Murine Ifit3 restricts the replication of Rabies virus both in vitro and in vivo

Rabies virus (RABV) infection can initiate the host immune defence response and induce an antiviral state characterized by the expression of interferon (IFN)-stimulated genes (ISGs), among which the family of genes of IFN-induced protein with tetratricopeptide repeats (Ifits) are prominent representatives. Herein, we demonstrated that the mRNA and protein levels of Ifit1, Ifit2 and Ifit3 were highly increased in cultured cells and mouse brains after RABV infection. Recombinant RABV expressing Ifit3, designated rRABV-Ifit3, displayed a lower pathogenicity than the parent RABV in C57BL/6 mice after intramuscular administration, and Ifit3-deficient mice exhibited higher susceptibility to RABV infection and higher mortality during RABV infection. Moreover, compared with their individual expressions, co-expression of Ifit2 and Ifit3 could more effectively inhibit RABV replication in vitro. These results indicate that murine Ifit3 plays an essential role in restricting the replication and reducing the pathogenicity of RABV. Ifit3 acts synergistically with Ifit2 to inhibit RABV replication, providing further insight into the function and complexity of the Ifit family.

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Role for ICAT in β-catenin-dependent nuclear signaling and cadherin functions

AJP Cell Physiology ◽

10.1152/ajpcell.00433.2003 ◽

2004 ◽

Vol 286 (4) ◽

pp. C747-C756 ◽

Cited By ~ 52

Author(s):

Cara J. Gottardi ◽

Barry M. Gumbiner

Keyword(s):

Cultured Cells ◽

Mdck Cells ◽

Primary Role ◽

Protein Levels ◽

Nuclear Signaling ◽

Endogenous Protein ◽

Hepatocyte Growth Factor Treatment

Inhibitor of β-catenin and TCF-4 (ICAT) is a 9-kDa polypeptide that inhibits β-catenin nuclear signaling by binding β-catenin and competing its interaction with the transcription factor TCF (T cell factor), but basic characterization of the endogenous protein and degree to which it alters other β-catenin functions is less well understood. At the subcellular level, we show that ICAT localizes to both cytoplasmic and nuclear compartments. In intestinal tissue, ICAT is upregulated in the mature, nondividing enterocyte population lining intestinal villi and is absent in the β-catenin/TCF signaling-active crypt region, suggesting that its protein levels may be inversely related with β-catenin signaling activity. However, ICAT protein levels are not altered by activation or inhibition of Wnt signaling in cultured cells, suggesting that ICAT expression is not a direct target of the Wnt/β-catenin pathway. In cells where β-catenin levels are elevated by Wnt, a fraction of this β-catenin pool is associated with ICAT, suggesting that ICAT may buffer the cell from increased levels of β-catenin. Distinct from TCF and cadherin, ICAT does not protect the soluble pool of β-catenin from degradation by the adenomatous polyposis coli containing “destruction complex.” Although ICAT inhibits β-catenin binding to the cadherin as well as TCF in vitro, stable overexpression of ICAT in Madin-Darby canine kidney (MDCK) epithelial cells shows no obvious alterations in the cadherin complex, suggesting that the ability of ICAT to inhibit β-catenin binding to the cadherin may be restricted in vivo. MDCK cells overexpressing ICAT do, however, exhibit enhanced cell scattering on hepatocyte growth factor treatment, suggesting a possible role in the regulation of dynamic rather than steady-state cell-cell adhesions. These findings confirm ICAT's primary role in β-catenin signaling inhibition and further suggest that ICAT may have consequences for cadherin-based adhesive function in certain circumstances, implying a broader role than previously described.

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Characterization of the small molecule ARC39, a direct and specific inhibitor of acid sphingomyelinase in vitro

The Journal of Lipid Research ◽

10.1194/jlr.ra120000682 ◽

2020 ◽

Vol 61 (6) ◽

pp. 896-910 ◽

Cited By ~ 5

Author(s):

Eyad Naser ◽

Stephanie Kadow ◽

Fabian Schumacher ◽

Zainelabdeen H. Mohamed ◽

Christian Kappe ◽

...

Keyword(s):

Cultured Cells ◽

Specific Inhibitor ◽

Acid Sphingomyelinase ◽

Intact Cells ◽

Protein Levels ◽

High Doses ◽

Hydrolysis Of

Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM’s catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.

