scholarly journals Relative sensitivity to inhibition by cimetidine and clonidine differentiates between the two types of Na+-H+ exchangers in cultured cells

1991 ◽  
Vol 280 (2) ◽  
pp. 317-322 ◽  
Author(s):  
S Ramamoorthy ◽  
C Tiruppathi ◽  
C N Nair ◽  
V B Mahesh ◽  
F H Leibach ◽  
...  
Keyword(s):  
Cell Line ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Membrane Vesicles ◽  
Type A ◽  
Relative Sensitivity ◽  
Type B ◽  
Opossum Kidney ◽  
Cell Line Hela ◽  
Inhibition Studies

Available evidence indicates that there are two types of Na(+)-H+ exchangers, type A (housekeeping type) and type B (epithelial or apical type), in mammalian cells. We have recently reported, using isolated membrane vesicles, that these two types can be differentiated by their relative sensitivities to inhibition by clonidine and cimetidine [Kulanthaivel, Leibach, Mahesh, Cragoe & Ganapathy (1990) J. Biol. Chem. 264, 1249-1252]. The present study was undertaken to determine whether this approach is also effective in cultured cells. The JAR human placental choriocarcinoma cell line and the opossum kidney (OK) cell line, when grown as confluent monolayer cultures on an impermeable plastic support, express Na(+)-H+ exchanger activity which is measurable by determining Na+ uptake into the cells from the culture medium. The JAR cell Na(+)-H+ exchanger was found to be about 100 times more sensitive to inhibition by dimethylamiloride than the OK cell Na(+)-H+ exchanger. Inhibition studies with clonidine and cimetidine were able to differentiate between these two exchangers very clearly. Cimetidine was 18 times more potent than clonidine in inhibiting the JAR cell Na(+)-H+ exchanger. In contrast, clonidine was at least 8 times more potent than cimetidine in inhibiting the Na(+)-H+ exchanger of the OK cell. The results show that the JAR cell expresses the type A Na(+)-H+ exchanger, whereas the OK cell expresses the type B Na(+)-H+ exchanger. This approach also proved to be very effective in correctly identifying the type of Na(+)-H+ exchanger in a third cell line (HeLa). It is concluded that the relative susceptibility to inhibition by clonidine and cimetidine offers an easy and efficient means of differentiating between the two types of Na(+)-H+ exchangers in cultured cells.

Studies on the use of cultured cells in a bioassay for parathyroid hormone

1994 ◽  
Vol 143 (2) ◽  
pp. 261-268
Author(s):  
A E Armston ◽  
P J Wood
Keyword(s):  
Parathyroid Hormone ◽  
Cyclic Amp ◽  
Cell Line ◽  
Cultured Cells ◽  
Biologically Active ◽  
Kidney Cell Line ◽  
Opossum Kidney ◽  
Pre Treatment ◽  
Set Up ◽  
Insufficient Sensitivity

Abstract Measurement of parathyroid hormone (PTH) is important for diagnosing hyper- and hypoparathyroidism. The move to two-site immunometric assays that detect the whole molecule has improved the discrimination of these conditions but these assays may be too restrictive because some PTH fragments that are biologically active may not be detected. In addition, PTH-like peptide of malignancy, an important cause of malignancy-associated hypercalcaemia, is not detected by the two-site assays. Experiments were performed to set up a simple, robust and inexpensive bioassay for PTH, exploiting a kidney cell line and using cyclic AMP or an eluted stain assay as the end point. Of the 12 cell lines tested, an opossum kidney (WOK) cell line showed the most promise. Despite optimization of the procedure to include pre-treatment with dexamethasone, insulin and PTH, followed by incubation in the presence of 5′ -guanylimidodiphosphate, isobutyl-1-methylxanthine and forskolin, the WOK cells showed insufficient sensitivity for use in a cultured cell bioassay for PTH in human serum. In addition, the cells were less sensitive to PTH-like peptide precluding their use for an assay for this molecule. Journal of Endocrinology (1994) 143, 261–268

scholarly journals THE POPULATION-DEPENDENT REQUIREMENT BY CULTURED MAMMALIAN CELLS FOR METABOLITES WHICH THEY CAN SYNTHESIZE

