scholarly journals Iron in cytosolic ferritin can be recycled through lysosomal degradation in human fibroblasts

1998 ◽  
Vol 336 (1) ◽  
pp. 201-205 ◽  
Author(s):  
Derek C. RADISKY ◽  
Jerry KAPLAN
Keyword(s):  
Human Fibroblasts ◽  
Dependent Manner ◽  
Lysosomal Degradation ◽  
Temperature Dependent ◽  
Intracellular Iron ◽  
Vitro Assay ◽  
Cellular Iron ◽  
Horse Spleen Ferritin

Examination of the mechanism of intracellular iron recovery from lysosomally-degraded ferritin in vivo has been complicated by the continuous flux of cellular iron through ferritin molecules. Here we incubated human fibroblasts with cationic ferritin, a derivative of horse spleen ferritin, as a technique for delivering immunologically distinct ferritin molecules directly to lysosomes. Using this method, we found increased endogenous ferritin levels after the cellular degradation of cationic ferritin, demonstrating that cells can utilize lysosomal ferritin to produce increased cytosolic ferritin levels. Further, using an in vitro assay, we showed that isolated lysosomes degrade endogenous ferritin in a time- and temperature-dependent manner. These results are consistent with a model in which cytosolic ferritin is taken into the lysosomes and degraded. The solubilized iron from the ferric core could then be transported across the lysosomal membrane back into the cytosol.

scholarly journals ATP:Mg2+ shapes condensate properties of rRNA-NPM1 in vitro nucleolus model and its partitioning of ribosomes

2021 ◽  
Author(s):  
N. Amy Yewdall ◽  
Alain A. M. André ◽  
Merlijn H. I. van Haren ◽  
Frank H. T. Nelissen ◽  
Aafke Jonker ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Enzymatic Reaction ◽  
Atp Depletion ◽  
Gel Point ◽  
Dependent Manner ◽  
Biophysical Properties ◽  
Temperature Dependent ◽  
Quantitative Fluorescence

Nucleoli have viscoelastic gel-like condensate dynamics that are not well represented in vitro. Nucleoli models, such as those formed by nucleophosmin 1 (NPM1) and ribosomal RNA (rRNA), exhibit condensate dynamics orders of magnitude faster than in vivo nucleoli. Here we show that an interplay between magnesium ions (Mg2+) and ATP governs rRNA dynamics, and this ultimately shapes the physical state of these condensates. Using quantitative fluorescence microscopy, we demonstrate that increased RNA compaction occurs in the condensates at high Mg2+ concentrations, contributing to the slowed RNA dynamics. At Mg2+ concentrations above 7 mM, rRNA is fully arrested and the condensates are gels. Below the critical gel point, NPM1-rRNA droplets age in a temperature-dependent manner, suggesting that condensates are viscoelastic materials, undergoing maturation driven by weak multivalent interactions. ATP addition reverses the dynamic arrest of rRNA, resulting in liquefaction of these gel-like structures. Surprisingly, ATP and Mg2+ both act to increase partitioning of NPM1-proteins as well as rRNA, which influences the partitioning of small client molecules. By contrast, larger ribosomes form a halo around NPM1-rRNA coacervates when Mg2+ concentrations are higher than ATP concentrations. Within cells, ATP levels fluctuate due to biomolecular reactions, and we demonstrate that a dissipative enzymatic reaction can control the biophysical properties of in vitro condensates through depletion of ATP. This enzymatic ATP depletion also reverses the formation of the ribosome halos. Our results illustrate how cells, by changing local ATP concentrations, may regulate the state and client partitioning of RNA-containing condensates such as the nucleolus.

scholarly journals Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level.

1996 ◽  
Vol 132 (3) ◽  
pp. 345-357 ◽  
Author(s):  
X M Wang ◽  
J G Peloquin ◽  
Y Zhai ◽  
J C Bulinski ◽  
G G Borisy
Keyword(s):  
Posttranslational Modifications ◽  
Glutathione Transferase ◽  
Cultured Cells ◽  
Human Fibroblasts ◽  
Cellular Level ◽  
Vitro Assay ◽  
Normal Functions ◽  
Projection Domain

Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT-dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.

scholarly journals Effect of Massoia (Massoia aromatica Becc.) Bark on the Phagocytic Activity of Wistar Rat Macrophages

