PKCalpha mediates acetylcholine-induced activation of TRPV4-dependent calcium influx in endothelial cells.
Acetylcholine/*pharmacology; Animals; Calcium Signaling/*drug effects; Carbazoles/pharmacology; Cells; Cultured; Endothelial Cells/*drug effects/enzymology; Enzyme Activation; Inbred C57BL; Knockout; Male; Mesenteric Arteries/drug effects/enzymology; Mice; Mutation; Nitric Oxide Synthase Type III/metabolism; Phosphorylation; Protein Kinase C-alpha/genetics/*metabolism; Protein Kinase Inhibitors/pharmacology; Tetradecanoylphorbol Acetate/pharmacology; Time Factors; Transfection; TRPV Cation Channels/*agonists/deficiency/genetics/metabolism; Vasodilation/*drug effects; Vasodilator Agents/*pharmacology
Transient receptor potential vanilloid channel 4 (TRPV4) is a polymodally activated nonselective cationic channel implicated in the regulation of vasodilation and hypertension. We and others have recently shown that cyclic stretch and shear stress activate TRPV4-mediated calcium influx in endothelial cells (EC). In addition to the mechanical forces, acetylcholine (ACh) was shown to activate TRPV4-mediated calcium influx in endothelial cells, which is important for nitric oxide-dependent vasodilation. However, the molecular mechanism through which ACh activates TRPV4 is not known. Here, we show that ACh-induced calcium influx and endothelial nitric oxide synthase (eNOS) phosphorylation but not calcium release from intracellular stores is inhibited by a specific TRPV4 antagonist, AB-159908. Importantly, activation of store-operated calcium influx was not altered in the TRPV4 null EC, suggesting that
Adapala Ravi K; Talasila Phani K; Bratz Ian N; Zhang David X; Suzuki Makoto; Meszaros J Gary; Thodeti Charles K
American journal of physiology. Heart and circulatory physiology
2011
2011-09
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajpheart.00142.2011" target="_blank" rel="noreferrer noopener">10.1152/ajpheart.00142.2011</a>
MicroRNA-602 and microRNA-608 regulate sonic hedgehog expression via target sites in the coding region in human chondrocytes.
Academic Medical Centers; Aged; Analysis of Variance; Animal; Animals; Anterior Cruciate Ligament/surgery; Blotting; Cells; Chondrocytes – Physiology; Chondrocytes/drug effects/*metabolism/pathology; Cultured; Disease Models; Female; Funding Source; Gene Expression Regulation/genetics/*physiology; Hedgehog Proteins/genetics/*metabolism; HEK293 Cells; Human; Humans; Immunohistochemistry; In Vitro Techniques; Interleukin-1beta/pharmacology; Knee/etiology/*metabolism/pathology; Male; Matrix Metalloproteinase 13/metabolism; MicroRNAs/genetics/*metabolism; Middle Age; Middle Aged; Ohio; Open Reading Frames/genetics/*physiology; Osteoarthritis; Osteoarthritis – Epidemiology; Osteoarthritis – Physiopathology; Polymerase Chain Reaction; Rabbits; Signal Transduction/genetics/physiology; T-Tests; Transfection; Up-Regulation/drug effects/genetics/physiology; Western
OBJECTIVE: Hedgehog (HH) signaling has recently been associated with cartilage degradation in osteoarthritis (OA). Because interleukin-1beta (IL-1beta) has been implicated as a principal instigator of OA, we sought to determine whether
Akhtar Nahid; Makki Mohammad S; Haqqi Tariq M
Arthritis & rheumatology (Hoboken, N.J.)
2015
2015-02
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/art.38952" target="_blank" rel="noreferrer noopener">10.1002/art.38952</a>
Butein Activates Autophagy Through AMPK/TSC2/ULK1/mTOR Pathway to Inhibit IL-6 Expression in IL-1beta Stimulated Human Chondrocytes.
AMP-Activated Protein Kinases/metabolism; AMPK; Articular/cytology/pathology; Autophagy; Autophagy-Related Protein 5/antagonists & inhibitors/genetics/metabolism; Autophagy-Related Protein-1 Homolog/metabolism; Autophagy/*drug effects; Butein; Cartilage; Cell Survival/drug effects; Cells; Chalcones/*pharmacology; Chondrocytes/cytology/drug effects/metabolism; Cultured; Humans; Inflammation; Interleukin-1beta/*pharmacology; Interleukin-6/genetics/*metabolism; Intracellular Signaling Peptides and Proteins/metabolism; mTOR; Osteoarthritis; Osteoarthritis/metabolism/pathology; Phosphorylation/drug effects; Reactive Oxygen Species/metabolism; RNA; RNA Interference; Signal Transduction/*drug effects; Small Interfering/metabolism; TOR Serine-Threonine Kinases/metabolism; TSC2; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins/metabolism; ULK1
BACKGROUND/AIMS: Butein (2',3,4,4'-Tetrahydroxychalcone), a polyphenol produced by several plants including Butea monoserpma, has been reported to exert potent anti-inflammatory activity but the mechanism remains unknown. In the present work we investigated the mechanism of Butein-mediated suppression of IL-6 expression in normal and human osteoarthritis (OA) chondrocytes under pathological conditions. METHODS: Expression level of interleukin-6 (IL-6) protein in OA cartilage was analyzed by immunohistochemistry using a validated antibody. Chondrocytes derived from normal or OA cartilage by enzymatic digestion were pretreated with Butein followed by stimulation with interleukin-1beta (IL-1beta) and the levels of IL-6 mRNA were quantified by TaqMan assay and the protein levels were measured by Western immunoblotting. Autophagy activation was determined by Western blotting and confocal microscopy. Autophagy was inhibited by siRNA mediated knockdown of ATG5. RESULTS: Expression of IL-6 protein was high in the OA cartilage compared to smooth cartilage from the same patient. OA chondrocytes and cartilage explants stimulated with IL-1beta showed high level expression of IL-6 mRNA and protein. Butein increased the phosphorylation of AMPKalphaThr-172, TSC2Ser-1387 and ULK1Ser-317 and inhibited the phosphorylation of mTORSer-2448 and its downstream target p70S6K and increased autophagy flux that correlated with the suppression of the IL-1beta mediated expression of IL-6 in normal and OA chondrocytes. In OA chondrocytes with siRNA-mediated knockdown of ATG5 expression, treatment with Butein failed to activate autophagy and abrogated the suppression of IL-1beta induced IL-6 expression. CONCLUSION: Our findings demonstrate for the first time that Butein activate autophagy in OA chondrocytes via AMPK/TSC2/ULK1/mTOR pathway. Additionally, activation of autophagy was essential to block the IL-1beta-induced expression of IL-6 in OA chondrocytes. These data support further studies to evaluate the use of Butein or compounds derived from it for the management of OA.
