scholarly journals Induction of calreticulin expression in HeLa cells by depletion of the endoplasmic reticulum Ca2+ store and inhibition of N-linked glycosylation

1996 ◽  
Vol 318 (2) ◽  
pp. 555-560 ◽  
Author(s):  
David H LLEWELLYN ◽  
Jonathan M. KENDALL ◽  
SHEIKH Naureen ◽  
Anthony K. F. CAMPBELL
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Transcriptional Activation ◽  
Prolonged Exposure ◽  
Transcriptional Start Site ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Primary Role ◽  
Transfected Cells ◽  
Promoter Construct

Calreticulin is now considered to be a multifunctional Ca2+-binding protein. Its primary role is as a Ca2+ storage protein within the lumen of the endoplasmic reticulum (ER), where it also seems to assist in the correct folding and assembly of proteins. We have investigated whether agents that affect these processes can alter calreticulin expression in HeLa cells. Perturbation of intracellular Ca2+ levels by prolonged exposure to either thapsigargin or ionomycin induced calreticulin mRNA, both in the presence and absence of extracellular Ca2+, consistent with the proposal that sustained depletion of the ER Ca2+ store can trigger these increases. The mechanism underlying the induction seems to be transcriptional up-regulation as both agents increased calreticulin promoter-driven firefly luciferase expression in transfected cells to the same degree as the observed increases in calreticulin mRNA. Experiments with a truncated promoter construct showed that the sequences that confer this inducibility reside within the 225 bp immediately upstream of the putative major transcriptional start site. We also examined the effect of tunicamycin, which inhibits N-linked glycosylation in the ER thereby interfering with protein processing. This caused increases in calreticulin mRNA greater than those with either thapsigargin or ionomycin, but failed to transactivate the calreticulin promoter. Thus either additional cis sequences that reside outside our promoter region are necessary for transcriptional activation by tunicamycin, or the increases in calreticulin mRNA occur post-transcriptionally. This suggests that there are probably different mechanisms by which calreticulin expression can be induced in response to agents that affect normal ER functioning.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

Temporal and spatial interrelationships of confronting cisternae to nuclear envelope

1992 ◽  
Vol 50 (1) ◽  
pp. 930-931
Author(s):  
John R. Palisano
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Tumor Cells ◽  
Hela Cells ◽  
Microscopic Investigation ◽  
Electron Microscopic Investigation ◽  
Serial Sections ◽  
Electron Microscopic ◽  
Fetal Rat ◽  
Temporal And Spatial

Although confronting cistemae (CC) have been observed in a variety of tumor cells and normal fetal rat, mouse, and human epithelial tissues, little is known about their origin or role in mitotic cells. While several investigators have suggested that CC arise from nuclear envelope (NE) folding back on itself during prophase, others have suggested that CC arise when fragments of NE pair with endoplasmic reticulum. An electron microscopic investigation of 0.25 um thick serial sections was undertaken to examine the origin of CC in HeLa cells.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

The Only Function of Grauzone Required for Drosophila Oocyte Meiosis Is Transcriptional Activation of the cortex Gene

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1831-1839
Author(s):  
Emily Harms ◽  
Tehyen Chu ◽  
Gwénola Henrion ◽  
Sidney Strickland
Keyword(s):  
Zinc Finger ◽  
Transcriptional Activation ◽  
Single Amino Acid ◽  
Primary Role ◽  
Extra Copy ◽  
Oocyte Meiosis ◽  
Zinc Finger Motifs ◽  
High Level ◽  
Transcriptional Pathway

Abstract The grauzone and cortex genes are required for the completion of meiosis in Drosophila oocytes. The grauzone gene encodes a C2H2-type zinc-finger transcription factor that binds to the cortex promoter and is necessary for high-level activation of cortex transcription. Here we define the region of the cortex promoter to which Grauzone binds and show that the binding occurs through the C-terminal, zinc-finger-rich region of the protein. Mutations in two out of the five grauzone alleles result in single amino acid changes within different zinc-finger motifs. Both of these mutations result in the inability of Grauzone to bind DNA effectively. To determine the mechanism by which Grauzone regulates meiosis, transgenic flies were produced with an extra copy of the cortex gene in homozygous grauzone females. This transgene rescued the meiosis arrest of embryos from these mutants and allowed their complete development, indicating that activation of cortex transcription is the primary role of Grauzone during Drosophila oogenesis. These experiments further define a new transcriptional pathway that controls the meiotic cell cycle in Drosophila oocytes.

scholarly journals Measurement of Effects of Antibiotics in Bioluminescent Staphylococcus aureus RN4220

2001 ◽  
Vol 45 (12) ◽  
pp. 3456-3461 ◽  
Author(s):  
Mervi Tenhami ◽  
Kaisa Hakkila ◽  
Matti Karp
Keyword(s):  
Staphylococcus Aureus ◽  
High Throughput Screening ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Light Emission ◽  
Detection System ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Luciferase Reporter Gene

