scholarly journals The Role of Golgi Morphology in Post-Alcohol Recovery of Hepatocytes: Observations in Cellular and Animal Models

2018 ◽  
Author(s):  
Carol A. Casey ◽  
Paul Thomes ◽  
Sonia Manca ◽  
Armen Petrosyan
Keyword(s):  
Matrix Protein ◽  
Protein Transport ◽  
Cultured Cells ◽  
Control Diet ◽  
Rat Hepatocytes ◽  
Recovery Phase ◽  
Compact Structure ◽  
Alcohol Recovery ◽  
Golgi Methods ◽  
Hepatic Proteins

Background: In hepatocytes and alcohol-metabolizing cultured cells, Golgi undergoes ethanol (EtOH)-induced disorganization. Periniclear and organized Golgi is important in liver homeostasis, but how the Golgi remains intact is unknown. Work from our laboratories showed that EtOH-altered cellular function could be reversed after alcohol removal; we wanted to determine whether this recovery would apply to Golgi. Methods: We used alcohol-metabolizing HepG2 (VA-13) cells (cultured with or without EtOH for 72 h) and rat hepatocytes (control and EtOH-fed (Lieber-DeCarli diet). For recovery, EtOH was removed and replenished with control medium (48 hours for VA-13 cells) or control diet (10 days for rats). Results: EtOH-induced Golgi disassembly was associated with de-dimerization of the largest Golgi matrix protein giantin, along with impaired transport of selected hepatic proteins. After recovery from EtOH, Golgi regained their compact structure, and alterations in giantin and protein transport were restored. In VA-13 cells, when we knocked down giantin, Rab6a GTPase or non-muscle Myosin IIB, minimal changes were observed in control conditions, but post-EtOH recovery was impaired. Conclusions: These data provide a link between Golgi organization and plasma membrane protein expression and identify several proteins whose expression is important to maintain Golgi structure during the recovery phase after EtOH administration.

scholarly journals Giantin Is Required for Post-Alcohol Recovery of Golgi in Liver Cells

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 150 ◽  
Author(s):  
Carol Casey ◽  
Paul Thomes ◽  
Sonia Manca ◽  
Armen Petrosyan
Keyword(s):  
Matrix Protein ◽  
Protein Transport ◽  
Cultured Cells ◽  
Control Diet ◽  
Rat Hepatocytes ◽  
Recovery Phase ◽  
Compact Structure ◽  
Alcohol Recovery ◽  
Golgi Organization ◽  
Hepatic Proteins

In hepatocytes and alcohol-metabolizing cultured cells, Golgi undergoes ethanol (EtOH)-induced disorganization. Perinuclear and organized Golgi is important in liver homeostasis, but how the Golgi remains intact is unknown. Work from our laboratories showed that EtOH-altered cellular function could be reversed after alcohol removal; we wanted to determine whether this recovery would apply to Golgi. We used alcohol-metabolizing HepG2 (VA-13) cells (cultured with or without EtOH for 72 h) and rat hepatocytes (control and EtOH-fed (Lieber–DeCarli diet)). For recovery, EtOH was removed and replenished with control medium (48 h for VA-13 cells) or control diet (10 days for rats). Results: EtOH-induced Golgi disassembly was associated with de-dimerization of the largest Golgi matrix protein giantin, along with impaired transport of selected hepatic proteins. After recovery from EtOH, Golgi regained their compact structure, and alterations in giantin and protein transport were restored. In VA-13 cells, when we knocked down giantin, Rab6a GTPase or non-muscle myosin IIB, minimal changes were observed in control conditions, but post-EtOH recovery was impaired. Conclusions: These data provide a link between Golgi organization and plasma membrane protein expression and identify several proteins whose expression is important to maintain Golgi structure during the recovery phase after EtOH administration.