Ansari Mohammad Y; Ahmad Nashrah; Haqqi Tariq M
Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
2018
2018
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1159/000493225" target="_blank" rel="noreferrer noopener">10.1159/000493225</a>
Interleukin-1beta induced Stress Granules Sequester COX-2 mRNA and Regulates its Stability and Translation in Human OA Chondrocytes.
*Protein Biosynthesis; Cells; Chondrocytes/drug effects/metabolism/pathology; Cultured; Cyclooxygenase 2/*genetics; Dinoprostone/genetics; Fluorescence; Gene Expression Regulation; Humans; Immunoprecipitation; In Situ Hybridization; Interleukin-1beta/administration & dosage/*genetics; Messenger/genetics; Nitric Oxide/metabolism; Osteoarthritis/drug therapy/*genetics/pathology; RNA
Enhanced and immediate expression of cyclooxygenase-2 (COX-2) mRNA is observed in
Ansari Mohammad Y; Haqqi Tariq M
Scientific reports
2016
2016-06
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1038/srep27611" target="_blank" rel="noreferrer noopener">10.1038/srep27611</a>
Methods and insights from the characterization of osteoprogenitor cells of bats (Mammalia: Chiroptera).
Animals; Bone Marrow Cells/cytology; Cell Differentiation/genetics; Cell Proliferation; Cells; Cellular Reprogramming; Chiroptera; Core Binding Factor Alpha 1 Subunit/genetics/metabolism; Cultured; Inbred C57BL; Mice; Osteoblasts/cytology/*metabolism; Osteocalcin/genetics/metabolism; Osteogenesis/*genetics; Real-Time Polymerase Chain Reaction; Stem Cells/*cytology/metabolism; Transcription Factors/genetics/metabolism
Osteoprogenitor cells contribute to the development and maintenance of skeletal tissues. Bats are unique model taxa whose cellular processes are poorly understood, especially in regards to skeletal biology. Forelimb bones of bats, unlike those of terrestrial mammals, bend during flight and function in controlled deformation. As a first step towards understanding the molecular processes governing deposition of this flexible bone matrix, we provide the first method for isolation and differentiation of cell populations derived from the bone marrow and cortical bone of bats, and compare results with those harvested from C57BL/6J mice. Osteogenic capacity of these cells was assessed via absolute quantitative real-time PCR (qPCR) and through quantification of in vitro mineral deposition. Results indicate the differentiated bone cells of bats display significantly lower gene expression of known osteogenic markers (Runt-related transcription factor (RUNX2), osteocalcin (BGLAP) and osterix (SP7)), and deposit a less-mineralized matrix compared with murine controls. By characterizing the in vitro performance of osteoprogenitor cells throughout differentiation and matrix production, this study lays the ground work for in vitro manipulations of bat stem and osteoprogenitor cells and extends our understanding of the cellular diversity across mammals that occupy different habitats.
Ball H C; Moussa F M; Mbimba T; Orman R; Safadi F F; Cooper L N
Stem cell research
2016
2016-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.scr.2016.05.009" target="_blank" rel="noreferrer noopener">10.1016/j.scr.2016.05.009</a>
Upregulation of thrombospondin-1 expression by leptin in vascular smooth muscle cells via JAK2- and MAPK-dependent pathways.
Animals; Cell Movement/physiology; Cells; Cultured; Gene Expression Regulation/physiology; Humans; Inbred C57BL; Janus Kinase 2/*biosynthesis; Knockout; Leptin/*physiology; Male; MAP Kinase Signaling System/*physiology; Mice; Muscle; Myocytes; Smooth; Smooth Muscle/enzymology/metabolism; Thrombospondin 1/*biosynthesis; Up-Regulation/*physiology; Vascular/enzymology/*metabolism
Hyperleptinemia, characteristic of diabetes and a hallmark feature of human obesity, contributes to the increased risk of atherosclerotic complications. However, molecular mechanisms mediating leptin-induced atherogenesis and gene expression in vascular cells remain incompletely understood. Accumulating evidence documents a critical role of a potent antiangiogenic and proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in atherosclerosis. Although previous studies reported elevated TSP-1 levels in both diabetic and obese patients and rodent models, there is no direct information on TSP-1 expression in vascular cells in response to leptin. In the present study, we show that leptin upregulates TSP-1 expression in cultured human aortic smooth muscle cells (HASMC) in vitro, and this increase occurs at the level of transcription, revealed by mRNA stability and TSP-1 promoter-reporter assays. Utilizing specific pharmacological inhibitors and siRNA approaches, we demonstrate that upregulation of TSP-1 expression by leptin is mediated by JAK2/ERK/JNK-dependent mechanisms. Furthermore, we report that while ERK and JNK are required for both the constitutive and leptin-induced expression of TSP-1, JAK-2 appears to be specifically involved in leptin-mediated TSP-1 upregulation. Finally, we found that increased HASMC migration and proliferation in response to leptin is significantly inhibited by a TSP-1 blocking antibody, thereby revealing the physiological significance of leptin-TSP-1 crosstalk. Taken together, these findings demonstrate, for the first time, that leptin has a direct regulatory effect on TSP-1 expression in HASMCs, underscoring a novel role of TSP-1 in hyperleptinemia-induced atherosclerotic complications.
Chavez Ronaldo J; Haney Rebecca M; Cuadra Rene H; Ganguly Rituparna; Adapala Ravi K; Thodeti Charles K; Raman Priya
American journal of physiology. Cell physiology
2012
2012-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajpcell.00008.2012" target="_blank" rel="noreferrer noopener">10.1152/ajpcell.00008.2012</a>
Nuclear receptor-mediated repression of human cholesterol 7alpha-hydroxylase gene transcription by bile acids.