ABSTRACT The spread of antibiotic resistance among pathogenic bacteria is a serious threat to humans and animals. Therefore, unnecessary use should be minimized, and new antimicrobial agents with novel mechanisms of action are needed. We have developed an efficient method for measuring the action of antibiotics which is applied to a gram-positive strain,Staphylococcus aureus RN4220. The method utilizes the firefly luciferase reporter gene coupled to the metal-induciblecadA promoter in a plasmid, pTOO24. Correctly timed induction by micromolar concentrations of antimonite rapidly triggers the luciferase gene transcription and translation. This sensitizes the detection system to the action of antibiotics, and especially for transcriptional and translational inhibitors. We show the results for 11 model antibiotics with the present approach and compare them to an analytical setup with a strain where luciferase expression is under the regulation of a constitutive promoter giving only a report of metabolic inhibition. The measurement of light emission from intact living cells is shown to correlate extremely well (r = 0.99) with the conventional overnight growth inhibition measurement. Four of the antibiotics were within a 20% concentration range and four were within a 60% concentration range of the drugs tested. This approach shortens the assay time needed, and it can be performed in 1 to 4 h, depending on the sensitivity needed. Furthermore, the assay can be automatized for high-throughput screening by the pharmaceutical industry.

scholarly journals Overexpression of CALNUC (Nucleobindin) Increases Agonist and Thapsigargin Releasable Ca2+ Storage in the Golgi

1999 ◽  
Vol 145 (2) ◽  
pp. 279-289 ◽  
Author(s):  
Ping Lin ◽  
Yong Yao ◽  
Robert Hofmeister ◽  
Roger Y. Tsien ◽  
Marilyn Gist Farquhar
Keyword(s):  
Endoplasmic Reticulum ◽  
Cho Cells ◽  
Binding Capacity ◽  
Binding Protein ◽  
Ip3 Receptor ◽  
Transfected Cells ◽  
Permeabilized Cells ◽  
Endoplasmic Reticulum Calcium ◽  
Golgi Protein

We previously demonstrated that CALNUC, a Ca2+-binding protein with two EF-hands, is the major Ca2+-binding protein in the Golgi by 45Ca2+ overlay (Lin, P., H. Le-Niculescu, R. Hofmeister, J.M. McCaffery, M. Jin, H. Henneman, T. McQuistan, L. De Vries, and M. Farquhar. 1998. J. Cell Biol. 141:1515–1527). In this study we investigated CALNUC's properties and the Golgi Ca2+ storage pool in vivo. CALNUC was found to be a highly abundant Golgi protein (3.8 μg CALNUC/mg Golgi protein, 2.5 × 105 CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca2+-binding site (Kd = 6.6 μM, binding capacity = 1.1 μmol Ca2+/μmol CALNUC). 45Ca2+ storage was increased by 2.5- and 3-fold, respectively, in HeLa cells transiently overexpressing CALNUC-GFP and in EcR-CHO cells stably overexpressing CALNUC. Deletion of the first EF-hand α helix from CALNUC completely abolished its Ca2+-binding capability. CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker, α-mannosidase II (Man II). Approximately 70% of the 45Ca2+ taken up by HeLa and CHO cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (Ca2+ pump) blocker. Stimulation of transfected cells with the agonist ATP or IP3 alone (permeabilized cells) also resulted in a significant increase in Ca2+ release from Golgi stores. By immunofluorescence, the IP3 receptor type 1 (IP3R-1) was distributed over the endoplasmic reticulum and codistributed with CALNUC in the Golgi. These results provide direct evidence that CALNUC binds Ca2+ in vivo and together with SERCA and IP3R is involved in establishment of the agonist-mobilizable Golgi Ca2+ store.

scholarly journals Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells

1991 ◽  
Vol 276 (3) ◽  
pp. 637-641 ◽  
Author(s):  
F F Craig ◽  
A C Simmonds ◽  
D Watmore ◽  
F McCapra ◽  
M R H White
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Intact Cells ◽  
Cos Cells ◽  
Escherichia Coli Cells ◽  
Luminescent Signal ◽  
The Difference ◽  
Kinetics Of

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.