Changes in rat mesentery interstitial matrix due to superfusate

1993 ◽  
Vol 265 (3) ◽  
pp. H852-H856 ◽  
Author(s):  
B. J. Barber ◽  
R. A. Babbitt ◽  
S. Dutta ◽  
S. Parameswaran
Keyword(s):  
Protein Content ◽  
Normal Saline ◽  
Protein Concentration ◽  
Matrix Protein ◽  
Protein Transport ◽  
Albumin Concentration ◽  
Sprague Dawley ◽  
Tissue Samples ◽  
Rat Mesentery ◽  
Sprague Dawley Rats

Animal preparations for microscopy often require a superfusate solution to cover surgically exposed tissue. There are few, if any, data concerning the effects of this solution on extravascular protein concentration and hydration. The effect of superfusion on mesenteric tissue in anesthetized male Sprague-Dawley rats was studied. Tissue samples were taken from nonsuperfused and superfused tissue and analyzed for hydration, albumin, and transferrin content. The mesenteric tissue interstitial matrix was rapidly altered by normal saline superfusate. After superfusion, there was a decrease (P < 0.01) in tissue albumin concentration from 1.17 +/- 0.27 to 0.10 +/- 0.08 g/dl (n = 9). Tissue hydration increased from 4.98 +/- 0.8 micrograms water/microgram dry wt in controls to 7.38 +/- 1.2 micrograms water/micrograms dry wt after superfusion. When a range of superfusate albumin concentrations was used (0, 1, 2, and 3 g/dl), tissue albumin concentration changed 0.59 +/- 0.09 g/dl for each gram per deciliter change in superfusate concentration (P < 0.0001). The large changes in interstitial matrix protein content and hydration suggest that superfusate solution effects need to be considered in microvascular protein transport experiments.

scholarly journals Insulin-responsive cultured foetal-rat hepatocytes. Their preparation and characterization

1984 ◽  
Vol 223 (1) ◽  
pp. 39-46 ◽  
Author(s):  
D C DeSante ◽  
L Little ◽  
D E Peavy ◽  
F Vinicor
Keyword(s):  
Monolayer Culture ◽  
Cultured Cells ◽  
Rat Hepatocytes ◽  
Dependent Manner ◽  
Deoxyribonuclease I ◽  
Haematopoietic Cells ◽  
Foetal Rat ◽  
The Relationship ◽  
Dose Dependent ◽  
Scatchard Plots

An improved non-perfusion method for the preparation of cultured foetal-rat hepatocytes is described. Digestion of the liver with collagenase and deoxyribonuclease I gave yields of 40 × 10(6) hepatocytes/g of liver. The plating efficiency of hepatocytes in medium with 10 microM-cortisol was 50%. Cell morphology and metabolism were maintained through 3 days of monolayer culture, with minimal contamination by haematopoietic cells or fibroblasts. The cultured cells bound and degraded 125I-insulin in a time- and dose-dependent manner. The estimated ED50 for competitive binding at 37 degrees C was 1.1 nM. Curvilinear Scatchard plots were observed, with estimates of 16 500 high-affinity sites (Kd = 813 pM) and 53 000 low-affinity sites (Kd = 23 nM) per cell. The cultured cells demonstrated a glycogenic response to insulin, with an estimated ED50 of 120 pM. The degree of glycogenic response to insulin varied with time in culture: 500% above basal on day 1, 200% on day 2, and only 150% on day 3. Cultured foetal cells also exhibited a time-dependent uptake of 2-aminoisobutyric acid, which, in contrast with previous reports with adult cells, was not stimulated by the presence of 10 nM-insulin. Cultured foetal hepatocytes may provide an interesting model with which to study the relationship between insulin-receptor binding and insulin action.

scholarly journals Measurement of the metabolism of cytochrome P-450 in cultured hepatocytes by a quantitative and specific immunochemical method

1982 ◽  
Vol 204 (1) ◽  
pp. 281-290 ◽  
Author(s):  
Sammye L. Newman ◽  
Joyce L. Barwick ◽  
Nabil A. Elshourbagy ◽  
Philip S. Guzelian
Keyword(s):  
Cultured Cells ◽  
De Novo ◽  
Internal Standard ◽  
Rat Hepatocytes ◽  
Cellular Protein ◽  
Small Samples ◽  
Cultured Hepatocytes ◽  
Immunochemical Method ◽  
Adult Rat ◽  
Cytochrome P 450

