scholarly journals Assembly and turnover of detyrosinated tubulin in vivo.

1987 ◽  
Vol 105 (1) ◽  
pp. 265-276 ◽  
Author(s):  
D R Webster ◽  
G G Gundersen ◽  
J C Bulinski ◽  
G G Borisy
Keyword(s):  
Dynamic Instability ◽  
Human Fibroblasts ◽  
Chinese Hamster ◽  
Living Cells ◽  
Complete Loss ◽  
Almost All ◽  
Turn Over ◽  
Fixed Times ◽  
Over Time

Detyrosinated (Glu) tubulin was prepared from porcine brain and microinjected into human fibroblasts and Chinese hamster ovary (CHO) cells. Glu tubulin assembled onto the ends of preexisting microtubules and directly from the centrosome within minutes of its microinjection. Incorporation into the cytoskeleton continued until almost all of the microtubules were copolymers of Glu and tyrosinated (Tyr) tubulin. However, further incubation resulted in the progressive and ultimately complete loss of Glu-staining microtubules. Glu tubulin injected into nocodazole-treated cells was converted to Tyr tubulin by a putative tubulin/tyrosine ligase activity. The observed decrease in staining with the Glu antibody over time was used to analyze microtubule turnover in microinjected cells. The mode of Glu disappearance was analyzed quantitatively by tabulating the number of Glu-Tyr copolymers and Tyr-only microtubules at fixed times after injection. The proportion of Glu-Tyr copolymers decreased progressively over time and no segmentally labeled microtubules were observed, indicating that microtubules turn over rapidly and individually. Our results are consistent with a closely regulated tyrosination-detyrosination cycle in living cells and suggest that microtubule turnover is mediated by dynamic instability.

scholarly journals Prevalence and Photobiology of Photosynthetic Dinoflagellate Endosymbionts in the Nudibranch Berghia stephanieae

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2200
Author(s):  
Ruben X. G. Silva ◽  
Paulo Cartaxana ◽  
Ricardo Calado
Keyword(s):  
Food Deprivation ◽  
Photosynthetic Efficiency ◽  
Rapid Decrease ◽  
Complete Loss ◽  
Symbiotic Association ◽  
Non Invasive ◽  
Sea Slugs ◽  
Non Destructive ◽  
Over Time

Berghia stephanieae is a stenophagous sea slug that preys upon glass anemones, such as Exaiptasia diaphana. Glass anemones host photosynthetic dinoflagellate endosymbionts that sea slugs ingest when consuming E. diaphana. However, the prevalence of these photosynthetic dinoflagellate endosymbionts in sea slugs appears to be short-lived, particularly if B.stephanieae is deprived of prey that host these microalgae (e.g., during bleaching events impacting glass anemones). In the present study, we investigated this scenario, along with food deprivation, and validated the use of a non-invasive and non-destructive approach employing chlorophyll fluorescence as a proxy to monitor the persistence of the association between sea slugs and endosymbiotic photosynthetic dinoflagellates acquired through the consumption of glass anemones. Berghia stephanieae deprived of a trophic source hosting photosynthetic dinoflagellate endosymbionts (e.g., through food deprivation or by feeding on bleached E. diaphana) showed a rapid decrease in minimum fluorescence (Fo) and photosynthetic efficiency (Fv/Fm) when compared to sea slugs fed with symbiotic anemones. A complete loss of endosymbionts was observed within 8 days, confirming that no true symbiotic association was established. The present work opens a new window of opportunity to rapidly monitor in vivo and over time the prevalence of associations between sea slugs and photosynthetic dinoflagellate endosymbionts, particularly during bleaching events that prevent sea slugs from incorporating new microalgae through trophic interactions.

scholarly journals Hydrophobic glycosides of N-acetylglucosamine can act as primers for polylactosamine synthesis and can affect glycolipid synthesis in vivo

1995 ◽  
Vol 307 (3) ◽  
pp. 791-797 ◽  
Author(s):  
D C A Neville ◽  
R A Field ◽  
M A J Ferguson
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Culture Medium ◽  
Linear Array ◽  
Chinese Hamster ◽  
Living Cells ◽  
Anomeric Configuration ◽  
Glycolipid Synthesis ◽  
Synthetic Capacity