2018 ◽  
Author(s):  
Triana Hertiani ◽  
Agustinus Yuswanto ◽  
Sylvia Utami Tunjung Pratiwi ◽  
Harlyanti Mashar
Keyword(s):  
Phagocytic Activity ◽  
Wistar Rats ◽  
In Vitro Assay ◽  
Phagocytic Index ◽  
Dependent Manner ◽  
Vitro Assay ◽  
Macrophage Cells ◽  
In Vivo Assay

Massoia (Massoia aromatica Becc., Lauraceae) bark has been widely used as a component of traditional Indonesian medicine. The indigenous people boil or steam the bark for traditional applications. Our preliminary research revealed the potency of Massoia essential oil and its major compound, C-10 Massoialactone as potential immunomodulator in vitro. However, no scientific evidence regarding its in vivo effects is available. Therefore, this study evaluated the potential immunomodulatory effects of Massoia bark infusion on the nonspecific immune response (phagocytosis) of Wistar rats. The aqueous extract of Massoia bark was obtained by boiling pulverized bark in water, and the C-10 massoialactone content of the extract was determined through Thin Layer Chromatography (TLC) densitometry. For the in vitro assay, macrophages were treated with the freeze-dried infusion at the concentrations of 2.5, 5, 10, 20, or 40 μg/mL media. For the in vivo assay, 2-month-old male Wistar rats were divided into 5 groups. The baseline group received distilled water at the dose of 1 mL/100 g BW with the immunostimulant herbal product “X” administered as the positive control at the dose of 0.54 mL/rat. The treatment groups received the infusion at a dose of 100, 300, or 500 mg/100 g BW. Treatments were given orally every day for 14 days. The ability of macrophage cells to phagocyte latex was determined as phagocytic index (PI) and was observed under microscopy with 300 macrophages. The in vitro study revealed that the phagocytic activity of the infusion-treated macrophages significantly increased in comparison with that of the control macrophages in a concentration-dependent manner. Among all treatment concentrations, the concentration of 40 μg/ml provided the highest activity with a PI value of 70.51% ± 1.11%. The results of the in vivo assay confirmed those of the in vitro assay. The results of the present study indicate that Massoia bark can increase the phagocytic activity of rat macrophage cells. Its potential as a naturally derived immunomodulatory agent requires further study.

scholarly journals DAB2IP inhibits p53 ubiquitin-mediated degradation by competitively binding to GRP75 and suppresses tumor malignancy in colon cancer

2021 ◽  
Author(s):  
Jie Shen ◽  
Shengjie Feng ◽  
Jiao Deng ◽  
Qingwen Huang ◽  
Dayong Zhao ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Animal Experiments ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Wild Type ◽  
Vitro Assay ◽  
Cell Invasiveness ◽  
The Stability

Increasing evidence has shown that DAB2IP acts as a tumor suppressor and plays an inhibition role in many tumors. However, the underlying mechanism is still uncertain. Our study shows that DAB2IP is positively associated with a better prognosis in colon cancer patients with wild-type TP53 expression. In vitro assay shows that DAB2IP elicits potent tumor-suppressive effects on inhibiting cell invasiveness, colony formation and promoting cell apoptosis in wild-type TP53 colon cancer cell lines. Subsequently, DAB2IP is demonstrated to up-regulate the stability of wild-type TP53 by inhibiting its degradation in a ubiquitin-proteasome-dependent manner. Using mass spectrometry profiling, we unveil that DAB2IP and p53 could both interact with the ubiquitin ligase-related protein, GRP75. Mechanistically, DAB2IP could competitively bind with GRP75, thus reducing GRP75-mediated p53 ubiquitination and degradation. Finally, animal experiments also reveal that DAB2IP inhibits the tumor progression in vivo. In conclusion, our study presents a novel function of DAB2IP in GRP75-driven wild-type p53 degradation, which provides a new insight in DAB2IP-induced tumor suppression and provides a novel molecular aspect of the p53 pathway.

scholarly journals Inhibition of Cytomegalovirus Replication with Extended-Half-Life Synthetic Ozonides

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Yiping Wang ◽  
Rupkatha Mukhopadhyay ◽  
Sujayita Roy ◽  
Arun Kapoor ◽  
Yu-Pin Su ◽  
...  
Keyword(s):  
Half Life ◽  
Cell Cycle Progression ◽  
Early Stage ◽  
Human Fibroblasts ◽  
Dependent Manner ◽  
Therapeutic Success ◽  
Concentration Dependent Manner ◽  
Malaria Therapy

