scholarly journals In Vivo Crystallization of Human IgG in the Endoplasmic Reticulum of Engineered Chinese Hamster Ovary (CHO) Cells

2011 ◽  
Vol 286 (22) ◽  
pp. 19917-19931 ◽  
Author(s):  
Haruki Hasegawa ◽  
John Wendling ◽  
Feng He ◽  
Egor Trilisky ◽  
Riki Stevenson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Cho Cells ◽  
Membrane Integrity ◽  
Chinese Hamster ◽  
High Concentration ◽  
Human Igg ◽  
Secretory Capacity

Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity.

Suppression of tumor formation in vivo by expression of the JE gene in malignant cells

1991 ◽  
Vol 11 (6) ◽  
pp. 3125-3131
Author(s):  
B J Rollins ◽  
M E Sunday
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Suppressive Effect ◽  
Tumor Formation ◽  
Host Animal ◽  
Early Growth Response ◽  
Discernible Effect ◽  
Early Growth Response Gene

The early growth response gene JE encodes a monocyte chemoattractant, MCP-1. The JE/MCP-1 protein attracts and stimulates human monocytes and induces monocyte-mediated inhibition of tumor cell growth in vitro. Expression of human or murine JE/MCP-1 in Chinese hamster ovary (CHO) cells completely suppressed their ability to form tumors in nude mice. Coinjection of JE/MCP-1-expressing cells with nonexpressing CHO cells or with HeLa cells also prevented tumor formation. Since JE/MCP-1 expression had no discernible effect on the tranformed phenotype of these cells in vitro, the suppressive effect depends on host animal factors. These factors are likely to be components of the inflammatory response, because JE/MCP-1-expressing cells elicited a predominantly monocytic infiltrate at the site of injection. Our results suggest that JE/MCP-1 protein may be useful in cancer therapy.

scholarly journals The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

1977 ◽  
Vol 73 (3) ◽  
pp. 601-615 ◽  
Author(s):  
RR Gould ◽  
GG Borisy
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Carbon Coated ◽  
Pericentriolar Material ◽  
And Function

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.

scholarly journals Interaction of Ipa proteins of Shigella flexneri with alpha5beta1 integrin promotes entry of the bacteria into mammalian cells.

1996 ◽  
Vol 183 (3) ◽  
pp. 991-999 ◽  
Author(s):  
M Watarai ◽  
S Funato ◽  
C Sasakawa
Keyword(s):  
Epithelial Cells ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Shigella Flexneri ◽  
Chinese Hamster ◽  
Invasive Capacity ◽  
Beta 1 Integrin ◽  
Cellular Components

Shigella is a genus of highly adapted bacterial pathogens that cause bacillary dysentery in humans. Bacteria reaching the colon invade intestinal epithelial cells by a process of bacterial-directed endocytosis mediated by the Ipa proteins: IpaB, IpaC, and IpaD of Shigella. The invasion of epithelial cells is thought to be a receptor-mediated phenomenon, although the cellular components of the host that interact with the Ipa proteins have not yet been identified. We report here that in a Shigella flexneri invasive system and Chinese hamster ovary (CHO) cell monolayers, the Ipa proteins were capable of interacting directly with alpha5beta1 integrin. The invasive capacity of S. flexneri for CHO cells increased as levels of alpha5beta1 integrin were elevated. When CHO cells were infected with S. flexneri, the tyrosine phosphorylation both of pp 125FAK, an integrin-regulated 125 K focal adhesion kinase, and of paxillin was stimulated. In contrast, an isogenic strain of S. flexneri that was defective in invasion owing to a mutation in its spa32 gene failed to induce such phosphorylation. Under in vitro and in vivo conditions, the released IpaB, IpaC, and IpaD proteins bound to alpha 5 beta 1 integrin in a manner different from that of soluble fibronectin but similar to that of the tissue form of fibronectin. At the site of attachment of S. flexneri to CHO cells, alpha5beta1 integrin converged with polymerization of actin. These data thus suggest that the capacity of Ipa proteins to interact with alpha5beta1 integrin may be an important Shigella factor in triggering the reorganization of actin cytoskeletons.

scholarly journals Unstructured mRNAs form multivalent RNA-RNA interactions to generate TIS granule networks