1962 ◽  
Vol 116 (1) ◽  
pp. 29-43 ◽  
Author(s):  
Harry Eagle ◽  
Karl Piez
Keyword(s):  
Population Density ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Free Medium ◽  
Experimental Conditions ◽  
Cellular Survival ◽  
Sustained Growth ◽  
Cell Line Hela ◽  
Specific Organ ◽  
Cell Densities

At least seven compounds synthesized by cultured cells in amounts which should suffice for sustained growth have nevertheless proved under certain conditions necessary for survival (asparagine, cystine, glutamine, homocystine inositol, pyruvate, serine). In every instance so far examined, that requirement has been population-dependent, disappearing at cell densities sufficiently large to bring the concentration in the medium and in the cellular pool to metabolically effective levels before the cells died of the specific deficiency. At population densities of less than 100 cells per ml, serine was required by all cultured cells so far studied. With more exigent strains, such as the RT6 strain of rabbit fibroblast and the P388 mouse leukemia, the serine requirement disappeared only at populations in excess of 50,000 and 150,000 cells per ml, respectively. The requirement for pyruvate by the latter cell as an alternative to serine also disappeared at that population density. In a cystine-free medium there were population-dependent requirements for cystine, homocystine, or serine, depending on the experimental conditions. With methionine and glucose as cystine precursors, the critical population density permitting cellular survival and growth was in excess of 200,000 cells/ml. The provision of homocystine as an intermediate reduced the critical population density to 10,000 to 60,000 cells/ml; with the further provision of serine, the critical cell concentration permitting growth was reduced to 50 to 500 cells/ml. Cells adapted to glutamic acid, and capable of utilizing it as a substitute for glutamine, nevertheless required exogeneous glutamine at cellular densities of less than 50,000 cells per ml. In some experiments, the provision of asparagine reduced the critical population density to 10,000 cells/ml, presumably because of its glutamine-sparing action. Inositol is required by most cell lines, despite their demonstrated capacity to synthesize it from glucose. With at least one cell line (HeLa), sustained growth was occasionally achieved in an inositol-free medium if the population density was maintained in excess of 240,000 cells/ml. The possible implications of these findings with respect to the loss of specific organ functions in dispersed cell culture are discussed.

scholarly journals Improved Split-GFP Systems for Visualizing Organelle Contact Sites in Yeast and Human Cells

2020 ◽  
Vol 8 ◽  
Author(s):  
Shinya Tashiro ◽  
Yuriko Kakimoto ◽  
Manatsu Shinmyo ◽  
Shintaro Fujimoto ◽  
Yasushi Tamura
Keyword(s):  
Hela Cell ◽  
Cell Line ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Early Stage ◽  
Human Cells ◽  
Yeast Cells ◽  
Hela Cell Line ◽  
Contact Sites ◽  
Low Levels

Inter-organelle contact sites have attracted a lot of attention as functionally specialized regions that mediate the exchange of metabolites, including lipids and ions, between distinct organelles. However, studies on inter-organelle contact sites are at an early stage and it remains enigmatic what directly mediates the organelle-organelle interactions and how the number and degree of the contacts are regulated. As a first step to answer these questions, we previously developed split-GFP probes that could visualize and quantify multiple inter-organelle contact sites in the yeast and human cultured cells. However, the split-GFP probes possessed a disadvantage of inducing artificial connections between two different organelle membranes, especially when overexpressed. In the present study, we developed a way to express the split-GFP probes whose expressions remained at low levels, with minimal variations between different yeast cells. Besides, we constructed a HeLa cell line in which the expression of the split-GFP probes could be induced by the addition of doxycycline to minimize the artificial effects. The improved split-GFP systems may be faithful tools to quantify organelle contact sites and screen new factors involved in organelle-organelle tethering in yeast and mammalian cells.