Humans; Animals; Rats; Cell Line; Transfection; Liver/metabolism; Reverse Transcriptase Polymerase Chain Reaction; DNA/metabolism; CHO Cells; Cricetinae; Cholesterol 7-alpha-Hydroxylase/*genetics; Bile Acids and Salts/*pharmacology; *Membrane Glycoproteins; *Hydroxysteroid Dehydrogenases; Caco-2 Cells; Carrier Proteins/genetics/physiology; DNA-Binding Proteins/drug effects/genetics/physiology; Gene Expression/*drug effects; Kidney; Luciferases/genetics; Recombinant Fusion Proteins/metabolism; Retinoid X Receptors; Taurocholic Acid/pharmacology; Transcription Factors/drug effects/genetics/physiology; Cultured; Receptors; RNA; Genetic/drug effects; Messenger/analysis; Transcription; Genetic; Tumor Cells; Promoter Regions; Embryo; Cytoplasmic and Nuclear/genetics/*physiology; Mammalian; Retinoic Acid/genetics/physiology
Hydrophobic bile acids strongly repressed transcription of the human cholesterol 7alpha-hydroxylase gene (CYP7A1) in the bile acid biosynthetic pathway in the liver. Farnesoid X receptor (FXR) repressed CYP7A1/Luc reporter activity in a transfection assay in human liver-derived HepG2 cells, but not in human embryonic kidney (HEK) 293 cells. FXR-binding activity was required for bile acid repression of CYP7A1 transcription despite the fact that FXR did not bind to the CYP7A1 promoter. FXR-induced liver-specific factors must be required for mediating bile acid repression. Bile acids and FXR repressed endogenous CYP7A1 but stimulated alpha-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) mRNA expression in HepG2 cells. Feeding of rats with chenodeoxycholic acid repressed CYP7A1, induced FTF, but had no effect on SHP mRNA expression in the liver. FTF strongly repressed CYP7A1 transcription in a dose-dependent manner, and SHP further inhibited CYP7A1 in HepG2 cells, but not in HEK 293 cells. FXR only moderately stimulated SHP transcription, whereas FTF strongly inhibited SHP transcription in HepG2 cells. Results revealed that FTF was a dominant negative factor that was induced by bile acid-activated FXR to inhibit both CYP7A1 and SHP transcription. Differential regulation of FTF and SHP expression by bile acids may explain the wide variation in CYP7A1 expression and the rate of bile acid synthesis and regulation in different species.
Chen W; Owsley E; Yang Y; Stroup D; Chiang J Y
Journal of lipid research
2001
2001-09
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Regulation of human sterol 27-hydroxylase gene (CYP27A1) by bile acids and hepatocyte nuclear factor 4alpha (HNF4alpha).
Humans; Cell Line; Transfection; Gene Expression Regulation/drug effects; Base Sequence; Binding Sites/genetics; Response Elements/genetics; Molecular Sequence Data; Mutation; Chenodeoxycholic Acid/pharmacology; Transcription Factors/genetics/*metabolism; Hepatocyte Nuclear Factor 4; Mutagenesis; *DNA-Binding Proteins; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Bile Acids and Salts/*pharmacology; Cholestanetriol 26-Monooxygenase; DNA/chemistry/genetics; Luciferases/genetics/metabolism; Phosphoproteins/genetics/*metabolism; Recombinant Fusion Proteins/genetics/metabolism; Steroid Hydroxylases/*genetics; DNA; Dose-Response Relationship; Drug; Cultured; Receptors; Tumor Cells; Cloning; Molecular; Sequence Analysis; Promoter Regions; Genetic/*genetics; Cytoplasmic and Nuclear/genetics/metabolism; Site-Directed
Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyses sterol side-chain oxidation of bile acid synthesis from cholesterol, and the first reaction of the acidic bile acid biosynthetic pathway. Hydrophobic bile acids suppress human CYP27A1 gene reporter activity when assayed in human hepatocellular blastoma HepG2 cells. Bile acids also inhibit CYP27A1 reporter activity in human embryonic kidney 293 cells. A putative bile acid response element (BARE) was mapped to a region downstream of nt -147 of the human CYP27A1 gene, within which a binding site for a liver-specific nuclear receptor, HNF4alpha, is identified. HNF4alpha strongly stimulates CYP27A1 gene transcription and mutation of its binding site markedly reduced promoter activity. Results suggest that human CYP27A1 gene transcription is suppressed by bile acids and HNF4alpha plays a pivotal role in transcriptional regulation of this gene.
Chen Wenling; Chiang John Y L
Gene
2003
2003-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Regulation of cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription by the liver orphan receptor (LXRalpha).
Humans; Animals; Binding Sites; Rats; Species Specificity; Transfection; Gene Expression Regulation/drug effects; Organ Specificity; Transcription Factors/genetics/metabolism; Cricetinae; Response Elements; Luciferases/genetics/metabolism; Retinoid X Receptors; Cholesterol 7-alpha-Hydroxylase/drug effects/*genetics/metabolism; Hydroxycholesterols; Liver/physiology; Lovastatin/pharmacology; Mevalonic Acid/metabolism/pharmacology; Nicotinic Acids/pharmacology; Polyisoprenyl Phosphates/pharmacology; Tetrahydronaphthalenes/pharmacology; Cells; Cultured; Receptors; Transcription; Genetic; Retinoic Acid/genetics/metabolism; Steroid/genetics/*metabolism
The cholesterol 7alpha-hydroxylase gene (CYP7A1) plays an important role in regulation of bile acid biosynthesis and cholesterol homeostasis. Oxysterol receptor, LXR, stimulates, whereas the bile acid receptor, FXR, inhibits CYP7A1 transcription. The goal of this study was to investigate the role of LXRalpha on the regulation of rat, human and hamster CYP7A1 transcription in its native promoter and cellular context. Cotransfection with LXRalpha and RXRalpha expression plasmids strongly stimulated rat CYP7A1/luciferase reporter activity in HepG2 cells and oxysterol was not required. However, LXRalpha had much less effect on hamster and no significant effect on human CYP7A1 promoter activity in HepG2 cells. In Chinese hamster ovary cells, cotransfection with LXRalpha stimulated reporter activity by less than 2-fold and addition of 22(R)-hydroxycholesterol caused a small but significant stimulation of rat, human and hamster CYP7A1 promoter activity. At least two direct repeats of AGGTCA-like sequences with 4-base spacing (DR4) and five-base spacing (DR5), in previously identified bile acid response elements of the rat CYP7A1 were able to bind LXRalpha/RXRalpha and confer LXRalpha stimulation. However, LXRalpha did not bind to the corresponding sequences of the human gene and bound weakly to hamster and mouse DR4 sequences. Therefore, rats and mice have the unusual capacity to convert cholesterol to bile acids by LXRalpha-mediated stimulation of CYP7A1 transcription, whereas other species do not respond to cholesterol and develop hypercholesterolemia on a diet high in cholesterol.
Chiang J Y; Kimmel R; Stroup D
Gene
2001
2001-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Farnesoid X receptor responds to bile acids and represses cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription.
Humans; Animals; Cricetinae; Cholesterol 7-alpha-Hydroxylase/*genetics; Response Elements; Bile Acids and Salts/*pharmacology; Transcription Factors/genetics/*physiology; Retinoid X Receptors; DNA-Binding Proteins/*physiology; Repressor Proteins/*physiology; Cultured; Receptors; Genetic; Tumor Cells; Promoter Regions; *Transcription; Cytoplasmic and Nuclear; Retinoic Acid/genetics
Cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription is repressed by bile acids. The goal of this study is to elucidate the mechanism of CYP7A1 transcription by bile acid-activated farnesoid X receptor (FXR) in its native promoter and cellular context and to identify FXR response elements in the gene. In Chinese hamster ovary cells transfected with retinoid X receptor alpha (RXRalpha)/FXR, only chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) were able to stimulate a heterologous promoter/reporter containing an ecdysone response element. In HepG2 cells, all bile acids (25 microM) were able to repress CYP7A1/luciferase reporter activity, and only CDCA and DCA further repressed reporter activity when cotransfected with RXRalpha/FXR. The concentration of CDCA required to inhibit 50% of reporter activity (IC(50)) was determined to be approximately 25 microM without FXR and 10 microM with FXR. Deletion analysis revealed that the bile acid response element located between nucleotides -148 and -128 was the FXR response element, but RXRalpha/FXR did not bind to this sequence. These results suggest that bile acid-activated FXR exerts its inhibitory effect on CYP7A1 transcription by an indirect mechanism, in contrast to the stimulation and binding of FXR to intestinal bile acid-binding protein gene promoter. Results also reveal that bile acid receptors other than FXR are present in HepG2 cells.