ABSTRACTArtesunate (AS), a semisynthetic artemisinin approved for malaria therapy, inhibits human cytomegalovirus (HCMV) replicationin vitro, but therapeutic success in humans has been variable. We hypothesized that the shortin vivohalf-life of AS may contribute to the different treatment outcomes. We tested novel synthetic ozonides with longer half-lives against HCMVin vitroand mouse cytomegalovirus (MCMV)in vivo. Screening of the activities of four ozonides against a pp28-luciferase-expressing HCMV Towne recombinant identified OZ418 to have the best selectivity; its effective concentration inhibiting viral growth by 50% (EC50) was 9.8 ± 0.2 µM, and cytotoxicity in noninfected human fibroblasts (the concentration inhibiting cell growth by 50% [CC50]) was 128.1 ± 8.0 µM. In plaque reduction assays, OZ418 inhibited HCMV TB40 in a concentration-dependent manner as well as a ganciclovir (GCV)-resistant HCMV isolate. The combination of OZ418 and GCV was synergistic in HCMV inhibitionin vitro. Virus inhibition by OZ418 occurred at an early stage and was dependent on the cell density at the time of infection. OZ418 treatment reversed HCMV-mediated cell cycle progression and correlated with the reduction of HCMV-induced expression of pRb, E2F1, and cyclin-dependent kinases 1, 2, 4, and 6. In an MCMV model, once-daily oral administration of OZ418 had significantly improved efficacy against MCMV compared to that of twice-daily oral AS. A parallel pharmacokinetic study with a single oral dose of OZ418 or AS showed a prolonged plasma half-life and higher unbound concentrations of OZ418 than unbound concentrations of AS. In summary, ozonides are proposed to be potential therapeutics, alone or in combination with GCV, for HCMV infection in humans.

scholarly journals Characterization of Histone Lysine β-hydroxybutyrylation in bovine tissues, cells, and cumulus-oocyte complexes

2021 ◽  
Author(s):  
Juliano Rodrigues Sangalli ◽  
Ricardo Perecin Nociti ◽  
Maite del Collado ◽  
Rafael Vilar Sampaio ◽  
Juliano Coelho da Silveira ◽  
...  
Keyword(s):  
Epigenetic Modification ◽  
Human Fibroblasts ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Epigenetic Mark ◽  
Dependent Manner ◽  
Histone Lysine ◽  
Entry Points

ABSTRACTBesides their canonical roles as energy sources, short-chain fatty acids act as metabolic regulators of gene expression through the histone post-translational modifications. The ketone body β-hydroxybutyrate (BHB) was shown to cause a novel type of epigenetic modification, Histone Lysine β-hydroxybutyrylation (Kbhb), associated with genes upregulated in starvation-responsive metabolic pathways. Dairy cows increase BHB in early lactation and its effects on cellular epigenome are largely unknown. To unravel these effects, we sought and identified that Kbhb is present in bovine tissues in vivo and further confirmed that this epigenetic mark is responsive to BHB in bovine and human fibroblasts cultured in vitro in a dose-dependent manner. We also demonstrated that the maturation of cumulus-oocyte complexes with high concentrations of BHB did not affect the competence to complete meiotic maturation neither to develop until blastocyst stage. BHB treatment strongly induced H3K9bhb in cumulus cells, but this modification was only faintly detected in oocytes. Profiling the transcriptome in cumulus cells indicated that BHB treatment altered the expression of 345 genes. The down-regulated genes are mainly involved in glycolysis and ribosome assembly pathways, while the up-regulated genes are involved in mitochondrial metabolism and oocyte development. The specific genes and pathways altered by BHB treatment will provide entry points to carry out functional experiments aiming to mitigate problems and improve fertility in cattle suffering metabolic disorders. A key goal for future work will be to understand mechanistically how BHB transmits signals from the environment to affect cellular functions and the bovine epigenome.Summary sentenceBeta-hydroxybutyrate induces Histone Lysine β-hydroxybutyrylation in fibroblasts and cumulus-oocyte complexes, it alters the transcriptome in cumulus cells, but does not affect oocyte’s competence to resume meiosis and develop until blastocyst stage.

scholarly journals Visualization of microRNA-21 Dynamics in Neuroblastoma Cells Using Magnetic Resonance Imaging Based on a microRNA-21-responsive Reporter Gene