We have defined conditions that permit quantitative and specific measurement of the metabolism of the major phenobarbital-inducible form of cytochrome P-450 protein in primary non-proliferating monolayer cultures of adult rat hepatocytes. Isolated antibodies specifically directed against phenobarbital cytochrome P-450 are used to immunoprecipitate the cytochrome from lysates of cultured hepatocytes pulse-labelled with [3H]leucine. Phenobarbital cytochrome P-450 protein is then isolated from the immunoprecipitate by electrophoresis on polyacrylamide gradient slab gels. Specificity of the assay for phenobarbital cytochrome P-450 was established by competition experiments involving other forms of purified cytochrome P-450 as well as by testing antibodies directed against these other forms of the cytochrome. Using purified phenobarbital cytochrome P-450, radiolabelled in both its haem and apoprotein portions, as an internal standard, we demonstrated that, with this immunoassay, recovery of cytochrome P-450 from microsomal samples is nearly complete. Basal rates of synthesis of phenobarbital cytochrome P-450 representing as little as 0.02–0.05% of total cellular protein synthesis were reliably and reproducibly detected in hepatocyte culture maintained in serum-free medium for 72h. Moreover, inclusion of phenobarbital in the culture medium for 96h stimulated not only synthesis de novo of phenobarbital cytochrome P-450 protein, but also accumulation of spectrally and catalytically active cytochrome P-450. Advantages of this immunoassay are that metabolism (synthesis or degradation) of the haem or protein of this important form of the cytochrome can be measured conveniently in the small samples available from cultured cells without the necessity of preparing subcellular fractions.

scholarly journals Identification of the growth factor–binding sequence in the extracellular matrix protein MAGP-1

2020 ◽  
Vol 295 (9) ◽  
pp. 2687-2697 ◽  
Author(s):  
Thomas J. Broekelmann ◽  
Nicholas K. Bodmer ◽  
Robert P. Mecham
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Matrix Protein ◽  
Transforming Growth Factor ◽  
Cultured Cells ◽  
Transforming Growth Factor Β ◽  
Bmp Signaling ◽  
Extracellular Matrix Protein ◽  
Surface Plasmon Resonance Analysis ◽  
Factor Binding

Microfibril-associated glycoprotein-1 (MAGP-1) is a component of vertebrate extracellular matrix (ECM) microfibrils that, together with the fibrillins, contributes to microfibril function. Many of the phenotypes associated with MAGP-1 gene inactivation are consistent with dysregulation of the transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling system. We have previously shown that full-length MAGP-1 binds active TGFβ-1 and some BMPs. The work presented here further defines the growth factor–binding domain of MAGP-1. Using recombinant domains and synthetic peptides, along with surface plasmon resonance analysis to measure the kinetics of the MAGP-1–TGFβ-1 interaction, we localized the TGFβ- and BMP-binding site in MAGP-1 to a 19-amino acid–long, highly acidic sequence near the N terminus. This domain was specific for binding active, but not latent, TGFβ-1. Growth factor activity experiments revealed that TGFβ-1 retains signaling activity when complexed with MAGP-1. Furthermore, when bound to fibrillin, MAGP-1 retained the ability to interact with TGFβ-1, and active TGFβ-1 did not bind fibrillin in the absence of MAGP-1. The absence of MAGP was sufficient to raise the amount of total TGFβ stored in the ECM of cultured cells, suggesting that the MAGPs compete with the TGFβ large latent complex for binding to microfibrils. Together, these results indicate that MAGP-1 plays an active role in TGFβ signaling in the ECM.

scholarly journals Evidence that binding of CTP:phosphocholine cytidylyltransferase to membranes in rat hepatocytes is modulated by the ratio of bilayer- to non-bilayer-forming lipids