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Curcumin induces paraoxonase 1 in cultured hepatocytes in vitro but not in mouse liver in vivo

British Journal Of Nutrition ◽

10.1017/s0007114510004356 ◽

2010 ◽

Vol 105 (2) ◽

pp. 167-170 ◽

Cited By ~ 17

Author(s):

Charlotte Schrader ◽

Christina Schiborr ◽

Jan Frank ◽

Gerald Rimbach

Keyword(s):

Cultured Cells ◽

Paraoxonase 1 ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Protein Levels ◽

Huh7 Cells ◽

Gene Assay ◽

B6c3f1 Mice

Paraoxonase 1 (PON1) is an enzyme that is mainly synthesised in the liver and protects LDL from oxidation, thereby exhibiting antiatherogenic properties. Using a luciferase reporter gene assay, we tested curcumin for its ability to induce PON1 in Huh7 hepatocytes in culture. Curcumin ( ≥ 10 μmol/l) dose-dependently induced PON1 transactivation in Huh7 cells. However, dietary supplementation of female B6C3F1 mice with curcumin (500 mg/kg diet) for 2 weeks did not increase the hepatic PON1 mRNA and protein levels. No curcumin was detectable in the plasma of the 12 h fasted mice. In conclusion, curcumin may be a potent PON1 inducer in cultured cells in vitro, but not in the liver of curcumin-fed mice because of its low concentrations in vivo.

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12-hydroxyheptadecatrienoic acid promotes epidermal wound healing by accelerating keratinocyte migration via the BLT2 receptor

Journal of Experimental Medicine ◽

10.1084/jem.20132063 ◽

2014 ◽

Vol 211 (6) ◽

pp. 1063-1078 ◽

Cited By ~ 70

Author(s):

Min Liu ◽

Kazuko Saeki ◽

Takehiko Matsunobu ◽

Toshiaki Okuno ◽

Tomoaki Koga ◽

...

Keyword(s):

Wound Healing ◽

Wound Closure ◽

Cultured Cells ◽

Leukotriene B4 ◽

Epidermal Keratinocytes ◽

Skin Wound Healing ◽

Wound Fluid ◽

Deficient Mice

Leukotriene B4 (LTB4) receptor type 2 (BLT2) is a G protein–coupled receptor (GPCR) for 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB4. Despite the well-defined proinflammatory roles of BLT1, the in vivo functions of BLT2 remain elusive. As mouse BLT2 is highly expressed in epidermal keratinocytes, we investigated the role of the 12-HHT/BLT2 axis in skin wound healing processes. 12-HHT accumulated in the wound fluid in mice, and BLT2-deficient mice exhibited impaired re-epithelialization and delayed wound closure after skin punching. Aspirin administration reduced 12-HHT production and resulted in delayed wound closure in wild-type mice, which was abrogated in BLT2-deficient mice. In vitro scratch assay using primary keratinocytes and a keratinocyte cell line also showed that the 12-HHT/BLT2 axis accelerated wound closure through the production of tumor necrosis factor α (TNF) and matrix metalloproteinases (MMPs). A synthetic BLT2 agonist accelerated wound closure in cultured cells as well as in C57BL/6J and diabetic mice. These results identify a novel mechanism underlying the action of the 12-HHT/BLT2 axis in epidermal keratinocytes and accordingly suggest the use of BLT2 agonists as therapeutic agents to accelerate wound healing, particularly for intractable wounds, such as diabetic ulcers.

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Targeted Deletion of Minpp1 Provides New Insight into the Activity of Multiple Inositol Polyphosphate Phosphatase In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.20.17.6496-6507.2000 ◽

2000 ◽

Vol 20 (17) ◽

pp. 6496-6507 ◽

Cited By ~ 41

Author(s):

Hongbo Chi ◽

Xiaonian Yang ◽

Paul D. Kingsley ◽

Regis J. O'Keefe ◽

J. Edward Puzas ◽

...

