scholarly journals HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation

2020 ◽  
Author(s):  
Soichiro Kumamoto ◽  
Atsuya Nishiyama ◽  
Yoshie Chiba ◽  
Ryota Miyashita ◽  
Chieko Konishi ◽  
...  
Keyword(s):  
Dna Ligase ◽  
Dependent Manner ◽  
Parp Activation ◽  
Okazaki Fragment ◽  
Free System ◽  
Adp Ribosylation ◽  
Okazaki Fragments ◽  
Egg Extracts ◽  
A Cell ◽  
Underlying Mechanisms

ABSTRACTDNA Ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1 which was recruited onto chromatins. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signalling promotes the recruitment of LIG3 on chromatins and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.

scholarly journals Monocyte dysfunction in patients with Gaucher disease: evidence for interference of glucocerebroside with superoxide generation [see comments]

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2646-2653 ◽  
Author(s):  
Y Liel ◽  
A Rudich ◽  
O Nagauker-Shriker ◽  
T Yermiyahu ◽  
R Levy
Keyword(s):  
Gaucher Disease ◽  
Reticuloendothelial System ◽  
Type I ◽  
Dependent Manner ◽  
Superoxide Generation ◽  
Free System ◽  
Nitroblue Tetrazolium Reduction ◽  
Nadph Oxidase Complex ◽  
A Cell ◽  
Nbt Reduction

Abstract Gaucher disease patients are occasionally affected by chronic or fulminant infections. Since Gaucher cells originate from tissue phagocytes, we studied the functional implications of glucocerbroside accumulation on phagocytes in Gaucher disease patients. Circulating monocytes and granulocytes from nine type I Gaucher disease patients, and matched controls, were studied. Evaluation of phagocytic activity included (1) maximal superoxide generation rates following stimulation by phorbol 12-myristate 13-acetate (PMA), opsonized zymosan (OZ), or formyl-methionyl-leucylphenylalanine (FMLP); (2) nitroblue tetrazolium reduction test (NBT); (3) chemotaxis toward FMLP; (4) phagocytosis of OZ particles; and (5) killing activity against Staphylococcus aureus. Superoxide generation in monocytes following PMA, OZ, and FMLP stimulation was significantly suppressed at 52% +/- 15%, 39% +/- 8%, and 51% +/- 11% of control, respectively. Superoxide generation in granulocytes was normal. NBT reduction, staphylococcal killing, and phagocytosis were also markedly decreased in monocytes, and normal in granulocytes. Mean chemotaxis rates were normal in both monocytes and granulocytes; however, decreased chemotactic rates were observed in some patients. The abnormality of superoxide generation could be reproduced in a dose- and time-dependent manner in normal circulating monocytes incubated with glucocerebroside. Superoxide generation in glucocerebroside-conditioned normal monocytes in a cell-free system showed normal superoxide generation, reflecting the integrity of the NADPH oxidase complex itself. These results demonstrate markedly compromised phagocytic functions in circulating monocytes in Gaucher disease patients. These abnormalities can be attributed to accumulation of glucocerebroside, since it could be reproduced in normal monocytes incubated with glucocerebroside. Similar abnormalities in Gaucher cells throughout the reticuloendothelial system could impair host defense, and may be of particular importance in the pathogenesis of osteomyelitis in Gaucher disease patients.

scholarly journals Ligand-dependent and -independent activation of the transcription factor γRF-1 in a cell-free system

1995 ◽  
Vol 310 (2) ◽  
pp. 461-467 ◽  
Author(s):  
C A Feghali ◽  
T M Wright
Keyword(s):  
Transcription Factor ◽  
Tyrosine Phosphatase ◽  
Cell Fractionation ◽  
Dependent Manner ◽  
Sodium Orthovanadate ◽  
Ifn Gamma ◽  
Free System ◽  
Cell Free System ◽  
A Cell