Chiang J Y; Kimmel R; Weinberger C; Stroup D
The Journal of biological chemistry
2000
2000-04
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Inhibition of human papillomavirus type 16 gene expression by nordihydroguaiaretic acid plant lignan derivatives.
Antiviral Agents/*pharmacology; Cultured; Female; Gene Expression Regulation; Genetic/drug effects; HIV Long Terminal Repeat/drug effects; Humans; Lignans/chemistry/pharmacology; Masoprocol/*analogs & derivatives/*pharmacology; Papillomaviridae/*genetics; Promoter Regions; Simian virus 40/genetics; Tumor Cells; Viral/*drug effects
Several methylated derivatives of a plant lignan, nordihydroguaiaretic acid (NDGA) were found to be potent anti-viral agents by suppressing Sp1 regulated transcription within the sexually transmitted viruses human immunodeficiency virus (HIV) and herpes simplex virus (HSV). A prominent Sp1 DNA binding site within many human papillomavirus (HPV) promoters has been noted to play an active role in HPV gene expression. In this report it is shown that the three NDGA derivatives, Mal.4, M(4)N, and tetra-acetyl NDGA can also inhibit gene expression from the early promoter P(97) of HPV16. The drug activity on gene expression was measured after DNA transfection of recombinant vector constructs linking the viral promoter and enhancer elements to the luciferase reporter gene. Using the specific luciferase activity as the indicator of gene expression, Mal.4 and M(4)N were found to be active in a dose dependent manner that is in the same range of concentrations reported for the promoters of HIV, HSV, and simian virus 40 (SV40) while tetra-acetyl NDGA was much more active in suppression of the HPV P(97) promoter activity than Mal.4 and M(4)N. The drugs showed limited to no effect on gene expression driven by the adenovirus major late promoter and the cytomegalovirus (CMV) promoter. Hence, such drug derivatives may be significant in the therapy of papillomavirus infections and their associated induced human cancers.
Craigo J; Callahan M; Huang R C; DeLucia A L
Antiviral research
2000
2000-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/s0166-3542(00)00089-9" target="_blank" rel="noreferrer noopener">10.1016/s0166-3542(00)00089-9</a>
Effects of bile acids and steroid/thyroid hormones on the expression of cholesterol 7 alpha-hydroxylase mRNA and the CYP7 gene in HepG2 cells.
Animals; Bile Acids and Salts/*pharmacology; Cells; Cholesterol 7-alpha-Hydroxylase/*genetics; Cultured; Enzymologic/*drug effects; Gene Expression Regulation; Genetic; Genetic/genetics; Humans; Luciferases/biosynthesis/genetics; Messenger/*biosynthesis; Promoter Regions; Rats; Recombinant Fusion Proteins/biosynthesis; RNA; Steroids/*pharmacology; Thyroid Hormones/*pharmacology; Transcription; Transfection
The expression of cholesterol 7 alpha-hydroxylase mRNA levels in confluent HepG2 cultures was reduced by tauro- or glyco-conjugates of deoxycholate and chenodeoxycholate, but not by cholate. Ursodeoxycholates, on the other hand, stimulated the mRNA level. The 5'-upstream regions of rat cholesterol 7 alpha-hydroxylase gene (CYP7) were fused to luciferase reporter gene and the constructs, p-3616/Luc, p-224/Luc and p-160/Luc, were transiently transfected into HepG2 cells. Tauro-conjugates of deoxycholate and chenodeoxycholate inhibited the transcriptional activities of the gene constructs in the confluent cells, but not in subconfluent cells. These results reveal that bile acid responsive elements are located in the -160 fragment and also between nt -3616 and -224. Thyroid and steroid hormones stimulated transcriptional activity expressed in the confluent cells and their responsive elements are located upstream of nt -224. It appears that adult phenotypes are responsible for bile acid feedback and hormone response in HepG2 cells.
Crestani M; Karam W G; Chiang J Y
Biochemical and biophysical research communications
1994
1994-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1006/bbrc.1994.1080" target="_blank" rel="noreferrer noopener">10.1006/bbrc.1994.1080</a>
Transcriptional activation of the cholesterol 7alpha-hydroxylase gene (CYP7A) by nuclear hormone receptors.
Animals; Rats; Transcription Factors/metabolism; Base Sequence; Molecular Sequence Data; DNA/metabolism; Cholesterol 7-alpha-Hydroxylase/*genetics; Hepatocyte Nuclear Factor 4; Mutagenesis; Luciferases/genetics; Retinoid X Receptors; Phosphoproteins/metabolism; COUP Transcription Factor II; COUP Transcription Factors; *Transcriptional Activation; Bile Acids and Salts/biosynthesis; DNA-Binding Proteins/metabolism; Hormones/*physiology; Oligonucleotide Probes/metabolism; Genes; Cultured; Receptors; Genetic; Cytoplasmic and Nuclear/*physiology; Tumor Cells; Reporter; Retinoic Acid/metabolism; Promoter Regions; Nucleic Acid; Site-Directed; *Receptors; Repetitive Sequences; Steroid
The gene encoding cholesterol 7alpha-hydroxylase (CYP7A), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene. The BARE II is located in nt -149/-118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 (-146- TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 (-139-AGTTCAaggccGGG TAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4)1, binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer. A hepatocyte nuclear factor-3 (HNF-3) binding site (-175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid. Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription.
Crestani M; Sadeghpour A; Stroup D; Galli G; Chiang J Y
Journal of lipid research
1998
1998-11
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Hormonal regulation of the cholesterol 7 alpha-hydroxylase gene (CYP7).
