ◽  
Relationship Of ◽  
The Relationship

ABSTRACTWe describe the design of a set of inhibitors to investigate the relationship between cyclin G associated kinase (GAK) and epidermal growth factor receptor (EGFR) in chordoma bone cancers. These compounds were characterized both in vitro and using in cell target engagement assays. The most potent chordoma inhibitors were further characterized in a kinome-wide screen demonstrating narrow spectrum profiles.

scholarly journals Characterization of Microtubule Destabilizing Drugs: A Quantitative Cell-Based Assay That Bridges the Gap between Tubulin Based- and Cytotoxicity Assays

Cancers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 5226
Author(s):  
Marie-Catherine Laisne ◽  
Sophie Michallet ◽  
Laurence Lafanechère
Keyword(s):  
Cell Viability ◽  
Microtubule Inhibitors ◽  
Cytotoxicity Assays ◽  
A Cell ◽  
Sustained Effort ◽  
Emergence Of Resistance ◽  
The Relationship ◽  
Kinetics Of

(1) Background: Microtubule depolymerizing agents (MDAs) are commonly used for cancer treatment. However, the therapeutic use of such microtubule inhibitors is limited by their toxicity and the emergence of resistance. Thus, there is still a sustained effort to develop new MDAs. During the characterization of such agents, mainly through in vitro analyses using purified tubulin and cytotoxicity assays, quantitative comparisons are mandatory. The relationship between the effect of the drugs on purified tubulin and on cell viability are not always direct. (2) Methods: We have recently developed a cell-based assay that quantifies the cellular microtubule content. In this study, we have conducted a systematic comparative analysis of the effect of four well-characterized MDAs on the kinetics of in vitro tubulin assembly, on the cellular microtubule content (using our recently developed assay) and on cell viability. (3) Conclusions: These assays gave complementary results. Additionally, we found that the drugs’ effect on in vitro tubulin polymerization is not completely predictive of their relative cytotoxicity. Their effect on the cellular microtubule content, however, is closely related to their effect on cell viability. In conclusion, the assay we have recently developed can bridge the gap between in vitro tubulin assays and cell viability assays.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

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