scholarly journals Transient activity of Golgi-like membranes as donors of vesicular stomatitis viral glycoprotein in vitro.

1981 ◽  
Vol 90 (3) ◽  
pp. 697-704 ◽  
Author(s):  
E Fries ◽  
J E Rothman
Keyword(s):  
G Protein ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Sucrose Density Gradient ◽  
Viral Glycoprotein ◽  
Golgi Membranes ◽  
Transient Activity ◽  
Vesicular Stomatitis

Previous reports demonstrated that the vesicular stomatitis viral glycoprotein (G protein), initially present in membranes of a Chinese hamster ovary mutant cell line (clone 15B) that is incapable of terminal glycosylation, can be transferred in vitro to exogenous Golgi membranes and there glycosylated (E. Fries and J. E. Rothman, 1980, Proc. Natl. Acad. Sci. U. S. A. 77:3870-3874; and J. E. Rothman and E. Fries, 1981, J. Cell Biol. 89:162-168). Here we present evidence that Golgi-like membranes serve as donors of G protein in this process. Pulse-chase experiments revealed that the donor activity of membranes is greatest at approximately 10 min of chase, a time when G protein has been shown to have arrived in Golgi stacks (J. E. Bergmann, K. T. Tokuyasu, and S. J. Singer, 1981, Proc. Natl. Acad. Sci. U. S. A. 78:1746-1750). Additional evidence that the G protein that is transferred to exogenous Golgi membranes in vitro had already entered the Golgi membranes in vivo was provided by observations that its oligosaccharides had already been trimmed, and that its distribution in a sucrose density gradient was coincident with that of enzymatic markers of Golgi membranes. The capacity of this Golgi-like membrane to serve as donor is transient, declining within 5 min after "trimming" in vivo as the G protein enters a "nontransferable" pool. The rapidity of the process suggests that both the "transferable" and "nontransferable" pools of G protein reside in Golgi-like membranes.

scholarly journals Enhanced Gene Transfer with Fusogenic Liposomes Containing Vesicular Stomatitis Virus G Glycoprotein

1998 ◽  
Vol 72 (7) ◽  
pp. 6159-6163 ◽  
Author(s):  
Akihiro Abe ◽  
Atsushi Miyanohara ◽  
Theodore Friedmann
Keyword(s):  
Gene Transfer ◽  
Vesicular Stomatitis Virus ◽  
Sucrose Density Gradient ◽  
Virus Envelope ◽  
Lipid Complex ◽  
Targeted Gene Delivery ◽  
Vesicular Stomatitis ◽  
Sucrose Density Gradient Sedimentation

ABSTRACT Exposure of Lipofectin-DNA complexes to the partially purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G) results in loss of serum-mediated inhibition and in enhanced efficiency of gene transfer. Sucrose density gradient sedimentation analysis indicated that the VSV-G associates physically with the DNA-lipid complex to produce a VSV-G liposome. The ability to incorporate surrogate viral or cellular envelope components such as VSV-G into liposomes may allow more-efficient and possibly targeted gene delivery by lipofection, both in vitro and in vivo.

scholarly journals Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system.

1987 ◽  
Vol 104 (3) ◽  
pp. 749-760 ◽  
Author(s):  
W E Balch ◽  
K R Wagner ◽  
D S Keller
Keyword(s):  
Endoplasmic Reticulum ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Free System ◽  
A Cell ◽  
Vesicular Stomatitis ◽  
High Mannose

Transport of the vesicular stomatitis virus-encoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (man GlcNAc2) ER form of G protein to the man5GlcNAc5 form by the cis Golgi enzyme alpha-mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the man5GlcNAc2 form without the appearance of the intermediate man GlcNAc2 oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi alpha-mannosidase I inhibitor 1-deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supernatant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro.

scholarly journals 160. Truncated Vesicular Stomatitis Virus G- Protein Improves Baculovirus Transduction Efficiency In Vitro and In Vivo

2006 ◽  
Vol 13 ◽  
pp. S63
Author(s):  
Minna U. Kaikkonen ◽  
Jani K. Raty ◽  
Kari J. Airenne ◽  
Thomas Wirth ◽  
Tommi Heikura ◽  
...  
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Transduction Efficiency ◽  
Vesicular Stomatitis

scholarly journals Truncated vesicular stomatitis virus G protein improves baculovirus transduction efficiency in vitro and in vivo

Gene Therapy ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 304-312 ◽  
Author(s):  
M U Kaikkonen ◽  
J K Räty ◽  
K J Airenne ◽  
T Wirth ◽  
T Heikura ◽  
...  
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Transduction Efficiency ◽  
Vesicular Stomatitis

scholarly journals Transport of protein between cytoplasmic membranes of fused cells: correspondence to processes reconstituted in a cell-free system.