Several hydrophobic glycosides of N-acetylglucosamine (GlcNAc) served as primers for polylactosamine synthesis when added to Chinese hamster ovary (CHO) cells. The modified glycosides, containing one to six lactosamine repeats in linear array, were sialylated and secreted into the culture medium. The relative efficiencies of the glycosides to serve as primers were dependent on the nature of the aglycone and on the anomeric configuration of the GlcNAc residue. The same compounds were tested for their effects on glycolipid synthesis in CHO cells. All of the beta-glycosides significantly inhibited the synthesis of the lactoseries glycolipid GM3 whereas the alpha-glycoside was inactive. The compound GlcNAc alpha 1-O-benzyl- was the most efficient primer of polylactosamine synthesis and had no effect on glycolipid synthesis. This compound may have potential for the assay of the polylactosamine synthetic capacity of living cells.

Decreasing oncoprotein 18/stathmin levels reduces microtubule catastrophes and increases microtubule polymer in vivo

1999 ◽  
Vol 112 (21) ◽  
pp. 3713-3722 ◽  
Author(s):  
B. Howell ◽  
H. Deacon ◽  
L. Cassimeris
Keyword(s):  
Antisense Oligonucleotides ◽  
Dynamic Instability ◽  
Fold Increase ◽  
Living Cells ◽  
Lung Cells ◽  
Slight Reduction ◽  
Level Measurement ◽  
Fold Reduction ◽  
Oncoprotein 18

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein which destabilizes microtubules. To characterize the function of Op18 in living cells, we used microinjection of anti-Op18 antibodies or antisense oligonucleotides to block either Op18 activity or expression in interphase newt lung cells. Anti-tubulin staining of cells microinjected with anti-Op18 and fixed 1–2 hours after injection showed an increase in total microtubule polymer. In contrast, microinjection of either non-immune IgG or anti-Op18 preincubated with bacterially-expressed Op18 had little effect on microtubule polymer level. Cells treated with Op18 antisense oligonucleotides for 4 days had (greater than or equal to)50% reduced levels of Op18 with no change in the soluble tubulin level. Measurement of MT polymer level in untreated, antisense or nonsense oligonucleotide treated cells demonstrated that reduced Op18 levels resulted in a 2.5-fold increase in microtubule polymer. Next, the assembly dynamics of individual microtubules at the peripheral regions of living cells were examined using video-enhanced contrast DIC microscopy. Microinjection of antibodies against oncoprotein 18 resulted in a 2.2-fold reduction in catastrophe frequency and a slight reduction in plus end elongation velocity compared to uninjected cells or cells microinjected with non-immune IgG. Preincubation of anti-Op18 antibody with recombinant Op18 greatly diminished the effects of the antibody. Similarly, treatment of cells with antisense oligonucleotides reduced catastrophes 2.5- to 3-fold compared to nonsense oligonucleotide treated or untreated cells. The other parameters of dynamic instability were unchanged after reducing Op18 with antisense oligonucleotides. These studies are consistent with Op18 functioning to regulate microtubule catastrophes during interphase in vivo.

scholarly journals Compartment-specific metabolome labeling enables the identification of subcellular fluxes that may serve as promising metabolic engineering targets in CHO cells

2021 ◽  
Author(s):  
Andy Wiranata Wijaya ◽  
Andreas Ulmer ◽  
Lara Hundsdorfer ◽  
Natascha Verhagen ◽  
Attila Teleki ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Malic Enzyme ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Metabolic Labeling ◽  
Ovary Cells ◽  
Cellular Analysis ◽  
Cellular Labeling ◽  
Almost All

Abstract13C labeling data are used to calculate quantitative intracellular flux patterns reflecting in vivo conditions. Given that approaches for compartment-specific metabolomics exist, the benefits they offer compared to conventional non-compartmented 13C flux studies remain to be determined. Using compartment-specific labeling information of IgG1-producing Chinese hamster ovary cells, this study investigated differences of flux patterns exploiting and ignoring metabolic labeling data of cytosol and mitochondria. Although cellular analysis provided good estimates for the majority of intracellular fluxes, half of the mitochondrial transporters, and NADH and ATP balances, severe differences were found for some reactions. Accurate flux estimations of almost all iso-enzymes heavily depended on the sub-cellular labeling information. Furthermore, key discrepancies were found for the mitochondrial carriers vAGC1 (Aspartate/Glutamate antiporter), vDIC (Malate/H+ symporter), and vOGC (α-ketoglutarate/malate antiporter). Special emphasis is given to the flux of cytosolic malic enzyme (vME): it could not be estimated without the compartment-specific malate labeling information. Interesting enough, cytosolic malic enzyme is an important metabolic engineering target for improving cell-specific IgG1 productivity. Hence, compartment-specific 13C labeling analysis serves as prerequisite for related metabolic engineering studies.