Animals; Rats; Gene Expression Regulation; Cell Count; Transfection; Base Sequence; Molecular Sequence Data; Phorbol Esters/pharmacology; Cholesterol 7-alpha-Hydroxylase/*genetics; Luciferases/genetics; Cyclic AMP/pharmacology; Hormones/*pharmacology; Second Messenger Systems/physiology; Cultured; Genetic; Tumor Cells; Cloning; Molecular; *Promoter Regions; *Transcription; Enzymologic/*physiology
The transcriptional regulation of the rat cholesterol 7 alpha-hydroxylase gene (CYP7) by hormones and signal transduction pathways was studied by transient transfection assay of the promoter activity. HepG2 cells were transfected with deletion mutants of the CYP7 upstream region linked to the luciferase reporter gene. The transcription of CYP7/luciferase chimeric genes was higher in confluent than in subconfluent cultures of HepG2 cells. Glucocorticoid receptors, in the presence of dexamethasone, up-regulated the CYP7 gene through two regions located between -3262 and -2803, and between -344 and -222, respectively. Thyroid hormones did not have any effect on the promoter activity. Insulin inhibited the promoter activity through sequences located between -344 and -222, and abolished the stimulation by dexamethasone. Hence, the insulin effect was dominant over that of glucocorticoids. Treatment of transfected HepG2 cells with phorbol
Crestani M; Stroup D; Chiang J Y
Journal of lipid research
1995
1995-11
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Pertubation of beta1 integrin function using anti-sense or function-blocking antibodies on corneal cells grown on fibronectin and tenascin.
Animals; Antisense/*pharmacology; Cattle; Cell Adhesion; Cell Culture Techniques/*methods; Cells; Chick Embryo; Chickens; Complementary/metabolism; Cornea/*cytology; Cultured; DNA; Dose-Response Relationship; Drug; Fibronectins/*metabolism; Fluorescence; Immunohistochemistry; Integrin beta1/*metabolism/*physiology; Integrins/metabolism; Microscopy; Oligonucleotides; Protein Binding; Retroviridae/genetics; Tenascin/*metabolism
During corneal development, neural crest derivatives from the periocular mesenchyme migrate into the cornea and differentiate into corneal fibroblasts. During this time, these cells interact with a variety of extracellular matrices for proper orientation and development. In the present studies, we have examined the interaction of beta(1) integrins on periocular mesenchyme cells (POM) and corneal fibroblasts (CF) with fibronectin and tenascin by perturbing the function of this integrin. POM and CF attached and spread to a much greater extent on fibronectin than on tenascin. An antibody against beta(1) integrin, CSAT, decreased spreading and attachment, and resulted in a lack of immuno-detectable beta(1) integrin in focal adhesions on fibronectin; few beta(1) positive focal adhesions were observed in cells grown on tenascin. An anti-sense retroviral construct decreased endogenous levels of beta(1) integrin protein, and caused decreased attachment and spreading as well as sparse, disorganized focal adhesions. These data indicate that in vitro, both POM and CF have beta(1) integrins that interact with fibronectin and allow them to attach and spread, while tenascin is anti-adhesive. Further studies using both of these experimental paradigms will clarify whether these interactions also occur in vivo.
Doane Kathleen J; Bhattacharya Raka; Marchant Jeff
Cell biology international
2002
1905-6
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1006/cbir.2001.0818" target="_blank" rel="noreferrer noopener">10.1006/cbir.2001.0818</a>
Basic transcription element binding protein (BTEB) transactivates the cholesterol 7 alpha-hydroxylase gene (CYP7A).
*Gene Expression Regulation; Animals; Bile Acids and Salts/genetics; Binding Sites/genetics; Carcinoma; Cholesterol 7-alpha-Hydroxylase/*genetics/metabolism; Complementary/analysis; Cultured; DNA; DNA-Binding Proteins/metabolism/*physiology; Electrophoresis; Enzyme Activation/genetics; Genetic; Hepatocellular; Humans; Kruppel-Like Transcription Factors; Liver/enzymology; Podophyllin/analogs & derivatives/metabolism; Podophyllotoxin/analogs & derivatives; Polyacrylamide Gel; Promoter Regions; Protein Binding/genetics; Rats; Transcription Factors/metabolism/*physiology; Tumor Cells
Cholesterol 7 alpha-hydroxylase catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids in the liver. Previously, we have identified two bile acid response elements located in nt -74 to -54 (BARE-I) and -148 to -118 (BARE-II) regions. The nucleotide sequences in these BAREs are highly conserved and shared a novel sequence, AGTTCAAG. To identify and isolate nuclear protein factors that bind to these BAREs, we have screened a human liver cDNA expression library with oligonucleotide probes containing the sequence from nt -149 to -127. Twenty positive clones were selected and purified. Partial nucleotide sequences of these clones were determined. Nucleotide homology search of DNA databases of the sequences of these clones revealed that sequence of one clone, G13, is identical to basic transcription element binding protein (BTEB), a GC box-binding protein of Sp1 family transcription factors known to regulate many cytochrome P450 genes. Electrophoretic mobility shift assays have identified a basic transcription element (BTE) in BARE-II and a Sp1 binding site located in the nt -100/-82 region of the CYP7A promoter. Transient transfection assays have confirmed that BTEB was able to transactivate the CYP7A promoter/luciferase chimergic gene.
Foti D; Stroup D; Chiang J Y
Biochemical and biophysical research communications
1998
1998-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1006/bbrc.1998.9759" target="_blank" rel="noreferrer noopener">10.1006/bbrc.1998.9759</a>
Trivalent chromium inhibits TSP-1 expression, proliferation, and O-GlcNAc signaling in vascular smooth muscle cells in response to high glucose in vitro.
Aorta/drug effects/metabolism; Cell Proliferation/*drug effects/genetics; Cells; Chromium/*pharmacology; Cultured; Fructosephosphates/metabolism; Genetic/drug effects/genetics; Glucose/*metabolism; Glutamine/genetics; Glycosylation/drug effects; Hexosamines/metabolism; Humans; Hyperglycemia/metabolism; Muscle; Myocytes; N-Acetylglucosaminyltransferases/genetics; O-glycosylation; Promoter Regions; reactive oxygen species; Reactive Oxygen Species/metabolism; Signal Transduction/*drug effects/genetics; Smooth; Smooth Muscle/*drug effects/metabolism; Thrombospondin 1/*antagonists & inhibitors/genetics; thrombospondin-1; Transcription; trivalent chromium; vascular smooth muscle cells; Vascular/*drug effects/metabolism
Trivalent chromium (Cr(3+)) is a mineral nutrient reported to have beneficial effects in glycemic and cardiovascular health. In vitro and in vivo studies suggest that Cr(3+) supplementation reduces the atherogenic potential and lowers the risk of vascular inflammation in diabetes. However, effects of Cr(3+) in vascular cells under conditions of hyperglycemia, characteristic of diabetes, remain unknown. In the present study we show that a therapeutically relevant concentration of Cr(3+) (100 nM) significantly downregulates a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in human aortic smooth muscle cells (HASMC) stimulated with high glucose in vitro. Promoter-reporter assays reveal that this downregulation of TSP-1 expression by Cr(3+) occurs at the level of transcription. The inhibitory effects of Cr(3+) on
Ganguly Rituparna; Sahu Soumyadip; Chavez Ronaldo J; Raman Priya
American journal of physiology. Cell physiology
2015
2015-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajpcell.00256.2014" target="_blank" rel="noreferrer noopener">10.1152/ajpcell.00256.2014</a>
Anti-atherogenic effect of trivalent chromium-loaded CPMV nanoparticles in human aortic smooth muscle cells under hyperglycemic conditions in vitro.