Evidence for deterministic chaos in aperiodic oscillations of proliferative activity in long-term cultured Fao hepatoma cells

2000 ◽  
Vol 113 (6) ◽  
pp. 1069-1074
Author(s):  
C. Wolfrom ◽  
N.P. Chau ◽  
J. Maigne ◽  
J.C. Lambert ◽  
B. Ducot ◽  
...  
Keyword(s):  
Cell Line ◽  
Mammalian Cells ◽  
Proliferative Activity ◽  
Cultured Cells ◽  
Hepatoma Cell ◽  
Proliferation Rate ◽  
Hepatoma Cell Line ◽  
Common Value ◽  
Non Linear

The proliferative activity of long-term cultured mammalian cells exhibits traits of a complex dynamic system, with a succession of spontaneous rises and falls in proliferation rate. We analyzed three successive series of proliferation data for the Fao hepatoma cell line in long-term cultures. In the three series the proliferation rate displayed apparently disordered oscillations, which each lasted about 3–5 passages, with variable amplitude and were therefore unpredictable. Such non-linear kinetics raises the major issue of whether these fluctuations are random, or determined and coordinated. We used a graphical method of analysis of the data, which demonstrated that all troughs of proliferation were mathematically related to a common value in each series. This common value was itself related to the maximum level of proliferation of the cell line. Non-linear analysis thus confirmed that the fluctuations in proliferation rate of tumoral Fao cells are, at least in part, determined. This pattern evokes chaotic dynamics and is evidence for the flexible coordination of the complex system linking positive and negative growth regulators in long-term cultured cells.

Biological and Morphological Studies on Low-Leukemia-Strain Mice with Hodgkin's-Like Neoplasia Induced by Serial Cell-Free Transmission of Leukemic Organs of SJL/J Strain Mice

1968 ◽  
Vol 26 ◽  
pp. 210-211
Author(s):  
S. Fujinaga ◽  
K. Maruyama ◽  
C.W. Williams ◽  
K. Sekhri ◽  
L. Dmochowski
Keyword(s):  
Tissue Culture ◽  
Type A ◽  
Reticulum Cell ◽  
Type B ◽  
Viral Origin ◽  
Serial Transfer ◽  
Strain Mouse ◽  
Morphological Studies ◽  
Cell Neoplasm ◽  
Free Material

Yumoto and Dmochowski (Cancer Res.27, 2098 (1967)) reported the presence of mature and immature type C leukemia virus particles in leukemic organs and tissues such as lymph nodes, spleen, thymus, liver, and kidneys of SJL/J strain mice with Hodgki's-like disease or reticulum cell neoplasm (type B). In an attempt to ascertain the possibility that this neoplasia may be of viral origin, experiments with induction and transmission of this neoplasm were carried out using cell-free extracts of leukemic organs from an SJL/J strain mouse with spontaneous disease.It has been possible to induce the disease in low-leukemia BALB/c and C3HZB strain mice and serially transfer the neoplasia by cell-free extracts of leukemic organs of these mice. Histological examination revealed the neoplasia to be of either reticulum cell-type A or type B. Serial transfer is now in its fifth passage. In addition leukemic spleen from another SJL/J strain mouse with spontaneous reticulum cell neoplasm (type A) was set up in tissue culture and is now in its 141st serial passage in vitro. Preliminary results indicate that cell-free material of 39th tissue culture passage can reproduce neoplasia in BALB/c mice.

Anticoagulant Activity of β2-Glycoprotein I Is Potentiated by a Distinct Subgroup of Anticardiolipin Antibodies

1992 ◽  
Vol 68 (03) ◽  
pp. 297-300 ◽  
Author(s):  
Monica Galli ◽  
Paul Comfurius ◽  
Tiziano Barbui ◽  
Robert F A Zwaal ◽  
Edouard M Bevers
Keyword(s):  
Human Plasma ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Type A ◽  
Lipid Vesicles ◽  
Dependent Manner ◽  
Type B ◽  
Distinct Subgroup ◽  
Glycoprotein I ◽  
Thrombin Formation