1984 ◽  
Vol 99 (1) ◽  
pp. 248-259 ◽  
Author(s):  
J E Rothman ◽  
L J Urbani ◽  
R Brands
Keyword(s):  
G Protein ◽  
Golgi Complex ◽  
Chinese Hamster ◽  
In Vitro Assays ◽  
Wild Type ◽  
Free System ◽  
Cell Free System ◽  
A Cell

Mixed monolayers containing vesicular stomatitis virus-infected Chinese hamster ovary clone 15B cells (lacking UDP-N-acetylglucosamine transferase I, a Golgi enzyme) and uninfected wild-type Chinese hamster ovary cells were formed. Extensive cell fusion occurs after the monolayer is exposed to a pH of 5.0. The vesicular stomatitis virus encoded membrane glycoprotein (G protein) resident in the rough endoplasmic reticulum (labeled with [35S]methionine) or Golgi complex (labeled with [3H]palmitate) of 15B cells at the time of fusion can reach Golgi complexes from wild-type cells after fusion; G protein present in the plasma membrane cannot. Transfer to wild-type Golgi complexes is monitored by the conversion of G protein to an endoglycosidase H-resistant form upon arrival, and also demonstrated by immunofluorescence microscopy. G protein in the Golgi complex of the 15B cells at the time of fusion exhibits properties vis a vis its transfer to an exogenous Golgi population identical to those found earlier in a cell-free system (Fries, E., and J. E. Rothman. 1981. J. Cell Biol., 90: 697-704). Specifically, pulse-chase experiments using the in vivo fusion and in vitro assays reveal the same two populations of G protein in the Golgi complex. The first population, consisting of G protein molecules that have just received their fatty acid, can transfer to a second Golgi population in vivo and in vitro. The second population, entered by G protein approximately 5 min after its acylation, is unavailable for this transfer, in vivo and in vitro. Presumably, this second population consists of those G-protein molecules that had already been transferred between compartments within the 15B Golgi population, in an equivalent process before cell fusion or homogenization for in vitro assays. Evidently, the same compartment boundary in the Golgi complex is detected by these two measurements. The surprisingly facile process of glycoprotein transit between Golgi stacks that occurs in vivo may therefore be retained in vitro, providing a basis for the cell-free system.

Calsenilin and CALP interact with the cytoplasmic tail of UDP-Gal:GA2/GM2/GD2 β-1,3-galactosyltransferase

2008 ◽  
Vol 412 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Cristián A. Quintero ◽  
Javier Valdez-Taubas ◽  
Mariana L. Ferrari ◽  
Sergio D. Haedo ◽  
Hugo J. F. Maccioni
Keyword(s):  
Calcium Binding ◽  
Chinese Hamster ◽  
Cytoplasmic Tail ◽  
Yeast Two Hybrid ◽  
Molecular Determinants ◽  
Vesicular Stomatitis ◽  
Two Hybrid ◽  
Hybrid Screening

GalT2 (UDP-Gal:GA2/GM2/GD2 β-1,3-galactosyltransferase) is a Golgi-resident type II membrane protein that participates in the synthesis of glycosphingolipids. The molecular determinants for traffic and localization of this and other glycosyltransferases are still poorly characterized. Considering the possibility that interactions with other proteins may influence these processes, in the present study we carried out a yeast two-hybrid screening using elements of the N-terminal domain of GalT2 as bait. In this screening, we identified calsenilin and its close homologue CALP (calsenilin-like protein), both members of the recoverin-NCS (neuronal calcium sensor) family of calcium-binding proteins. In vitro, GalT2 binds to immobilized recombinant CALP, and CALP binds to immobilized peptides with the GalT2 cytoplasmic tail sequence. GalT2 and calsenilin interact physically when co-expressed in CHO (Chinese-hamster ovary)-K1 cells. The expression of CALP or calsenilin affect Golgi localization of GalT2, and of two other glycosyltransferases, SialT2 (CMP-NeuAc:GM3 sialyltransferase) and GalNAcT (UDP-GalNAc:lactosylceramide/GM3/GD3 β1-4 N-acetylgalactosaminyltransferase), by redistributing them from the Golgi to the ER (endoplasmic reticulum), whereas the localization of the VSV-G (G-protein of the vesicular stomatitis virus) or the Golgin GM130 was essentially unaffected. Conversely, the expression of GalT2 affects the localization of calsenilin and CALP by shifting a fraction of the molecules from being mostly diffuse in the cytosol, to clustered structures in the perinuclear region. These combined in vivo and in vitro results suggest that CALP and calsenilin are involved in the trafficking of Golgi glycosyltransferases.

scholarly journals Distinct Mechanisms of Clathrin-independent Endocytosis Have Unique Sphingolipid Requirements

2006 ◽  
Vol 17 (7) ◽  
pp. 3197-3210 ◽  
Author(s):  
Zhi-Jie Cheng ◽  
Raman Deep Singh ◽  
Deepak K. Sharma ◽  
Eileen L. Holicky ◽  
Kentaro Hanada ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Function ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Lipid Vesicles ◽  
Dominant Negative ◽  
Rho Proteins

Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.