3D microscopic analysis of molecular distributions in vivo: Modulation of hexokinase localization

1991 ◽  
Vol 49 ◽  
pp. 394-395
Author(s):  
R.M. Lynch ◽  
W. Carrington ◽  
K.E. Fogarty ◽  
F.S. Fay
Keyword(s):  
Turnover Rate ◽  
Imaging Techniques ◽  
Cell Metabolism ◽  
Microscopic Analysis ◽  
Living Cells ◽  
Mitochondrial Outer Membrane ◽  
Quantitative Image ◽  
Metabolic Conditions ◽  
Over Time

Several enzymes involved in cell metabolism appear to associate with subcellular structures in a dynamic fashion; i.e., their binding is modulated during changes in metabolic rate. Our current focus of investigation is on the modulation of binding of hexokinase isozyme I (HKI) to mitochondria in vivo. Binding of HKI to the mitochondrial outer membrane is proposed to provide kinetic advantages to the enzyme, thereby elevating turnover rate. Moreover, binding of the enzyme may be modulated, and therefore, is suggested to be involved in regulating glycolysis. Recently, we have used quantitative confocal microscopy to demonstrate that HKI is localized to mitochondria in astrocytes even though fractionation studies of these cells indicate that most HKI is not bound. In order to understand the dynamics of association of HKI with mitochondria under different metabolic conditions, we have used 3D microscopic imaging techniques to acquire information regarding the distribution of fluorophore labeled HKI and quantitative image analysis to monitor changes in this distribution over time in living cells.

scholarly journals Mir-21 Suppression Promotes Mouse Hepatocarcinogenesis

Cancers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 4983
Author(s):  
Marta Correia de Sousa ◽  
Nicolas Calo ◽  
Cyril Sobolewski ◽  
Monika Gjorgjieva ◽  
Sophie Clément ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Mouse Liver ◽  
Potential Therapeutic Target ◽  
Stat3 Signaling ◽  
Gene And Protein Expression ◽  
Genetic Deletion ◽  
Almost All ◽  
Over Time

The microRNA 21 (miR-21) is upregulated in almost all known human cancers and is considered a highly potent oncogene and potential therapeutic target for cancer treatment. In the liver, miR-21 was reported to promote hepatic steatosis and inflammation, but whether miR-21 also drives hepatocarcinogenesis remains poorly investigated in vivo. Here we show using both carcinogen (Diethylnitrosamine, DEN) or genetically (PTEN deficiency)-induced mouse models of hepatocellular carcinoma (HCC), total or hepatocyte-specific genetic deletion of this microRNA fosters HCC development—contrasting the expected oncogenic role of miR-21. Gene and protein expression analyses of mouse liver tissues further indicate that total or hepatocyte-specific miR-21 deficiency is associated with an increased expression of oncogenes such as Cdc25a, subtle deregulations of the MAPK, HiPPO, and STAT3 signaling pathways, as well as alterations of the inflammatory/immune anti-tumoral responses in the liver. Together, our data show that miR-21 deficiency promotes a pro-tumoral microenvironment, which over time fosters HCC development via pleiotropic and complex mechanisms. These results question the current dogma of miR-21 being a potent oncomiR in the liver and call for cautiousness when considering miR-21 inhibition for therapeutic purposes in HCC.

The Glycosaminoglycan of Recombinant Human Soluble Thrombomodulin Affects Antithrombotic Activity in a Rat Model of Tissue Factor-Induced Disseminated Intravascular Coagulation

1992 ◽  
Vol 67 (03) ◽  
pp. 366-370 ◽  
Author(s):  
Katsuhiko Nawa ◽  
Teru Itani ◽  
Mayumi Ono ◽  
Katsu-ichi Sakano ◽  
Yasumasa Marumoto ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Disseminated Intravascular Coagulation ◽  
Rat Model ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Soluble Thrombomodulin ◽  
Intravascular Coagulation ◽  
Platelet Counts ◽  
Recombinant Human Soluble Thrombomodulin