gamma RF-1 is a recently identified transcription factor induced by interferon-gamma (IFN-gamma) which binds to a unique palindromic enhancer, gamma RE-1, in the promoter of the mig gene. This paper describes the ligand-dependent and ligand-independent activation of gamma RF-1 in a cell-free system. gamma RF-1 activity was induced by IFN-gamma in a time-dependent manner from 5 to 60 min in lysates prepared from the human monocytic leukaemia line THP-1 and the human epidermoid carcinoma line A431. The activation of gamma RF-1 in vitro required both ATP and an inhibitor of tyrosine phosphatases (sodium orthovanadate or pervanadate). In the presence of limiting concentrations (micromolar) of ATP, activation was also dependent upon stimulation with IFN-gamma, whereas at millimolar concentrations of ATP, gamma RF-1 was activated by either sodium orthovanadate or pervanadate in the absence of ligand. Based on cell fractionation studies, both membrane and cytosol components were essential for activation of gamma RF-1 in vitro. Consistent with a role for one or more tyrosine kinases in the activation of gamma RF-1, its DNA binding activity was blocked by monoclonal anti-phosphotyrosine antibodies and by the tyrosine kinase inhibitors genistein, lavendustin A and herbimycin A. A comparison with recently described pathways of IFN-mediated transcription factor regulation indicates that the in vitro activation of gamma RF-1 is unique, requiring both membrane and cytosol fractions and inhibition of endogenous tyrosine phosphatase activity.

A Cell-Free Assay To Screen for Compounds That Modulate the Fanconi/BRCA Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4351-4351
Author(s):  
Stacie Stone ◽  
Alexandra Sobeck ◽  
Igor Landais ◽  
Alexis LaChapelle ◽  
Maureen E. Hoatlin
Keyword(s):  
Dna Damage ◽  
Cancer Susceptibility ◽  
Bone Marrow Failure ◽  
Current Model ◽  
S Phase ◽  
Mobility Shift ◽  
Western Blots ◽  
Free System ◽  
Egg Extracts ◽  
A Cell

Abstract Fanconi anemia is a multi-gene cancer susceptibility and bone marrow failure syndrome. In the current model, at least eight proteins (FANCA, -B-C,-E, -F, -G, -L, -M) are part of a nuclear complex that is required for the S phase and DNA-damage dependent monoubiquitination of FANCD2. This event is thought to functionally link the FA complex proteins to major breast cancer susceptibility proteins BRCA1, BRCA2 (FANCD1), and the BRCA1-associated helicase Brip1(FANCJ). An understanding of the function of the FA protein network is incomplete not only because some FA proteins are still unidentified, but also because the functions of individual proteins may be interdependent and are difficult to assess out of context with the entire FA network. We recently developed a cell-free system to evaluate the function of the Fanconi/BRCA pathway proteins in an S phase context in Xenopus egg extracts (Sobeck, et al. 2006). Egg extracts are naturally cell-cycle synchronized and mimic the complex interplay of proteins that support cellular DNA replication and regulated DNA damage checkpoint activation. Intricate protein interactions can be assayed in egg extracts, even without knowing each of the components if there is a measurable endpoint. We tested the hypothesis that the mobility shift of FANCD2 could be used as an endpoint in cell-free assays to determine FA pathway function. We found that an antibody specific for the Xenopus FANCD2 protein detected a single band of the expected size in western blots of proteins separated by SDS-PAGE from unstimulated egg extracts. Addition of DNA substrates to extracts resulted in the appearance of a slower mobility form of FANCD2, consistent with the monoubiquitinated FANCD2-L isoform observed in human cells following DNA damage. We measured inhibition or stimulation of xFANCD2-L in the presence of a series of candidate compounds. We found compounds that inhibit FANCD2-L, including curcumin, which was also identified in a cell-based assay as an inhibitor of FANCD2-L (Chirnomas, et al., 2006). Thus, this cell-free assay successfully mirrors the outcome obtained with a small molecule inhibitor of the FA/BRCA pathway in cell-based assays. This new approach is an improvement relative to cell-based screens because the extracts are fully synchronized, which maximizes the sensitivity of detection of S-phase events. Moreover, cell-free screens are rapid, inexpensive and well suited for semi- or high-throughput methods to identify small molecules that modulate the FA/BRCA DNA-damage response pathway.

scholarly journals The Traditional Medicinal Plants Cuphea calophylla, Tibouchina kingii, and Pseudelephantopus spiralis Attenuate Inflammatory and Oxidative Mediators