Formation and Structure of Casein Micelles in Lactating Mammary Tissue

1970 ◽  
Vol 28 ◽  
pp. 150-151
Author(s):  
Robert J. Carroll ◽  
Marvin P. Thompson ◽  
Harold M. Farrell
Keyword(s):  
Size Distribution ◽  
G Protein ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Colloidal System ◽  
Casein Micelles ◽  
The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

scholarly journals Akt kinase LANCL2 functions as a key driver in EGFR-mutant lung adenocarcinoma tumorigenesis

2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Yuqing Lou ◽  
Jianlin Xu ◽  
Yanwei Zhang ◽  
Wei Zhang ◽  
Xueyan Zhang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Line ◽  
Quantitative Polymerase Chain Reaction ◽  
Mutant Cell ◽  
A549 Cells ◽  
The Cancer Genome Atlas ◽  
Akt Kinase ◽  
Egfr Expression

AbstractEpidermal growth factor receptor (EGFR) is a key oncogene in lung adenocarcinoma (LUAD). Resistance to EGFR tyrosine kinase inhibitors is a major obstacle for EGFR-mutant LUAD patients. Our gene chip array, quantitative polymerase chain reaction validation, and shRNA-based high-content screening identified the Akt kinase lanthionine synthetase C-like protein 2 (LANCL2) as a pro-proliferative gene in the EGFR-mutant LUAD cell line PC9. Therefore, we investigated whether LANCL2 plays a role in promoting cell proliferation and drug resistance in EGFR-mutant LUAD. In silico clinical correlation analysis using the Cancer Genome Atlas Lung Adenocarcinoma dataset revealed a positive correlation between LANCL2 and EGFR expression and an inverse relationship between LANCL2 gain-of-function and survival in LUAD patients. The EGFR-mutant LUAD cell lines PC9 and HCC827 displayed higher LANCL2 expression than the non-EGFR-mutant cell line A549. In addition, LANCL2 was downregulated following gefitinib+pemetrexed combination therapy in PC9 cells. LANCL2 knockdown reduced proliferation and enhanced apoptosis in PC9, HCC827, and A549 cells in vitro and suppressed murine PC9 xenograft tumor growth in vivo. Notably, LANCL2 overexpression rescued these effects and promoted gefitinib + pemetrexed resistance in PC9 and HCC827 cells. Pathway analysis and co-immunoprecipitation followed by mass spectrometry of differentially-expressed genes in LANCL2 knockdown cells revealed enrichment of several cancer signaling pathways. In addition, Filamin A and glutathione S-transferase Mu 3 were identified as two novel protein interactors of LANCL2. In conclusion, LANCL2 promotes tumorigenic proliferation, suppresses apoptosis, and promotes gefitinib+pemetrexed resistance in EGFR-mutant LUAD cells. Based on the positive association between LANCL2, EGFR, and downstream Akt signaling, LANCL2 may be a promising new therapeutic target for EGFR-mutant LUAD.

scholarly journals Evaluation of Fab and F(ab′)2 Fragments and Isotype Variants of a Recombinant Human Monoclonal Antibody against Shiga Toxin 2

2010 ◽  
Vol 78 (3) ◽  
pp. 1376-1382 ◽  
Author(s):  
Donna E. Akiyoshi ◽  
Abhineet S. Sheoran ◽  
Curtis M. Rich ◽  
L. Richard ◽  
Susan Chapman-Bonofiglio ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Shiga Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Human Monoclonal Antibody ◽  
Chinese Hamster ◽  
The Body ◽  
Neutralization Activity ◽  
Shiga Toxin 2

ABSTRACT 5C12 HuMAb is a human monoclonal antibody against the A subunit of Shiga toxin 2 (Stx2). We have previously shown that 5C12 HuMAb effectively neutralizes the cytotoxic effects of this toxin by redirecting its transport within the cell and also by neutralizing the toxin's ability to inhibit protein synthesis. The 5C12 HuMAb and its recombinant IgG1 version protect mice at a dose of 0.6 μg against a lethal challenge of Stx2. The contribution of the Fc region to this observed neutralization activity of the 5C12 antibody against Stx2 was investigated in this study. Using recombinant DNA technology, 5C12 isotype variants (IgG1, IgG2, IgG3, and IgG4) and antibody fragments [Fab, F(ab′)2] were expressed in Chinese hamster ovary cells and evaluated in vitro and in vivo. All four 5C12 isotype variants showed protection in vitro, with the IgG3 and IgG4 variants showing the highest protection in vivo. The Fab and F(ab′)2 fragments also showed protection in vitro but no protection in the mouse toxicity model. Similar results were obtained for a second HuMAb (5H8) against the B subunit of Stx2. The data suggest the importance of the Fc region for neutralization activity, but it is not clear if this is related to the stability of the full-length antibody or if the Fc region is required for effective elimination of the toxin from the body.

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