2020 ◽  
Author(s):  
Weirui Ma ◽  
Gang Zhen ◽  
Wei Xie ◽  
Christine Mayr
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Complex Formation ◽  
Major Site ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Protein Complex Formation ◽  
Unstructured Regions

SummaryThe TIS granule network is a constitutively expressed membraneless organelle that concentrates mRNAs with AU-rich elements and interacts with the major site of protein synthesis, the rough endoplasmic reticulum. Most known biomolecular condensates are sphere-like, but TIS granules have a mesh-like morphology. Through in vivo and in vitro reconstitution experiments we discovered that this shape is generated by extensive intermolecular RNA-RNA interactions. They are mostly accomplished by mRNAs with large unstructured regions in their 3′UTRs that we call intrinsically disordered regions (IDRs). As AU-rich RNA is a potent chaperone that suppresses protein aggregation and is overrepresented in mRNAs with IDRs, our data suggests that TIS granules concentrate mRNAs that assist protein folding. In addition, the proximity of translating mRNAs in TIS granule networks may enable co-translational protein complex formation.

scholarly journals Salmonella enterica serotype Typhimurium DT104 ArtA-dependent modification of pertussis toxin-sensitive G proteins in the presence of [32P]NAD

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3710-3718 ◽  
Author(s):  
Ikuo Uchida ◽  
Ryoko Ishihara ◽  
Kiyoshi Tanaka ◽  
Eiji Hata ◽  
Sou-ichi Makino ◽  
...  
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Cho Cells ◽  
Salmonella Enterica ◽  
Phage Type ◽  
Chinese Hamster ◽  
Adp Ribosylation ◽  
Dependent Modification

Salmonella enterica serotype Typhimurium (S. Typhimurium) definitive phage type (DT) 104 has become a widespread cause of human and other animal infections worldwide. The severity of clinical illness in S. Typhimurium DT104 outbreaks suggests that this strain possesses enhanced virulence. ArtA and ArtB – encoded by a prophage in S. Typhimurium DT104 – are homologues of components of pertussis toxin (PTX), including its ADP-ribosyltransferase subunit. Here, we show that exposing DT104 to mitomycin C, a DNA-damaging agent, induced production of prophage-encoded ArtA/ArtB. Pertussis-sensitive G proteins were labelled in the presence of [32P]NAD and ArtA, and the label was released by HgCl2, which is known to cleave cysteine-ADP-ribose bonds. ADP-dependent modification of G proteins was markedly reduced in in vitro-synthesized ArtA6Arg-Ala and ArtA115Glu-Ala, in which alanine was substituted for the conserved arginine at position 6 (necessary for NAD binding) and the predicted catalytic glutamate at position 115, respectively. A cellular ADP-ribosylation assay and two-dimensional electrophoresis showed that ArtA- and PTX-induced ADP-ribosylation in Chinese hamster ovary (CHO) cells occur with the same type of G proteins. Furthermore, exposing CHO cells to the ArtA/ArtB-containing culture supernatant of DT104 resulted in a clustered growth pattern, as is observed in PTX-exposed CHO cells. Hydrogen peroxide, an oxidative stressor, also induced ArtA/ArtB production, suggesting that these agents induce in vivo synthesis of ArtA/ArtB. These results, taken together, suggest that ArtA/ArtB is an active toxin similar to PTX.

scholarly journals Suppression of tumor formation in vivo by expression of the JE gene in malignant cells.

1991 ◽  
Vol 11 (6) ◽  
pp. 3125-3131 ◽  
Author(s):  
B J Rollins ◽  
M E Sunday
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Suppressive Effect ◽  
Tumor Formation ◽  
Host Animal ◽  
Early Growth Response ◽  
Discernible Effect ◽  
Early Growth Response Gene

The early growth response gene JE encodes a monocyte chemoattractant, MCP-1. The JE/MCP-1 protein attracts and stimulates human monocytes and induces monocyte-mediated inhibition of tumor cell growth in vitro. Expression of human or murine JE/MCP-1 in Chinese hamster ovary (CHO) cells completely suppressed their ability to form tumors in nude mice. Coinjection of JE/MCP-1-expressing cells with nonexpressing CHO cells or with HeLa cells also prevented tumor formation. Since JE/MCP-1 expression had no discernible effect on the tranformed phenotype of these cells in vitro, the suppressive effect depends on host animal factors. These factors are likely to be components of the inflammatory response, because JE/MCP-1-expressing cells elicited a predominantly monocytic infiltrate at the site of injection. Our results suggest that JE/MCP-1 protein may be useful in cancer therapy.