SummaryPlasmas of 16 patients positive for both IgG anticardiolipin (aCL) antibodies and lupus anticoagulant (LA) antibodies were subjected to adsorption with liposomes containing cardiolipin. In 5 of these plasmas both the anticardiolipin and the anticoagulant activities were co-sedimented with the liposomes in a dose-dependent manner, whereas in the remaining cases only the anticardiolipin activity could be removed by the liposomes, leaving the anticoagulant activity (LA) in the supernatant plasma. aCL antibodies purified from the first 5 plasmas were defined as aCL-type A, while the term aCL-type B was used for antibodies in the other 11 plasmas, from which 2 were selected for this study.Prolongation of the dRVVT was produced by affinity-purified aCL-type A antibodies in plasma of human as well as animal (bovine, rat and goat) origin. aCL-type B antibodies were found to be devoid of anticoagulant activity, while the corresponding supernatants containing LA IgG produced prolongation of the dRVVT only in human plasma.These anticoagulant activities of aCL-type A and of LA IgG's were subsequently evaluated in human plasma depleted of β2-glycoprotein I (β2-GPI), a protein which was previously shown to be essential in the binding of aCL antibodies to anionic phospholipids. Prolongation of the dRVVT by aCL-type A antibodies was abolished using β2-GPI deficient plasma, but could be restored upon addition of β2-GPI. In contrast, LA IgG caused prolongation of the dRVVT irrespective of the presence or absence of β2-GPI.Since β2-GPI binds to negatively-charged phospholipids and impedes the conversion of prothrombin by the factor Xa/Va enzyme complex (Nimpf et al., Biochim Biophys Acta 1986; 884: 142–9), comparison was made of the effect of aCL-type A and aCL-type B antibodies on the rate of thrombin formation in the presence and absence of β2-GPI. This was measured in a system containing highly purified coagulation factors Xa, Va and prothrombin and lipid vesicles composed of 20 mole% phosphatidylserine and 80 mole% phosphatidylcholine. No inhibition on the rate of thrombin formation was observed with both types of aCL antibodies when either β2-GPI or the lipid vesicles were omitted. Addition of β2-GPI to the prothrombinase assay in the presence of lipid vesicles causes a time-dependent inhibition which was not affected by the presence of aCL-type B or non-specific IgG. In contrast, the presence of aCL-type A antibodies dramatically increased the anticoagulant effect of β2-GPI. These data indicate that the anticoagulant activity of aCL-type A antibodies in plasma is mediated by β2-GPI.

Feeding behaviour and bioerosion: the ecological role of the rock-boring urchin, Echinometra mathaei (de Blainville, 1825), in Okinawa reef flat

2013 ◽  
Vol 1 (1) ◽  
pp. 10
Author(s):  
Noar Muda Satyawan ◽  
Shelly Tutupoho ◽  
Yusli Wardiatno ◽  
Makoto Tsuchiya
Keyword(s):  
Feeding Behaviour ◽  
Reef Flat ◽  
Type A ◽  
Gut Contents ◽  
Biological Processes ◽  
Type B ◽  
Night Time ◽  
Echinometra Mathaei ◽  
Benthic Ecosystems

