scholarly journals Differences in the autocatalytic cleavage of pro-PC2 and pro-PC3 can be attributed to sequences within the propeptide and Asp310 of pro-PC2

1998 ◽  
Vol 334 (3) ◽  
pp. 531-537 ◽  
Author(s):  
Kathleen SCOUGALL ◽  
Neil A. TAYLOR ◽  
Joanne L. JERMANY ◽  
Kevin DOCHERTY ◽  
Kathleen I. J. SHENNAN
Keyword(s):  
Secretory Pathway ◽  
Cell Types ◽  
Structural Features ◽  
Neuroendocrine Cells ◽  
Acidic Ph ◽  
Ph Optimum ◽  
Equivalent Position ◽  
Optimum Ph ◽  
Pro Region

PC2 and PC3 are subtilisin-like proteases involved in the maturation of prohormones and proneuropeptides within neuroendocrine cells. They are synthesized as zymogens that undergo autocatalytic maturation within the secretory pathway. Maturation of pro-PC2 is slow (t½ > 8 h), exhibits a pH optimum of 5.5 and is dependent on calcium (K0.5 2 mM), while pro-PC3 maturation is relatively rapid (t½ 15 min), exhibits a neutral pH optimum and is not calcium dependent. These differences in the rates and optimal conditions for activation of the proteases may contribute to the diversity of products generated by these proteases in different cell types. Although highly similar, there are two major differences between pro-PC2 and pro-PC3: the presence of an aspartate at position 310 in pro-PC2 compared with asparagine at the equivalent position in pro-PC3 (and all other members of the subtilisin family), and the N-terminal propeptides, which exhibit low sequence identity (30%). With a view to establishing the structural features that might be responsible for these differences in the maturation of pro-PC2 and pro-PC3, Asp310 in pro-PC2 was mutated to Asn, and Asn309 in pro-PC3 was mutated to Asp. Chimaeric proteins were also made consisting of the pro-region of PC2 fused to the mature portion of PC3 and the pro-region of PC3 fused to the mature region of PC2. The wild-type and mutant DNA constructs were then transcribed and translated in an in vitro system capable of supporting maturation of pro-PC2 and pro-PC3. The results demonstrated that Asp310 of pro-PC2 is responsible for the acidic pH optimum for maturation. Thus changing Asp310 to Asn shifted the pH optimum for maturation to pH 7.0. However, changing Asn309 of pro-PC3 to Asp had no effect on the optimum pH for maturation of pro-PC3. A chimaeric construct containing the propeptide of pro-PC2 attached to PC3 shifted the pH optimum for maturation from pH 7.0 to 6.0 and slowed down the rate of maturation (t½ > 8 h). When attached to PC2, the pro-region of pro-PC3 had no effect on the optimum pH for maturation (pH 5.5–6.0), but it did accelerate the rate of maturation (t½ 2 h). These results demonstrate that Asp310 and the pro-region of pro-PC2 contribute to the acidic pH optimum and low rate of maturation of this zymogen relative to its closely related homologue PC3.

Neuroendocrine secretory protein 7B2: structure, expression and functions

2001 ◽  
Vol 357 (2) ◽  
pp. 329-342 ◽  
Author(s):  
Majambu MBIKAY ◽  
Nabil G. SEIDAH ◽  
Michel CHRÉTIEN
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Secretory Protein ◽  
Neuroendocrine Cells ◽  
High Similarity ◽  
Granule Formation ◽  
Proteolytic Activation ◽  
Polyproline Motif

7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2–7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin (‘ACTH’) hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation.

scholarly journals Murine clusterin: molecular cloning and mRNA localization of a gene associated with epithelial differentiation processes during embryogenesis

1993 ◽  
Vol 122 (5) ◽  
pp. 1119-1130 ◽  
Author(s):  
LE French ◽  
A Chonn ◽  
D Ducrest ◽  
B Baumann ◽  
D Belin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Cell Types ◽  
Branching Morphogenesis ◽  
Structural Features ◽  
Heparin Binding ◽  
Binding Domains ◽  
Cell Layers