1993 ◽  
Vol 291 (2) ◽  
pp. 419-427 ◽  
Author(s):  
H Jamil ◽  
G M Hatch ◽  
D E Vance
Keyword(s):  
Phospholipase A2 ◽  
Phospholipase C ◽  
Cultured Cells ◽  
Normal Values ◽  
Common Factor ◽  
Rat Hepatocytes ◽  
Cellular Membranes ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Cellular Concentration

The mechanism by which phospholipase C (PLC) digestion of cultured cells mediates binding of CTP:phosphocholine cytidylyltransferase to cellular membranes was investigated. Incubation of choline-depleted rat hepatocytes with PLC caused a translocation of enzyme from cytosol to membranes concomitant with a decrease in the concentration of phosphatidylcholine with no effect on the concentration of other phospholipids. Removal of PLC and supplementation with choline restored the amount of phosphatidylcholine in the cells and translocated cytidylyltransferase to the cytosol. However, when phosphatidylcholine levels were decreased by incubation with phospholipase A2 (PLA2), there was no significant redistribution of cytidylyltransferase activity. With PLA2 the concentration of phosphatidylethanolamine, as well as of phosphatidylcholine, was significantly decreased. Since PLC, but not phospholipase A2, raised the cellular concentration of diacylglycerol, possibly diacylglycerol mediated the binding of cytidylyltransferase to membranes. This possibility was examined, but is unlikely, since addition of lysophosphatidylcholine to PLC-treated cells restored the concentration of phosphatidylcholine and released cytidylyltransferase into the cytosol, but did not lower diacylglycerol levels to normal values. Studies in vitro, incubations of cells with choline analogues and a survey of the literature suggested that the over-riding common factor in regulation of cytidylyltransferase binding to membranes may be the ratio of bilayer to non-bilayer lipids in that membrane.

scholarly journals Phenotypic Consequences of Rearranging the P, M, and G Genes of Vesicular Stomatitis Virus

1999 ◽  
Vol 73 (6) ◽  
pp. 4705-4712 ◽  
Author(s):  
L. Andrew Ball ◽  
Craig R. Pringle ◽  
Brian Flanagan ◽  
Victoria P. Perepelitsa ◽  
Gail W. Wertz
Keyword(s):  
Gene Expression ◽  
Vesicular Stomatitis Virus ◽  
Gene Order ◽  
Gene Rearrangement ◽  
Matrix Protein ◽  
Cultured Cells ◽  
Growth Potential ◽  
Wild Type Virus ◽  
Ancestral Gene ◽  
Vesicular Stomatitis

ABSTRACT The nonsegmented negative-strand RNA viruses (orderMononegavirales) include many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site at the 3′ end of the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwise unaltered. The central three genes in the gene order, which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G, were rearranged into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mice. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3′ promoter. Some of the viruses with rearranged genomes replicated as well or slightly better than wild-type virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and death following inoculation varied. These data show that despite the highly conserved gene order of the Mononegavirales, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the Mononegavirales may result from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a consequence, gene rearrangement should be irreversible and provide an approach for constructing viruses with novel phenotypes.

scholarly journals A GRASP55-rab2 effector complex linking Golgi structure to membrane traffic

2001 ◽  
Vol 155 (6) ◽  
pp. 877-884 ◽  
Author(s):  
Benjamin Short ◽  
Christian Preisinger ◽  
Roman Körner ◽  
Robert Kopajtich ◽  
Olwyn Byron ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Matrix Protein ◽  
Protein Transport ◽  
Coiled Coil ◽  
Membrane Traffic ◽  
Secretory Protein ◽  
Rab Gtpase ◽  
Rab Proteins ◽  
Golgi Structure ◽  
Golgi Matrix

Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family. The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae. Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins. Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport. These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.