Protein synthesis in a fish heart: responses to increased power output

1988 ◽  
Vol 137 (1) ◽  
pp. 565-587 ◽  
Author(s):  
D. F. Houlihan ◽  
C. Agnisola ◽  
A. R. Lyndon ◽  
C. Gray ◽  
N. M. Hamilton
Keyword(s):  
Protein Synthesis ◽  
Cardiac Output ◽  
Oxygen Consumption ◽  
Power Output ◽  
Coronary Vessels ◽  
High Concentration ◽  
Fish Heart ◽  
Bulbus Arteriosus

The effects of exercise on the rates of protein synthesis in the chambers of the trout heart were investigated in vitro and in vivo. An in vitro rainbow trout heart preparation was developed which permitted perfusion of the coronary supply to the compact region of the ventricular muscle. This preparation was used to examine the mechanical responses to preload pressures, the oxygen consumption at different power outputs and the rates of protein synthesis in the various heart components. By increasing preload pressure it was possible to double cardiac output, oxygen consumption and power output without changing heart rate. Mechanical efficiency of the hearts was approximately 20%. Perfusion of the coronary vessels improved cardiac output. Protein synthesis was measured in isolated hearts by the incorporation of [3H]phenylalanine added at high concentration (1.35 mmol l-1) to the perfusion medium. The various chambers of the heart showed marked differences in their rates of protein synthesis. Increasing cardiac output and power output in vitro by twofold over 20 min increased the fractional rate of protein synthesis by approximately 2.5-fold in the atrium and ventricle but did not affect the rates in the bulbus arteriosus. Perfusion of the coronary vessels significantly increased the rates of protein synthesis of the compact layer of the ventricle. In vivo there were no significant differences in the fractional protein synthesis rates between the atrium and ventricle; slow-speed continuous swimming over 40 min (1.5 body lengths s-1) caused an increase in the rates of protein synthesis in all the chambers except the bulbus arteriosus. The stimulation in the fractional rates of protein synthesis by approximately 32% was not as great as in vitro. Both in vivo and in vitro the increased rates of protein synthesis occurred without any change in RNA to protein ratios, indicating an improved activity of protein synthesis per unit of RNA. It is concluded that short-term increases in cardiac contractility, possibly acting through the mechanical stretch on the cardiac muscle, stimulated protein synthesis, particularly in the ventricle, through increased ribosomal activity.

scholarly journals Toxicity Studies of Lactobacillus plantarum PS128TM Isolated from Spontaneously Fermented Mustard Greens

Foods ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 668
Author(s):  
Po-Lin Liao ◽  
Chien-Chen Wu ◽  
Tai-Ying Chen ◽  
Ying-Chieh Tsai ◽  
Wu-Shun Peng ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Toxicity Tests ◽  
Human Consumption ◽  
Probiotic Properties ◽  
Icr Mouse ◽  
In Vivo Testing

Probiotics are extensively available to consumers; however, the use of probiotics may not always be safe, and there are few reports on their side effects, including those of Lactobacillus. Lactobacillus plantarum strain PS128TM isolated from spontaneously fermented mustard greens in Taiwan was recently reported to exhibit probiotic properties. In this study, we aimed to assess the safety of strain PS128TM for use in humans via examining genotoxic and oral toxic effects using in vitro and in vivo testing. Five strains of Salmonella typhimurium were evaluated by the Ames test; no signs of increased reverse mutation were observed following exposure to PS128TM. Additional testing of Chinese hamster ovary (CHO) cells exposed to PS128TM revealed that the incidence of chromosomal aberrations in CHO cells had not increased. PS128TM treatment also did not affect the proportion of immature to total erythrocytes or the number of micronuclei in the immature erythrocytes of ICR mice. Moreover, following a 28 day study involving repeated oral dose toxicity tests (2400, 400, and 40 mg/kg body weight) utilizing an ICR mouse model, no observable adverse level (NOAEL) was found at any of the doses. PS128TM was sensitive to antibiotics; however, genes related to the production of biogenic amines were absent. While further research is required, these toxicological assessments suggest that PS128TM could be safe for human consumption.

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