Clusterin is a broadly distributed glycoprotein constitutively expressed by various tissues and cell types, that has been shown to be involved in cell-cell adhesion and expressed during cellular differentiation in vitro. To assess the suggested participation of clusterin in these processes in vivo, we have cloned the cDNA encoding murine clusterin and studied the cellular distribution of clusterin mRNA during murine embryogenesis. Sequence analysis of the cDNA encoding murine clusterin revealed 92 and 75% sequence identity with the rat and human cDNAs, respectively, and conservation of the predicted structural features which include alpha-helical regions and heparin-binding domains. From 12.5 d of development onwards, the clusterin gene is widely expressed in developing epithelia, and selectively localized within the differentiating cell layers of tissues such as the developing skin, tooth, and duodenum where proliferating and differentiating compartments are readily distinguished. In addition, transient and localized clusterin gene expression was detected in certain morphogenetically active epithelia. In the lung, abundant gene transcripts were detected in cuboidal epithelial cells of the terminal lung buds during branching morphogenesis, and in the kidney, clusterin gene expression in the epithelial cells of comma and S-shaped bodies coincided with the process of polarization. Our results demonstrate the in vivo expression of the clusterin gene by differentiating epithelial cells during murine embryogenesis, and provide novel evidence suggesting that clusterin may be involved in the differentiation and morphogenesis of certain epithelia.

scholarly journals In vitro generation of human pluripotent stem cell derived lung organoids

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Briana R Dye ◽  
David R Hill ◽  
Michael AH Ferguson ◽  
Yu-Hwai Tsai ◽  
Melinda S Nagy ◽  
...  
Keyword(s):  
Human Lung ◽  
Upper Airway ◽  
Fetal Lung ◽  
Cell Types ◽  
Structural Features ◽  
In Vitro Models ◽  
Basal Cells ◽  
3 Dimensional ◽  
Organ Systems

Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here, we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids, which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung, organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing, we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles, suggesting that HLOs are an excellent model to study human lung development, maturation and disease.

scholarly journals Surface-Coated Polylactide Fiber Meshes as Tissue Engineering Matrices with Enhanced Cell Integration Properties

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Matthias Schnabelrauch ◽  
Ralf Wyrwa ◽  
Henrike Rebl ◽  
Claudia Bergemann ◽  
Birgit Finke ◽  
...  
Keyword(s):  
Bone Cells ◽  
Polymer Network ◽  
Fiber Surface ◽  
Cell Types ◽  
Structural Features ◽  
Tissue Response ◽  
Hydrophobic Nature ◽  
Positively Charged

Poly(L-lactide-co-D/L-lactide)-based fiber meshes resembling structural features of the native extracellular matrix have been prepared by electrospinning. Subsequent coating of the electrospun fibers with an ultrathin plasma-polymerized allylamine (PPAAm) layer after appropriate preactivation with continuous O2/Ar plasma changed the hydrophobic nature of the polylactide surface into a hydrophilic polymer network and provided positively charged amino groups on the fiber surface able to interact with negatively charged pericellular matrix components. In vitro cell experiments using different human cell types (epithelial origin: gingiva and uroepithelium; bone cells: osteoblasts) revealed that the PPAAm-activated surfaces promoted the occupancy of the meshes by cells accompanied by improved initial cell spreading. This nanolayer is stable in its cell adhesive characteristics also afterγ-sterilization. An in vivo study in a rat intramuscular implantation model demonstrated that the local inflammatory tissue response did not differ between PPAAm-coated and untreated polylactide meshes.

Protein phosphatases types 2Cα and 2Cβ in apoptosis

2006 ◽  
Vol 34 (6) ◽  
pp. 1370-1375 ◽  
Author(s):  
S. Klumpp ◽  
M.-C. Thissen ◽  
J. Krieglstein
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Unsaturated Fatty Acids ◽  
Initial Step ◽  
Cell Types ◽  
Structural Features ◽  
Apoptotic Effect ◽  
Endothelial Cell Death ◽  
Protein Phosphatase Type

This mini-review highlights the involvement of PP2C (protein phosphatase type 2C) family members α and β in apoptosis. The activity of these isoenzymes can be stimulated by unsaturated fatty acids with special structural features, e.g. oleic acid. Those fatty acids capable of activating PP2Cα and PP2Cβ in vitro induce apoptosis in various cell types as shown here for neurons and endothelial cells. Using RNA interference to reduce the amount of PP2Cα and PP2Cβ results in cells significantly less susceptible to the apoptotic effect of oleic acid. Increased endothelial cell death is considered to be an initial step of atherogenesis. Thus activation of PP2C by physiological unbound (‘free’) unsaturated fatty acids (liberated from lipoproteins) could represent a crucial mechanism in the development of atherosclerosis.

scholarly journals α-Synuclein Delays Endoplasmic Reticulum (ER)-to-Golgi Transport in Mammalian Cells by Antagonizing ER/Golgi SNAREs

2010 ◽  
Vol 21 (11) ◽  
pp. 1850-1863 ◽  
Author(s):  
Nandhakumar Thayanidhi ◽  
Jared R. Helm ◽  
Deborah C. Nycz ◽  
Marvin Bentley ◽  
Yingjian Liang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Direct Action ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Early Secretory Pathway ◽  
Golgi Transport ◽  
Protein Receptors

Toxicity of human α-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson's disease has not been established. We tested elements of this hypothesis by expressing human α-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant α-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble α-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble α-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that α-synuclein antagonizes SNARE function. Ykt6 reversed α-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified α-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble α-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway.