Use of Thick Histochemical Preparations, Whole Mount Cultured Cells or Thick Sections from Embedded Tissues, for Three-Dimensional Observation by Ultrahigh Voltage Electron Microscopy

1990 ◽  
Vol 48 (3) ◽  
pp. 184-185
Author(s):  
Tetsuji Nagata ◽  
Nobuteru Usuda ◽  
Hongjun Ma
Keyword(s):  
Electron Microscopy ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Rat Hepatocytes ◽  
Wistar Rat ◽  
Specific Cell ◽  
Cell Organelles ◽  
Voltage Electron ◽  
Culture Cells ◽  
Whole Mount

We have observed very thick biological specimens, both whole mount cultured cells and thick sections from embedded tissues, which were stained with histochemical reactions for specific cell organelles, by means of high or ultrahigh voltage electron microscopy (UHVEM).Thick histochemical specimens, both whole mount cultured cells and semithin sections from embedded tissues were used. Two cell strains in culture, one established CHO-K1 cell line and the other primary culture cells of adult Wistar rat hepatocytes were used. Culture cells were seeded onto formval coated gold meshes and incubated in CO2 incubator. CHO-K1 cells were cultured in Ham’s F12 medium containing HRP (1 mg/ml), while rat hepatocytes were in L-15 medium containing Clofibrate (0.2 mM). The cultured cells were stained with DAB reaction, dried in a critical point dryer (Hitachi HCP-1). Thick sections (0.2-1.0 μm) from DEHP fed rat livers, fixed in 2.5% glutaraldehyde, stained with DAB reaction, postfixed in 1% osmium tetroxide and embedded in Epon, or semithin Epon sections (0.2μm) from the pancreases of 3H-thymidine or 3H-uridine injected mice, fixed doubly, radioautographed with Sakura NR-H2 emulsion.

scholarly journals Clinically-identified C-terminal mutations in fibulin-3 are prone to misfolding and destabilization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
DaNae R. Woodard ◽  
Emi Nakahara ◽  
John D. Hulleman
Keyword(s):  
Matrix Protein ◽  
Cultured Cells ◽  
Open Angle Glaucoma ◽  
Culture Conditions ◽  
Glycosylation Site ◽  
Age Related Macular Degeneration ◽  
Extracellular Matrix Protein ◽  
Macular Dystrophy ◽  
Alternative Mechanism ◽  
Age Related

AbstractDistinct mutations in the secreted extracellular matrix protein, fibulin-3 (F3), have been associated with a number of ocular diseases ranging from primary open angle glaucoma to cuticular age-related macular degeneration to a rare macular dystrophy, Malattia Leventinese (ML). The R345W F3 mutation that causes ML leads to F3 misfolding, inefficient secretion and accumulation at higher intracellular steady state levels in cultured cells. Herein, we determined whether fifteen other clinically-identified F3 mutations also led to similar levels of misfolding and secretion defects, which might provide insight into their potential pathogenicity. Surprisingly, we found that only a single F3 variant, L451F, presented with a significant secretion defect (69.5 ± 2.4% of wild-type (WT) F3 levels) and a corresponding increase in intracellular levels (226.8 ± 25.4% of WT F3 levels). Upon follow-up studies, when this conserved residue (L451) was mutated to a charged (Asp or Arg) or bulky (Pro, Trp, Tyr) residue, F3 secretion was also compromised, indicating the importance of small side chains (Leu, Ala, or Gly) at this residue. To uncover potential inherent F3 instability not easily observed under typical culture conditions, we genetically eliminated the sole stabilizing N-linked glycosylation site (N249) from select clinically-identified F3 mutants. This removal exacerbated R345W and L451F secretion defects (19.8 ± 3.0% and 12.4 ± 1.2% of WT F3 levels, respectively), but also revealed a previously undiscovered secretion defect in another C-terminal variant, Y397H (42.0 ± 10.1% of WT F3 levels). Yet, glycan removal did not change the relative secretion of the N-terminal mutants tested (D49A, R140W, I220F). These results highlight the uniqueness and molecular similarities between the R345W and L451F variants and also suggest that previously identified disease-associated mutations (e.g., R140W) are indistinguishable from WT with respect to secretion, hinting that they may lead to disease by an alternative mechanism.

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