*Comovirus; Aorta/*metabolism; Atherosclerosis/*drug therapy/therapy; Azo Compounds/chemistry; Cell Proliferation; Cells; Chlorides/*chemistry; Chromium Compounds/*chemistry; Cultured; Cytokines/metabolism; Drug Delivery Systems; Electron; Fluorescence; Glucose/chemistry; Humans; Hyperglycemia/*metabolism; Lipids/chemistry; Microscopy; Myocytes; Nanoparticles/chemistry; NF-kappa B/metabolism; Proliferating Cell Nuclear Antigen/chemistry; Smooth Muscle/*metabolism; Spectrophotometry; Transforming Growth Factor beta/metabolism; Transmission; Ultraviolet
Atherosclerosis, a major macrovascular complication associated with diabetes, poses a tremendous burden on national health care expenditure. Despite extensive efforts, cost-effective remedies are unknown. Therapies for atherosclerosis are challenged by a lack of targeted drug delivery approaches. Toward this goal, we turn to a biology-derived drug delivery system utilizing nanoparticles formed by the plant virus, Cowpea mosaic virus (CPMV). The aim herein is to investigate the anti-atherogenic potential of the beneficial mineral nutrient, trivalent chromium, loaded CPMV nanoparticles in human aortic smooth muscle cells (HASMC) under hyperglycemic conditions. A non-covalent loading protocol is established yielding CrCl3-loaded CPMV (CPMV-Cr) carrying 2000 drug molecules per particle. Using immunofluorescence microscopy, we show that CPMV-Cr is readily taken up by HASMC in vitro. In glucose (25 mM)-stimulated cells, 100 nM CPMV-Cr inhibits HASMC proliferation concomitant to attenuated proliferating cell nuclear antigen (PCNA, proliferation marker) expression. This is accompanied by attenuation in high glucose-induced phospho-p38 and pAkt expression. Moreover, CPMV-Cr inhibits the expression of pro-inflammatory cytokines, transforming growth factor-beta (TGF-beta) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), in glucose-stimulated HASMCs. Finally glucose-stimulated lipid uptake is remarkably abrogated by CPMV-Cr, revealed by Oil Red O staining. Together, these data provide key cellular evidence for an atheroprotective effect of CPMV-Cr in vascular smooth muscle cells (VSMC) under hyperglycemic conditions that may promote novel therapeutic ventures for diabetic atherosclerosis.
Ganguly Rituparna; Wen Amy M; Myer Ashley B; Czech Tori; Sahu Soumyadip; Steinmetz Nicole F; Raman Priya
Nanoscale
2016
2016-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1039/c6nr00398b" target="_blank" rel="noreferrer noopener">10.1039/c6nr00398b</a>
Scanning electron microscopy and transmission electron microscopy aspects of synergistic antitumor activity of vitamin C - vitamin K3 combinations against human prostatic carcinoma cells.
Humans; Male; Antineoplastic Agents/*pharmacology; Drug Synergism; Coloring Agents; Ascorbic Acid/*pharmacology; Carcinoma/*drug therapy/pathology; Cell Membrane/drug effects/ultrastructure; Cytoskeleton/drug effects/ultrastructure; Mitochondria/drug effects/ultrastructure; Prostatic Neoplasms/*drug therapy/pathology; Tetrazolium Salts; Thiazoles; Vitamin K/*pharmacology; Microscopy; Electron; Cultured; Scanning; Tumor Cells; Drug Therapy; Combination
A MTT/formazan assay was used to evaluate the antitumor activity of vitamin C (Vit C), vitamin K3 (Vit K3), or vitamin C:vitamin K3 combinations against a human prostatic carcinoma cell line (DU145). Both Vit C and Vit K3 alone exhibited antitumor activity, but only at elevated doses. When Vit C and Vit K3 were combined at a C:K3 ratio of 100:1 and administered to the carcinoma cells, the 50% cytotoxic concentrations (CD50) of the vitamins decreased 10- to 60-fold. Subsequently, the DU145 cells were examined with transmission and scanning electron microscopy (TEM and SEM) following a 1 hour treatment with Vit C, Vit K3, or Vit C/K3 combined at their 50% cytotoxic dose. Our morphological data suggest that vitamin treatment with individual vitamins affects the cytoskeleton, the mitochondria, and other membranous components of the cell. Treatment with the vitamin combination appears to potentiate the effects of the individual vitamin treatment. Specifically, there are abundant necrotic cells. The surviving cells display morphological defects characteristic of cell injury.
Gilloteaux J; Jamison J M; Venugopal M; Giammar D; Summers J L
Scanning microscopy
1995
1995-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae-induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation.
*Airway Remodeling; *fibronectin; *myofibroblasts; *PAI-1; *PI3K; *TGF-beta1; Adult; Animals; Asthma/etiology/genetics/*metabolism/pathology; Cells; Cultured; Dermatophagoides farinae/immunology; Extracellular Matrix/*metabolism/pathology; Fibroblasts/metabolism; Fibronectins/*metabolism; Humans; Inbred C57BL; Male; Mice; Phosphatidylinositol 3-Kinases/metabolism; Plasminogen Activator Inhibitor 1/metabolism; Transforming Growth Factor beta/metabolism; TRPV Cation Channels/genetics/*metabolism
Contributions of mechanical signals to airway remodeling during asthma are poorly understood. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, has been implicated in cardiac and pulmonary fibrosis; however, its role in asthma remains elusive. Employing a Dermatophagoides farinae-induced asthma model, we report here that TRPV4-knockout mice were protected from D. farinae-induced airway remodeling. Furthermore, lung fibroblasts that were isolated from TRPV4-knockout mice showed diminished differentiation potential compared with wild-type mice. Fibroblasts from asthmatic lung exhibited increased TRPV4 activity and enhanced differentiation potential compared with normal human lung fibroblasts. Of interest, TGF-beta1 treatment enhanced TRPV4 activation in a
Gombedza Farai; Kondeti Vinay; Al-Azzam Nosayba; Koppes Stephanie; Duah Ernest; Patil Prachi; Hexter Madison; Phillips Daniel; Thodeti Charles K; Paruchuri Sailaja
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
2017
2017-04
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1096/fj.201601045R" target="_blank" rel="noreferrer noopener">10.1096/fj.201601045R</a>
Prostate apoptosis response-4 enhances secretion of amyloid beta peptide 1-42 in human neuroblastoma IMR-32 cells by a caspase-dependent pathway.
