scholarly journals Endoplasmic reticulum resident proteins of normal human dermal fibroblasts are the major targets for oxidative stress induced by hydrogen peroxide

2002 ◽  
Vol 366 (3) ◽  
pp. 825-830 ◽  
Author(s):  
Dennis van der VLIES ◽  
Eward H.W. PAP ◽  
Jan Andries POST ◽  
Julio E. CELIS ◽  
Karel W.A. WIRTZ
Keyword(s):  
Hydrogen Peroxide ◽  
Endoplasmic Reticulum ◽  
Laser Scanning ◽  
Dermal Fibroblasts ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Human Dermal Fibroblasts ◽  
Normal Human ◽  
Covalently Linked ◽  
Normal Human Dermal Fibroblasts

The membrane-permeable fluorescein-labelled tyramine conjugate (acetylTyrFluo) was used to identify the proteins of normal human dermal fibroblasts most susceptible to oxidation by hydrogen peroxide [Van der Vlies, Wirtz and Pap (2001) Biochemistry 40, 7783—7788]. By exposing the cells to H2O2 (0.1mM for 10min), TyrFluo was covalently linked to target proteins. TyrFluo-labelled and [35S]Met-labelled cell lysates were mixed and subjected to two-dimensional PAGE. After Western blotting the 35S-labelled proteins were visualized by autoradiography and the TyrFluo-labelled proteins by using anti-fluorescein antibody. The TyrFluo-labelled proteins were matched with the 35S-labelled proteins and identified by comparison with our mastermap of proteins. Protein disulphide isomerase (PDI), IgG-binding protein (BiP), calnexin, endoplasmin and glucose-regulated protein 58 (endoplasmic reticulum protein 57/GRP58) were identified as targets of oxidation. All these proteins reside in the endoplasmic reticulum and are part of the protein folding machinery. In agreement, confocal laser scanning microscopy showed co-localization of TyrFluo-labelled proteins and the KDEL receptor ERD-2, a marker for the endoplasmic reticulum.

scholarly journals Effects of GABA on the expression of type I collagen gene in normal human dermal fibroblasts

2016 ◽  
Vol 81 (2) ◽  
pp. 376-379 ◽  
Author(s):  
Eriko Uehara ◽  
Hideki Hokazono ◽  
Takako Sasaki ◽  
Hidekatsu Yoshioka ◽  
Noritaka Matsuo
Keyword(s):  
Type I Collagen ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Collagen Gene ◽  
Human Dermal Fibroblasts ◽  
Normal Human ◽  
Normal Human Dermal Fibroblasts

scholarly journals Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins

2003 ◽  
Vol 77 (9) ◽  
pp. 5401-5414 ◽  
Author(s):  
Maria Dimitrova ◽  
Isabelle Imbert ◽  
Marie Paule Kieny ◽  
Catherine Schuster
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Interactions ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nonstructural Proteins

ABSTRACT Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.

Visualization of the endoplasmic reticulum in living buds and branches of the moss Funaria hygrometrica by confocal laser scanning microscopy

Development ◽  
1990 ◽  
Vol 109 (4) ◽  
pp. 753-764
Author(s):  
M.M. McCauley ◽  
P.K. Hepler
Keyword(s):  
Endoplasmic Reticulum ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Side Branch ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Funaria Hygrometrica ◽  
Bud Formation

Caulonemata of the moss Funaria hygrometrica were vitally stained with the fluorescent, lipophilic carbocyanine dye DiOC6(3) and examined via confocal laser scanning microscopy. Although DiOC6(3) stained nearly all of the organelles, cortical endoplasmic reticulum (ER) could be resolved under favorable conditions and appeared as a network of irregular polygons, interspersed with lamellar cisternae in some cell types. The pattern of cortical ER was examined first during side initial formation and then in young branches and buds. The ER network extends into the outgrowth of a developing side initial, keeping pace with elongation of the outgrowth. Prior to the cell division that cuts off the outgrowth from the underlying cell, the network in the outgrowth becomes tighter, i.e. the polygons become smaller. If the side initial develops as a branch, this somewhat tighter ER network is maintained in the tip-growing side branch. If the side initial develops as a bud, dramatic changes in both the configuration and the quantity of the ER network occur. Coincident with the apical swelling that marks the first visible sign of bud formation, the network becomes increasingly tighter until eventually the polygonal configuration is barely discernible. The increased coverage of the bud cortex by the ER network demonstrates that a significant increase in the quantity of membranes also takes place during bud formation in Funaria.

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