Keyword(s):

Enzyme Activity ◽

Cellular Proliferation ◽

Bone Development ◽

Physiological Activity ◽

Inositol Polyphosphate ◽

Aberrant Expression ◽

Deficient Mice ◽

Insight Into

ABSTRACT Multiple inositol polyphosphate phosphatase (Minpp1) metabolizes inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6) with high affinity in vitro. However, Minpp1 is compartmentalized in the endoplasmic reticulum (ER) lumen, where access of enzyme to these predominantly cytosolic substrates in vivo has not previously been demonstrated. To gain insight into the physiological activity of Minpp1,Minpp1-deficient mice were generated by homologous recombination. Tissue extracts from Minpp1-deficient mice lacked detectable Minpp1 mRNA expression and Minpp1 enzyme activity. Unexpectedly, Minpp1-deficient mice were viable, fertile, and without obvious defects. Although Minpp1 expression is upregulated during chondrocyte hypertrophy, normal chondrocyte differentiation and bone development were observed inMinpp1-deficient mice. Biochemical analyses demonstrate that InsP5 and InsP6 are in vivo substrates for ER-based Minpp1, as levels of these polyphosphates inMinpp1-deficient embryonic fibroblasts were 30 to 45% higher than in wild-type cells. This increase was reversed by reintroducing exogenous Minpp1 into the ER. Thus, ER-based Minpp1 plays a significant role in the maintenance of steady-state levels of InsP5 and InsP6. These polyphosphates could be reduced below their natural levels by aberrant expression in the cytosol of a truncated Minpp1 lacking its ER-targeting N terminus. This was accompanied by slowed cellular proliferation, indicating that maintenance of cellular InsP5 and InsP6 is essential to normal cell growth. Yet, depletion of cellular inositol polyphosphates during erythropoiesis emerges as an additional physiological activity of Minpp1; loss of this enzyme activity in erythrocytes from Minpp1-deficient mice was accompanied by upregulation of a novel, substitutive inositol polyphosphate phosphatase.

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Differential regulation of the retinoblastoma family of proteins during cell proliferation and differentiation

Biochemical Journal ◽

10.1042/bj3330645 ◽

1998 ◽

Vol 333 (3) ◽

pp. 645-654 ◽

Cited By ~ 46

Author(s):

Judit GARRIGA ◽

Ana LIMÓN ◽

Xavier MAYOL ◽

Sushil G. RANE ◽

Jeffrey H. ALBRECHT ◽

...

Keyword(s):

Cell Proliferation ◽

Protein Expression ◽

Cultured Cells ◽

Protein Levels ◽

Pocket Protein ◽

Cell Proliferation And Differentiation ◽

Mouse Tissues ◽

Proliferation And Differentiation

In the present study we have analysed the regulation of pocket protein expression and post-transcriptional modifications on cell proliferation and differentiation, both in vivo and in vitro. There are marked changes in pocket protein levels during these transitions, the most striking differences being observed between p130 and p107. The mechanisms responsible for regulating pocket protein levels seem to be dependent on both cell type and pocket protein, in addition to their dependence on the cell growth status. Changes in retinoblastoma protein and p107 levels are independent of their state of phosphorylation. However, whereas p130 phosphorylation to forms characteristic of quiescent/differentiated cells results in the accumulation of p130 protein, phosphorylation of p130 to one or more forms characteristic of cycling cells is accompanied by down-regulation of its protein levels. We also show here that the phosphorylation status and protein levels of p130 and p107 are regulated in vivo as in cultured cells. In vivo, changes in p130 forms are correlated with changes in E2F complexes. Moreover, the modulation of p130 and p107 status during cell differentiation in vitro is consistent with the patterns of protein expression and phosphorylation status found in mouse tissues. Thus in addition to the direct disruption of pocket protein/E2F complexes induced by cyclin/cyclin-dependent kinase, the results we report here indicate that the differential modulation of pocket protein levels constitutes a major mechanism that regulates the pool of each pocket protein that is accessible to E2F and/or other transcription factors.

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Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100053711 ◽

1982 ◽

Vol 40 ◽

pp. 210-211

Author(s):

M.J. Murphy ◽

R.R. Price ◽

J.C. Sloman

Keyword(s):

Cultured Cells ◽

Human Tumor ◽

Cell Type ◽

Tumor Mass ◽

Initial Cell ◽

Cloning Assay ◽

Human Tumor Cloning ◽

The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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Preparation of cultured astrocytes for x-ray microanalysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156924 ◽

1989 ◽

Vol 47 ◽

pp. 988-989

Author(s):

N.K.R. Smith ◽

K.E. Hunter ◽

P. Mobley ◽

L.P. Felpel

Keyword(s):

Cultured Cells ◽

Elemental Content ◽

Elemental Distribution ◽

Sprague Dawley ◽

X Ray ◽

Cultured Astrocytes ◽

Freeze Dried ◽

Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651541 ◽

1993 ◽

Vol 69 (01) ◽

pp. 021-024 ◽

Cited By ~ 20

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.

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