The actions of N-terminal fragments of corticotrophin on steroidogenesis in dispersed rat adrenal cells in vitro

1986 ◽  
Vol 109 (2) ◽  
pp. 275-278 ◽  
Author(s):  
G. P. Vinson ◽  
B. J. Whitehouse ◽  
A. Bateman ◽  
A. Dell ◽  
S. M. Laird
Keyword(s):  
Sodium Intake ◽  
Physiological Role ◽  
Cell Types ◽  
Structural Features ◽  
Zona Glomerulosa ◽  
Minimum Effective Concentration ◽  
Adrenal Cells ◽  
Aldosterone Production

ABSTRACT The finding that the rat adrenal zona glomerulosa cell shows specific sensitivity to stimulation by α-MSH and related peptides has been confirmed both in vivo and in vitro, raising the possibility that α-MSH may have a physiological role in the control of glomerulosa function and aldosterone secretion. To define more closely the structural features which confer the specificity of the glomerulosa response, other ACTH derived peptides have been tested for their specificity of actions on rat adrenal cells in vitro. The peptides tested were ACTH(5–24), ACTH(1–12), ACTH(1–14), ACTH(1–15), ACTH1–16) and ACTH(1–17). Their actions were compared with those of α-MSH and ACTH(1–24). All of the ACTH-derived peptides stimulated glomerulosa corticosterone production with sensitivities similar to that of α-MSH; minimum effective concentration was 10 nmol/l. Also, like α-MSH, the shorter ACTH peptides stimulated aldosterone production only relatively weakly in these cells from animals on normal sodium intake. Only ACTH(5–24), ACTH(1–16) and ACTH(1–17) stimulated fasciculata/reticularis cells at concentrations up to 1 μmol/l. The actions of all of the shorter peptides were thus unlike those of ACTH(1–24) which stimulates both cell types with approximately equal sensitivity, and which furthermore strongly stimulates aldosterone production. The data suggest that the 18–24 region of the ACTH molecule contains the signal for a fasciculata/ reticularis response, and the region 1–13 that for glomerulosa specificity. They confirm the view that, in the rat, α-MSH itself may be the specific pituitary glomerulosa-stimulating agent which much experimental work has predicted. They also indicate that synthetic ACTH(1–17) analogues should be used with caution. J. Endocr. (1986) 109, 275–278

Study of the Molecular Architecture of Keratin Filaments by Electron Microscopy

1982 ◽  
Vol 40 ◽  
pp. 118-119
Author(s):  
U. Aebi ◽  
P. Rew ◽  
T.-T. Sun
Keyword(s):  
Electron Microscopy ◽  
Structural Organization ◽  
Cell Types ◽  
Structural Features ◽  
Molecular Architecture ◽  
The Past ◽  
Keratin Filaments ◽  
Different Types ◽  
10 Nm Filaments ◽  
Different Cell Types

Various types of intermediate-sized (10-nm) filaments have been found and described in many different cell types during the past few years. Despite the differences in the chemical composition among the different types of filaments, they all yield common structural features: they are usually up to several microns long and have a diameter of 7 to 10 nm; there is evidence that they are made of several 2 to 3.5 nm wide protofilaments which are helically wound around each other; the secondary structure of the polypeptides constituting the filaments is rich in ∞-helix. However a detailed description of their structural organization is lacking to date.

Some Structural Features of Metaphase Chromosomes of Mice and Men

1967 ◽  
Vol 25 ◽  
pp. 190-191
Author(s):  
Godfrey C. Hoskins ◽  
Betty B. Hoskins
Keyword(s):  
Electron Microscopy ◽  
Electron Micrographs ◽  
Structural Features ◽  
Metaphase Chromosomes ◽  
The Future ◽  
Of Mice And Men ◽  
Mouse Cells ◽  
Human And Mouse

Metaphase chromosomes from human and mouse cells in vitro are isolated by micrurgy, fixed, and placed on grids for electron microscopy. Interpretations of electron micrographs by current methods indicate the following structural features.Chromosomal spindle fibrils about 200Å thick form fascicles about 600Å thick, wrapped by dense spiraling fibrils (DSF) less than 100Å thick as they near the kinomere. Such a fascicle joins the future daughter kinomere of each metaphase chromatid with those of adjacent non-homologous chromatids to either side. Thus, four fascicles (SF, 1-4) attach to each metaphase kinomere (K). It is thought that fascicles extend from the kinomere poleward, fray out to let chromosomal fibrils act as traction fibrils against polar fibrils, then regroup to join the adjacent kinomere.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

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