2018 ◽  
Vol 2018 ◽  
pp. 1-11
Author(s):  
Ana María Ramírez-Atehortúa ◽  
Lorena Morales-Agudelo ◽  
Edison Osorio ◽  
Oscar J. Lara-Guzmán
Keyword(s):  
Antioxidant Activities ◽  
Free System ◽  
Oxidative Markers ◽  
Aerial Parts ◽  
Traditional Medicinal Plants ◽  
A Cell ◽  
Anti Inflammatory ◽  
Underlying Mechanisms ◽  
Cox 1

Aerial parts of Cuphea calophylla, Tibouchina kingii, and Pseudelephantopus spiralis have been used in Colombian traditional medicine for inflammation. However, the underlying mechanisms that could explain the anti-inflammatory actions remain unknown. This study aimed to elucidate the anti-inflammatory and cytoprotective effects of hydroalcoholic extracts from C. calophylla (HECC), T. kingii (HETK), and P. spiralis (HEPS) in LPS-stimulated THP-1 macrophages. Reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA) were monitored as inflammatory and oxidative markers. The inhibition of lipoxygenase (LOX) and cyclooxygenase (COX) activities in a cell-free system were also investigated. Antioxidant activities were determined using standard in vitro methods. All extracts inhibited the NO, ROS, and MDA levels. HETK showed the highest ROS production inhibition and the highest antioxidant values, whereas HETK and HEPS significantly decreased the cytotoxicity mediated by LPS. The release of MDA was reduced significantly by all extracts. Moreover, the catalytic activity of LOX was inhibited by HECC and HETK. HECC was a more potent reducer of COX-2 activity. All extracts effectively suppressed COX-1 activity. In summary, these results suggest that HECC, HEPS, and HETK possess anti-inflammatory properties. Therefore, these plants could provide a valuable source of natural bioactive compounds for the treatment of inflammatory-related diseases.

scholarly journals Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Tomoyuki Iwasaki ◽  
Naoe Kaneko ◽  
Yuki Ito ◽  
Hiroyuki Takeda ◽  
Tatsuya Sawasaki ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Early Onset ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Blau Syndrome ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Early Onset Sarcoidosis

Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.

scholarly journals Cell cycle regulation of dynein association with membranes modulates microtubule-based organelle transport.

1996 ◽  
Vol 133 (3) ◽  
pp. 585-593 ◽  
Author(s):  
J Niclas ◽  
V J Allan ◽  
R D Vale
Keyword(s):  
Cell Cycle ◽  
Membrane Surface ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Microtubule Motor ◽  
A Cell ◽  
Cycle Dependent ◽  
Intermediate Chain

Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle-dependent manner, the dynein light intermediate chain incorporated approximately 12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycle-dependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes.

scholarly journals The fate of methylmercury through the formation of bismethylmercury sulfide as an intermediate in mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yumi Abiko ◽  
Yusuke Katayama ◽  
Wenyang Zhao ◽  
Sawako Horai ◽  
Kenji Sakurai ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Intraperitoneal Administration ◽  
Distal Colon ◽  
The Body ◽  
Gas Chromatography Mass Spectrometry ◽  
Dependent Manner ◽  
Free System ◽  
A Cell

AbstractA previous study by our group indicated that methylmercury (MeHg) is biotransformed to bismethylmercury sulfide [(MeHg)2S)] by interaction with reactive sulfur species (RSS) produced in the body. In the present study, we explored the transformation of MeHg to (MeHg)2S in the gut and the subsequent fate of (MeHg)2S in vitro and in vivo. An ex vivo experiment suggested the possibility of the extracellular transformation of MeHg to (MeHg)2S in the distal colon, and accordingly, the MeHg sulfur adduct was detected in the intestinal contents and feces of mice administered MeHg, suggesting that (MeHg)2S is formed through reactions between MeHg and RSS in the gut. In a cell-free system, we found that (MeHg)2S undergoes degradation in a time-dependent manner, resulting in the formation of mercury sulfide and dimethylmercury (DMeHg), as determined by X-ray diffraction and gas chromatography/mass spectrometry, respectively. We also identified DMeHg in the expiration after the intraperitoneal administration of (MeHg)2S to mice. Thus, our present study identified a new fate of MeHg through (MeHg)2S as an intermediate, which leads to conversion of volatile DMeHg in the body.