*Intracellular Signaling Peptides and Proteins; Amyloid beta-Peptides/*biosynthesis/metabolism; Apoptosis Regulatory Proteins; Apoptosis/*physiology; Carrier Proteins/genetics/*physiology; Caspases/*metabolism; Cultured; Enzyme Activation; Humans; Kinetics; Leucine Zippers; Neuroblastoma; Peptide Fragments/*biosynthesis/metabolism; Recombinant Proteins/metabolism; Time Factors; Transfection; Tumor Cells
Prostate apoptosis response-4 (Par-4) is a leucine zipper protein that promotes neuronal cell death in Alzheimer's disease (AD). Neuronal degeneration in AD may result from extracellular accumulation of amyloid beta peptide (Abeta) 1-42. To examine the effect of Par-4 on Abeta secretion and to reconcile amyloid/apoptosis hypotheses of AD, we generated IMR-32 cell lines that overexpress Par-4 and/or its leucine zipper domain. Overexpression of Par-4 did not significantly affect levels of the endogenously expressed beta amyloid precursor protein but drastically increased the Abeta(1-42)/Abeta(total) ratio in the conditioned media about 6-8 h after trophic factor withdrawal. Time course analysis of caspase activation reveals that Par-4 overexpression exacerbated caspase activation, which is detectable within 2 h after trophic factor withdrawal. Furthermore, inhibition of caspase activity by the broad spectrum caspase inhibitor BD-fmk significantly attenuated the Par-4-induced increase in Abeta 1-42 production. In addition, the effects of Par-4 on secretion of Abeta 1-42 were consistently blocked by co-expression of the leucine zipper domain, indicating that the effect of Par-4 on Abeta secretion may require its interaction with other protein(s). These results suggest that Par-4 increases secretion of Abeta 1-42 largely through a caspase-dependent pathway after apoptotic cascades are initiated.
Guo Q; Xie J; Chang X; Du H
The Journal of biological chemistry
2001
2001-05
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M010996200" target="_blank" rel="noreferrer noopener">10.1074/jbc.M010996200</a>
Mechanism of vitamin D receptor inhibition of cholesterol 7alpha-hydroxylase gene transcription in human hepatocytes.
Base Sequence; Calcitriol/drug effects/genetics/*physiology; Cell Line; Cells; Cholesterol 7-alpha-Hydroxylase/*genetics; Cultured; DNA Primers; Electrophoretic Mobility Shift Assay; Gene Knockdown Techniques; Genetic/*physiology; Hepatocytes/*drug effects/enzymology; Humans; Immunoprecipitation; Lithocholic Acid/pharmacology; Messenger/genetics; Polymerase Chain Reaction; Receptors; RNA; Small Interfering; Transcription; Tumor; Two-Hybrid System Techniques
Lithocholic acid (LCA) is a potent endogenous vitamin D receptor (VDR) ligand. In cholestasis, LCA levels increase in the liver and intestine. The objective of this study is to test the hypothesis that VDR plays a role in inhibiting cholesterol 7alpha-hydroxylase (CYP7A1) gene expression and bile acid synthesis in human hepatocytes. Immunoblot analysis has detected VDR proteins in the nucleus of the human hepatoma cell line HepG2 and human primary hepatocytes. 1alpha, 25-Dihydroxy-vitamin D(3) or LCA acetate-activated VDR inhibited CYP7A1 mRNA expression and bile acid synthesis, whereas small interfering RNA to VDR completely abrogated VDR inhibition of CYP7A1 mRNA expression in HepG2 cells. Electrophoretic mobility shift assay and mutagenesis analyses have identified the negative VDR response elements that bind VDR/retinoid X receptor alpha in the human CYP7A1 promoter. Mammalian two-hybrid, coimmunoprecipitation, glutathione
Han Shuxin; Chiang John Y L
Drug metabolism and disposition: the biological fate of chemicals
2009
2009-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1124/dmd.108.025155" target="_blank" rel="noreferrer noopener">10.1124/dmd.108.025155</a>
The effect of various insufflation gases on tumor implantation in an animal model.
*Carbon Dioxide; *Helium; *Neoplasm Transplantation; *Nitrous Oxide; *Peritoneal Cavity; *Pneumoperitoneum; Adenocarcinoma/pathology; Animals; Artificial; Cultured; Inbred F344; Injections; Intraperitoneal; Peritoneal Neoplasms/pathology; Rats; Tumor Cells
OBJECTIVE: The expanded use of laparoscopy has led to reports of tumor dissemination and spread to laparoscopic port sites. We previously showed that carbon dioxide insufflation produced tumor dissemination compared with laparotomy. It is unknown whether the type of gas used influences this dissemination. Although carbon dioxide is commonly used during laparoscopy, helium and nitrous oxide could be used. This study was undertaken to compare the effects of carbon dioxide, helium, and nitrous oxide gas on tumor spread in an animal model to determine whether the type of gas used for the pneumoperitoneum would affect tumor spread. STUDY DESIGN: Viable MATB mammary adenocarcinoma cells were injected into the peritoneal cavity of Fisher 344 rats (1 x 10(5) cells/rat). The animals were then divided into three groups. Four 18-gauge angiocaths were inserted into each of four quadrants of the peritoneal cavity, and induction of pneumoperitoneum with helium, nitrous oxide, or carbon dioxide gas was done at approximately 7 mm Hg for 2 hours. Animals were killed at 7 days and the ventral peritoneal wall and abdominal cavity were examined for evidence of tumor formation. Tumor implants were counted for each of the four quadrants on the anterior peritoneal wall and for the total abdominal cavity. RESULTS: A total of 39 rats were studied with 13 animals per group. The total number of implants was calculated for the groups: carbon dioxide, n = 57; helium, n = 62; nitrous oxide, n = 66. There was no significant difference in the total number of implants according to the type of gas or the size of implants. When implants that were \textless5 mm were analyzed, the four quadrants of the anterior peritoneum showed a random difference in tumor disbursement between one quadrant in the helium (n = 15) and one quadrant in the nitrous group (n = 23). However, when the three gasses were compared with all four quadrants, there was no statistical significance (carbon dioxide, n = 35; helium, n = 38; nitrous oxide, n = 44). CONCLUSION: There is no difference in tumor implantation when with use of carbon dioxide, helium, or nitrous oxide gas in an animal model. Thus, carbon dioxide insufflation does not appear to increase tumor spread compared with other gasses.
Hopkins Michael P; von Gruenigen Vivian; Haller Nairmeen Awad; Holda Sheri
American journal of obstetrics and gynecology
2002
2002-10
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1067/mob.2002.126644" target="_blank" rel="noreferrer noopener">10.1067/mob.2002.126644</a>
Type VI collagen increases cell survival and prevents anti-beta 1 integrin-mediated apoptosis.