Erosion rate on corals due to activities of other biota is called bioerosion. The rock-boring urchin, Echinometra mathaei, when it is abundant, plays a significant role in benthic ecosystems, including biological processes like coral erosion. During feeding, E. mathaei erodes calcium carbonate besides grazing on algae living on coral, so it plays an important role in both organic and inorganic carbons in coral reefs. The urchin E. mathaei actively feeds during the night time (nocturnal grazer). Although in Okinawa four types (A-D) of the urchin exist, the research only focused on the types A and B. Type A of E. mathaei produced 0.44951 g feces per day on average while type B produced 0.38030 g feces per day. CaCO3 analysis in feces and gut contents showed bioerosion rate of E. mathaei type A was 0.64492 g/individu/day, and 0.54436 g/individu/day in type B. There were no significant differences in bioerosion impact of E. mathaei type A and B© Laju erosi pada karang yang disebabkan oleh biota, dikenal dengan bioerosi. Bulu babi jenis Echinometra mathaei, ketika melimpah, menjadi sangat berpengaruh terhadap ekosistem bentik termasuk proses biologi seperti erosi karang. Selama aktivitas makan, E. mathaei menggerus kalsium karbonat dalam proporsi yang besar di samping alga yang tumbuh menempel pada karang sehingga memiliki peran penting dalam siklus karbon organik dan anorganik di ekosistem terumbu karang. Bulu babi E. mathaei aktif mencari makan pada malam hari (nocturnal grazer). Meskipun di Okinanawa ada 4 tipe (A-D), pada eksperimen kali ini memfokuskan pada tipe A dan B saja. Tipe A E. mathaei rata-rata memproduksi 0,44951 g feses/hari dan tipe B memproduksi 0,38030 g feses/hari. Berdasarkan analisis CaCO3 yang dilakukan pada feses dan isi lambung, laju bioerosi yang disebabkan oleh E. mathaei tipe A sebesar 0,64492 g/individu/hari sedangkan tipe B sebesar 0,54436 g/individu/hari. Tidak terdapat perbedaan dampak bioerosi yang signifikan antara E. mathaei tipe A dan B©

Identification and Expression Analysis of CD73 Inhibitors in Cervical Cancer

2020 ◽  
Vol 16 ◽  
Author(s):  
Jamshed Iqbal ◽  
Ayesha Basharat ◽  
Sehrish Bano ◽  
Syed Mobasher Ali Abid ◽  
Julie Pelletier ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Western Blot ◽  
Hela Cell ◽  
Cell Line ◽  
Cell Apoptosis ◽  
Immunofluorescence Staining ◽  
Cell Cycle Analysis ◽  
Hela Cell Line ◽  
Cell Line Hela

Aims: The present study was conducted to examine the inhibitory effects of synthesized sulfonylhydrazones on the expression of CD73 (ecto-5′-NT). Background: CD73 (ecto-5′-NT) represents the most significant class of ecto-nucleotidases which are mainly responsible for dephosphorylation of adenosine monophosphate to adenosine. Inhibition of CD73 played an important role in the treatment of cancer, autoimmune disorders, precancerous syndromes, and some other diseases associated with CD73 activity. Objective: Keeping in view the significance of CD73 inhibitor in the treatment of cervical cancer, a series of sulfonylhydrazones (3a-3i) derivatives synthesized from 3-formylchromones were evaluated. Methods: All sulfonylhydrazones (3a-3i) were evaluated for their inhibitory activity towards CD73 (ecto-5′-NT) by the malachite green assay and their cytotoxic effect was investigated on HeLa cell line using MTT assay. Secondly, most potent compound was selected for cell apoptosis, immunofluorescence staining and cell cycle analysis. After that, CD73 mRNA and protein expression were analyzed by real-time PCR and Western blot. Results: Among all compounds, 3h, 3e, 3b, and 3c were found the most active against rat-ecto-5′-NT (CD73) enzyme with IC50 (µM) values of 0.70 ± 0.06 µM, 0.87 ± 0.05 µM, 0.39 ± 0.02 µM and 0.33 ± 0.03 µM, respectively. These derivatives were further evaluated for their cytotoxic potential against cancer cell line (HeLa). Compound 3h and 3c showed the cytotoxicity at IC50 value of 30.20 ± 3.11 µM and 86.02 ± 7.11 µM, respectively. Furthermore, compound 3h was selected for cell apoptosis, immunofluorescence staining and cell cycle analysis which showed promising apoptotic effect in HeLa cells. Additionally, compound 3h was further investigated for its effect on expression of CD73 using qRT-PCR and western blot. Conclusion: Among all synthesized compounds (3a-3i), Compound 3h (E)-N'-((6-ethyl-4-oxo-4H-chromen-3-yl) methylene)-4-methylbenzenesulfonohydrazide was identified as most potent compound. Additional expression studies conducted on HeLa cell line proved that this compound successfully decreased the expression level of CD73 and thus inhibiting the growth and proliferation of cancer cells.

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