Apigetrin Promotes Cell Autophagy by Activating the Endoplasmic Reticulum Stress in Human Cervical Cancer Hela Cells

2021 ◽  
Vol 20 (1) ◽  
pp. 56-63
Author(s):  
Li Jiang ◽  
Zhi-Cheng Yao ◽  
Miao-Miao Liu ◽  
Run-Hui Ma ◽  
Kiran Thakur
Keyword(s):  
Cervical Cancer ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Hela Cells ◽  
Fruits And Vegetables ◽  
Radical Scavenging ◽  
Anticancer Activities ◽  
Treatment Results ◽  
Metastasis Rate ◽  
The Relationship

Cervical cancer has always been the top malignant cancer among female cancers in the world. Due to its recurrence, metastasis rate, and drug resistance, the treatment results of cervical cancer have been unsatisfactory. Apigetrin is present in a variety of fruits and vegetables and has been reported to have antioxidant, free radical scavenging, anti-inflammatory, and anticancer activities. Therefore, this study focuses on the effect of apigetrin on the autophagy of cervical cancer HeLa cells based on the previous research. The results showed that apigetrin can enhance the autophagy fluorescence of light chain 3B (LC3B), and further combined with quantitative real-time PCR (qPCR) and Western blotting found that the expression of autophagy-related genes and proteins p-mTOR, Beclin1, and LC3B increased, while the expression of AMPK, ULK1, and p62 decreased. In addition, apigetrin also promoted the release of Ca2+, the PERK/eIF2α/ATF4/chop, and IRE1α pathways activate endoplasmic reticulum (ER) stress. The addition of 4PBA proved that ER stress promoted autophagy in HeLa cells. Finally, the addition of the 3-MA indicates the relationship between autophagy and apoptosis in HeLa cells. Our results indicate that apigetrin has a certain anticancer potential and can be used as a drug adjuvant and food additive for the prevention and treatment of cervical cancer.

scholarly journals Evaluation of Transduction Properties of an Adenovirus Vector in Neonatal Mice

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Shunsuke Iizuka ◽  
Fuminori Sakurai ◽  
Kahori Shimizu ◽  
Kazuo Ohashi ◽  
Shin-ichiro Nakamura ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Adenovirus Vector ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Cmv Promoter ◽  
Neonatal Mice ◽  
Neonatal Heart ◽  
Neonatal Liver ◽  
Neonates And Infants

In gene therapy for congenital disorders, treatments during neonate and infant stages are promising. Replication-incompetent adenovirus (Ad) vectors have been used in gene therapy studies of genetic disorders; however, the transduction properties of Ad vectors in neonates and infants have not been fully examined. Accordingly, this study examined the properties of Ad vector-mediated transduction in neonatal mice. A first-generation Ad vector containing a cytomegalovirus (CMV) promoter-driven luciferase expression cassette was administered to neonatal mice on the second day of lifeviaretro-orbital sinus. The highest Ad vector genome copy numbers and transgene expression were found in the neonatal liver. The neonatal heart exhibited the second highest levels of transgene expression among the organs examined. There was an approximately 1500-fold difference in the transgene expression levels between the adult liver and heart, while the neonatal liver exhibited only an approximately 30-fold higher level of transgene expression than the neonatal heart. A liver-specific promoter for firefly luciferase expression conferred a more than 100-fold higher luciferase expression in the liver relative to the other organs. No apparent hepatotoxicity was observed in neonatal mice following Ad vector administration. These findings should provide valuable information for gene therapy using Ad vectors in neonates and infants.

scholarly journals Brucella abortus Transits through the Autophagic Pathway and Replicates in the Endoplasmic Reticulum of Nonprofessional Phagocytes

1998 ◽  
Vol 66 (12) ◽  
pp. 5711-5724 ◽  
Author(s):  
Javier Pizarro-Cerdá ◽  
Stéphane Méresse ◽  
Robert G. Parton ◽  
Gisou van der Goot ◽  
Alberto Sola-Landa ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Brucella Abortus ◽  
Cathepsin D ◽  
Bacterial Strains ◽  
Infected Cells ◽  
Bacterial Replication ◽  
Protein Disulfide ◽  
Intracellular Pathway ◽  
Replication Compartment

ABSTRACT Brucella abortus is an intracellular pathogen that replicates within a membrane-bounded compartment. In this study, we have examined the intracellular pathway of the virulent B. abortus strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells. At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1. At ∼1 h postinoculation, bacteria are located within a compartment positive for the lysosome-associated membrane proteins (LAMPs) and the endoplasmic reticulum (ER) marker sec61β but negative for the mannose 6-phosphate receptors and cathepsin D. Interestingly, this compartment is also positive for the autophagosomal marker monodansylcadaverin, suggesting that S2308 and S19 are located in autophagic vacuoles. At 24 h after inoculation, attenuated S19 is degraded in lysosomes, while virulent S2308 multiplies within a LAMP- and cathepsin D-negative but sec61β- and protein disulfide isomerase-positive compartment. Furthermore, treatment of infected cells with the pore-forming toxin aerolysin from Aeromonas hydrophila causes vacuolation of the bacterial replication compartment. These results are compatible with the hypothesis that pathogenic B. abortus exploits the autophagic machinery of HeLa cells to establish an intracellular niche favorable for its replication within the ER.

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