scholarly journals Spindle assembly in egg extracts of the Marsabit clawed frog, Xenopus borealis

2018 ◽  
Author(s):  
Maiko Kitaoka ◽  
Rebecca Heald ◽  
Romain Gibeaux
Keyword(s):  
Spindle Assembly ◽  
Morphometric Variation ◽  
Free System ◽  
Egg Extract ◽  
Egg Extracts ◽  
A Cell ◽  
Clawed Frog ◽  
Xenopus Borealis

ABSTRACTEgg extracts of the African clawed frog Xenopus laevis have provided a cell-free system instrumental in elucidating events of the cell cycle, including mechanisms of spindle assembly. Comparison with extracts from the diploid Western clawed frog, Xenopus tropicalis, which is smaller at the organism, cellular and subcellular levels, has enabled the identification of spindle size scaling factors. We set out to characterize the Marsabit clawed frog, Xenopus borealis, which is intermediate in size between the two species, but more recently diverged in evolution from X. laevis than X. tropicalis. X. borealis eggs were slightly smaller than those of X. laevis, and slightly smaller spindles were assembled in egg extracts. Interestingly, microtubule distribution across the length of the X. borealis spindles differed from both X. laevis and X. tropicalis. Extract mixing experiments revealed common scaling phenomena among Xenopus species, while characterization of spindle factors katanin, TPX2, and Ran indicate that X. borealis spindles possess both X. laevis and X. tropicalis features. Thus, X. borealis egg extract provides a third in vitro system to investigate interspecies scaling and spindle morphometric variation.

scholarly journals Monocyte dysfunction in patients with Gaucher disease: evidence for interference of glucocerebroside with superoxide generation [see comments]

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2646-2653
Author(s):  
Y Liel ◽  
A Rudich ◽  
O Nagauker-Shriker ◽  
T Yermiyahu ◽  
R Levy
Keyword(s):  
Gaucher Disease ◽  
Reticuloendothelial System ◽  
Type I ◽  
Dependent Manner ◽  
Superoxide Generation ◽  
Free System ◽  
Nitroblue Tetrazolium Reduction ◽  
Nadph Oxidase Complex ◽  
A Cell ◽  
Nbt Reduction

Gaucher disease patients are occasionally affected by chronic or fulminant infections. Since Gaucher cells originate from tissue phagocytes, we studied the functional implications of glucocerbroside accumulation on phagocytes in Gaucher disease patients. Circulating monocytes and granulocytes from nine type I Gaucher disease patients, and matched controls, were studied. Evaluation of phagocytic activity included (1) maximal superoxide generation rates following stimulation by phorbol 12-myristate 13-acetate (PMA), opsonized zymosan (OZ), or formyl-methionyl-leucylphenylalanine (FMLP); (2) nitroblue tetrazolium reduction test (NBT); (3) chemotaxis toward FMLP; (4) phagocytosis of OZ particles; and (5) killing activity against Staphylococcus aureus. Superoxide generation in monocytes following PMA, OZ, and FMLP stimulation was significantly suppressed at 52% +/- 15%, 39% +/- 8%, and 51% +/- 11% of control, respectively. Superoxide generation in granulocytes was normal. NBT reduction, staphylococcal killing, and phagocytosis were also markedly decreased in monocytes, and normal in granulocytes. Mean chemotaxis rates were normal in both monocytes and granulocytes; however, decreased chemotactic rates were observed in some patients. The abnormality of superoxide generation could be reproduced in a dose- and time-dependent manner in normal circulating monocytes incubated with glucocerebroside. Superoxide generation in glucocerebroside-conditioned normal monocytes in a cell-free system showed normal superoxide generation, reflecting the integrity of the NADPH oxidase complex itself. These results demonstrate markedly compromised phagocytic functions in circulating monocytes in Gaucher disease patients. These abnormalities can be attributed to accumulation of glucocerebroside, since it could be reproduced in normal monocytes incubated with glucocerebroside. Similar abnormalities in Gaucher cells throughout the reticuloendothelial system could impair host defense, and may be of particular importance in the pathogenesis of osteomyelitis in Gaucher disease patients.

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