SummaryPrevious studies on recombinant human soluble thrombomodulin (rsTM) from Chinese hamster ovary cells revealed that rsTM was expressed as two proteins that differed functionally in vitro due to the presence (rsTMp) or absence (rsTMa) of chondroitin-4-sulfate. The current study evaluates the in vivo behavior of rsTM in rats and in a rat model of tissue factor-induced disseminated intravascular coagulation (DIC). rsTMp was more potent than rsTMa for prolongation of the activated partial thromboplastin time (APTT) and their in vivo half-lives determined by ELISA were 20 min for rsTMp and 5.0 h for rsTMa. Injection of a tissue factor suspension (5 mg/kg) resulted in DIC as judged by decreased platelet counts and fibrinogen concentrations, prolonged APTT, and increased fibrin and fibrinogen degradation products (FDP) levels. A bolus injection of either rsTM (0.2 mg/kg) 1 min before induction of DIC essentially neutralized effects on platelets, fibrinogen, and FDP levels, and had only a moderate effect on APTT prolongation. The dose of anticoagulant to inhibit the drop in platelet counts by 50% (ED50) was 0.2 mg/kg rsTMa, 0.07 mg/kg rsTMp, and 7 U/ kg heparin. The effect of increasing concentrations of rsTM and heparin on bleeding times were compared in experiments involving incision of the rat tail. Doubling of the bleeding times occurred at 5 mg/kg rsTMa, 3 mg/kg rsTMp or 90 U/kg heparin. These values represent a 25-fold increase over the ED50 for rsTMa, 43-fold for rsTMp and 13-fold for heparin. These results suggest that rsTMp is a potent anticoagulant to inhibit the platelet reduction when injected prior to the induction of DIC in rats.

The Development of Homologous (Canine/Anti-Canine) Antibodies in Dogs with Haemophilia A (Factor VIII Deficiency): A Ten-Year Longitudinal Study

1993 ◽  
Vol 69 (01) ◽  
pp. 021-024 ◽  
Author(s):  
Shawn Tinlin ◽  
Sandra Webster ◽  
Alan R Giles
Keyword(s):  
Factor Viii ◽  
Canine Model ◽  
Significant Inhibition ◽  
Human Condition ◽  
Haemophilia A ◽  
Variable Expression ◽  
The Family ◽  
Over Time

SummaryThe development of inhibitors to factor VIII in patients with haemophilia A remains as a serious complication of replacement therapy. An apparently analogous condition has been described in a canine model of haemophilia A (Giles et al., Blood 1984; 63:451). These animals and their relatives have now been followed for 10 years. The observation that the propensity for inhibitor development was not related to the ancestral factor VIII gene has been confirmed by the demonstration of vertical transmission through three generations of the segment of the family related to a normal (non-carrier) female that was introduced for breeding purposes. Haemophilic animals unrelated to this animal have not developed functionally significant factor VIII inhibitors despite intensive factor VIII replacement. Two animals have shown occasional laboratory evidence of factor VIII inhibition but this has not been translated into clinical significant inhibition in vivo as assessed by clinical response and F.VIII recovery and survival characteristics. Substantial heterogeneity of inhibitor expression both in vitro and in vivo has been observed between animals and in individual animals over time. Spontaneous loss of inhibitors has been observed without any therapies designed to induce tolerance, etc., being instituted. There is also phenotypic evidence of polyclonality of the immune response with variable expression over time in a given animal. These observations may have relevance to the human condition both in determining the pathogenetic factors involved in this condition and in highlighting the heterogeneity of its expression which suggests the need for caution in the interpretation of the outcome of interventions designed to modulate inhibitor activity.

Globalization and interconfessional communications

2001 ◽  
pp. 13-17
Author(s):  
Serhii Viktorovych Svystunov
Keyword(s):  
21St Century ◽  
Global Economy ◽  
Modern World ◽  
The World ◽  
Target Set ◽  
Almost All ◽  
Over Time ◽  
Rapid Appearance

In the 21st century, the world became a sign of globalization: global conflicts, global disasters, global economy, global Internet, etc. The Polish researcher Casimir Zhigulsky defines globalization as a kind of process, that is, the target set of characteristic changes that develop over time and occur in the modern world. These changes in general are reduced to mutual rapprochement, reduction of distances, the rapid appearance of a large number of different connections, contacts, exchanges, and to increase the dependence of society in almost all spheres of his life from what is happening in other, often very remote regions of the world.

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

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