2021 ◽  
Author(s):  
Guangcheng Bao ◽  
Jun Sun ◽  
Jie Huang ◽  
Helin Zheng ◽  
Jie Wei ◽  
...  
Keyword(s):  
Magnetic Resonance Imaging ◽  
Magnetic Resonance ◽  
Reporter Gene ◽  
Dependent Manner ◽  
Resonance Imaging ◽  
Reporter System ◽  
Intracellular Iron ◽  
Responsive Gene

Abstract Background: MicroRNAs (miRs) have been shown to be closely associated with the occurrence and development of tumors and to have potential as diagnostic and therapeutic targets. The detection and quantification of miRs by noninvasive imaging technology is crucial for deeply understanding their biological functions. Our aim was to develop a novel miR-21-responsive gene reporter system for magnetic resonance imaging (MRI) visualization of the miR-21 dynamics in neuroblastoma.Methods: The reporter gene ferritin heavy chain (FTH1) was modified by the addition of 3 copies of the sequence completely complementary to miR-21 (3xC_miR-21) to its 3'-untranslated region (3' UTR) and transduced into SK-N-SH cells to obtain SK-N-SH/FTH1-3xC_miR-21 cells. Then, the antagomiR-21 was delivered into cells by graphene oxide functionalized with polyethylene glycol and dendrimer. Before and after antagomiR-21 delivery, FTH1 expression, MRI contrast and intracellular iron uptake were assayed in vitro and in vivo.Results: In the SK-N-SH/FTH1-3xC_miR-21 cells, FTH1 expression was in an “off” state due to the combination of intratumoral miR-21 with the 3' UTR of the reporter gene. AntagomiR-21 delivered into the cells bound to miR-21 and thereby released it from the 3' UTR of the reporter gene, thus “switching on” FTH1 expression in a dose-dependent manner. This phenomenon resulted in intracellular iron accumulation and allowed MRI detection in vitro and in vivo.Conclusions: MRI based on the miR-21-responsive gene reporter may be a potential method for visualization of the endogenous miR-21 dynamics.

scholarly journals MAP6-F Is a Temperature Sensor That Directly Binds to and Protects Microtubules from Cold-induced Depolymerization

2012 ◽  
Vol 287 (42) ◽  
pp. 35127-35138 ◽  
Author(s):  
Christian Delphin ◽  
Denis Bouvier ◽  
Maxime Seggio ◽  
Emilie Couriol ◽  
Yasmina Saoudi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Temperature Sensor ◽  
Cold Sensitivity ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Microtubule Network ◽  
Wide Range ◽  
In Cells

Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

scholarly journals Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer’s disease

2021 ◽  
Author(s):  
Wen-Dai Bao ◽  
Pei Pang ◽  
Xiao-Ting Zhou ◽  
Fan Hu ◽  
Wan Xiong ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Memory Impairment ◽  
Iron Homeostasis ◽  
Critical Role ◽  
Gene Set Enrichment Analysis ◽  
Molecular Characteristics ◽  
Intracellular Iron

AbstractIron homeostasis disturbance has been implicated in Alzheimer’s disease (AD), and excess iron exacerbates oxidative damage and cognitive defects. Ferroptosis is a nonapoptotic form of cell death dependent upon intracellular iron. However, the involvement of ferroptosis in the pathogenesis of AD remains elusive. Here, we report that ferroportin1 (Fpn), the only identified mammalian nonheme iron exporter, was downregulated in the brains of APPswe/PS1dE9 mice as an Alzheimer’s mouse model and Alzheimer’s patients. Genetic deletion of Fpn in principal neurons of the neocortex and hippocampus by breeding Fpnfl/fl mice with NEX-Cre mice led to AD-like hippocampal atrophy and memory deficits. Interestingly, the canonical morphological and molecular characteristics of ferroptosis were observed in both Fpnfl/fl/NEXcre and AD mice. Gene set enrichment analysis (GSEA) of ferroptosis-related RNA-seq data showed that the differentially expressed genes were highly enriched in gene sets associated with AD. Furthermore, administration of specific inhibitors of ferroptosis effectively reduced the neuronal death and memory impairments induced by Aβ aggregation in vitro and in vivo. In addition, restoring Fpn ameliorated ferroptosis and memory impairment in APPswe/PS1dE9 mice. Our study demonstrates the critical role of Fpn and ferroptosis in the progression of AD, thus provides promising therapeutic approaches for this disease.

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