Animals; Antibodies; Apoptosis/*drug effects/physiology; Cell Nucleus/drug effects/metabolism; Cell Survival/drug effects/physiology; Cells; Chick Embryo; Collagen/*pharmacology; Cornea/drug effects/embryology/ultrastructure; Culture Media/chemistry/pharmacology; Cultured; DNA Fragmentation/drug effects; Extracellular Matrix/physiology; Eye/drug effects/embryology/ultrastructure; Fibroblasts/drug effects/metabolism/ultrastructure; Genetic Techniques; Integrins/immunology; Monoclonal/*drug effects/pharmacology
Cell-matrix interactions are important in the development of the avian cornea. Type VI collagen is present within the periocular mesenchyme prior to the migration of cells into the corneal stroma and is abundant in the mature stroma. Whether the interaction of cells with type VI collagen is essential for cellular survival in the cornea is not known. In the present study, we examined the interaction of corneal cells with type VI collagen in vitro to determine if it can increase cell proliferation and decrease apoptosis. In vivo analysis demonstrated that apoptosis occurs in the periocular region during early stages of avian corneal development, but in fully mature corneas apoptosis only occurs in the corneal epithelium and not in the stroma. In vitro analysis examined the importance of beta 1 integrin interactions with type VI collagen in mature corneal fibroblasts and the precursor cells. Using an anti-beta 1 integrin blocking antibody, CSAT, integrin/matrix interactions were disrupted. Results indicated that viability of both corneal fibroblasts and periocular mesenchyme cells was greater on type VI collagen than on type I collagen or BSA-blocked glass. In addition, less apoptosis was observed for both cell types on type VI collagen when beta 1 integrin–matrix interactions were disrupted. These data indicated that these cells require intact beta 1 interactions with type I collagen and with BSA-coated glass controls to remain viable. Thus, type VI collagen may play a role in the rescue of corneal cells from anti-beta 1 integrin-induced apoptosis by increasing cell survival, probably via a non-beta 1 integrin-dependent mechanism.
Howell S J; Doane K J
Experimental cell research
1998
1998-05
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1006/excr.1998.4051" target="_blank" rel="noreferrer noopener">10.1006/excr.1998.4051</a>
Inhibition of dopamine and choline acetyltransferase concentrations in rat CNS neurons by rat alpha 1- and alpha 2-macroglobulins.
alpha-Macroglobulins/administration & dosage/*pharmacology; Animals; Cells; Choline O-Acetyltransferase/*antagonists & inhibitors; Corpus Striatum/drug effects/*enzymology/metabolism; Cultured; Dopamine Antagonists/*pharmacology; Dopamine/analysis/*metabolism; Enzyme Activation/drug effects; Injections; Intraventricular; Male; Neurons/drug effects/*enzymology/metabolism; Rats; Serotonin/pharmacology; Sprague-Dawley; Stereotaxic Techniques
Previous studies have implicated human alpha-2-macroglobulin (alpha2M) as a potential regulator of neuronal development and function. Rat alpha-1-macroglobulin (alpha1M) and acute-phase alpha-2-macroglobulin (alpha2M) are murine homologues of human alpha2M. In this report, we tested the effect of intracranially infused serotonin-activated rat alpha1M (5HT-alpha1M) on the concentration of dopamine (DA) in the corpus striatum in vivo and the effect of
Hu Y Q; Liebl D J; Dluzen D E; Koo P H
Journal of neuroscience research
1998
1998-02
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/(SICI)1097-4547(19980215)51:4%3C541::AID-JNR14%3E3.0.CO;2-6" target="_blank" rel="noreferrer noopener">10.1002/(SICI)1097-4547(19980215)51:4%3C541::AID-JNR14%3E3.0.CO;2-6</a>
Experimental use of fibrin glue to induce site-directed osteogenesis from cultured periosteal cells.
*Fibrin Tissue Adhesive; *Osteogenesis; Animals; Bone and Bones/chemistry/cytology/diagnostic imaging; Cattle; Cells; Cultured; Experimental; Implants; Injections; Mice; Non-programmatic; Nude; Osteopontin; Periosteum/*cytology; Radiography; Sialoglycoproteins/analysis
The purpose of this study was to determine whether a combination of fibrin glue and cultured periosteal cells will result in new bone formation at heterotopic sites in nude mice. Growing cells and developing matrices surrounding periosteal explants from the diaphyses of radii of newborn calves were minced and mixed with fibrin glue in a syringe. The cell/matrix-fibrin glue admixture was then injected into the subcutaneous space on the dorsum of athymic nude mice. After 12 weeks of implantation, gross morphology and histologic investigations showed newly formed bone structures in all cell/matrix-fibrin glue admixtures, but none in fibrin glue injected alone and used as control samples. Osteopontin, a protein important in bone development, was identified by a Western blot assay of the cell/matrix-fibrin glue composite. This study supports the feasibility of initiating site-directed formation of bone structures at heterotopic tissue sites by means of injection of cultured periosteal cells and matrix in a fibrin glue carrier.
Isogai N; Landis W J; Mori R; Gotoh Y; Gerstenfeld L C; Upton J; Vacanti J P
Plastic and reconstructive surgery
2000
2000-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1097/00006534-200003000-00019" target="_blank" rel="noreferrer noopener">10.1097/00006534-200003000-00019</a>
Alignment of inducible vascular progenitor cells on a micro-bundle scaffold improves cardiac repair following myocardial infarction.
*Cardiovascular regeneration; *Ischemic heart diseases; *Micro-bundle scaffold; *Myocardial infarction; *Neovascularization; *Stem cells; *Tissue Scaffolds; *Vascular progenitor cells; Animal; Animals; Cell Differentiation; Cell Proliferation; Cell Survival; Cells; Coculture Techniques; Cultured; Disease Models; Endothelial Progenitor Cells/metabolism/*transplantation; Fibroblast Growth Factor 2/metabolism; Lactic Acid/*chemistry; Muscle; Myocardial Infarction/metabolism/pathology/physiopathology/*surgery; Myocardium/metabolism/*pathology; Myocytes; Paracrine Communication; Phenotype; Physiologic; Polyglycolic Acid/*chemistry; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Signal Transduction; Smooth; Smooth Muscle/metabolism/*transplantation; Sprague-Dawley; Time Factors; Tissue Engineering/*methods; Vascular Endothelial Growth Factor A/metabolism; Vascular/metabolism/*transplantation; Ventricular Remodeling
Ischemic heart disease is still the leading cause of death even with the advancement of pharmaceutical therapies and surgical procedures. Early vascularization in the ischemic heart is critical for a better outcome. Although stem cell therapy has great potential for cardiovascular regeneration, the ideal cell type and delivery method of cells have not been resolved. We tested a new approach of stem cell therapy by delivery of induced vascular progenitor cells (iVPCs) grown on polymer micro-bundle scaffolds in a rat model of myocardial infarction. iVPCs partially reprogrammed from vascular endothelial cells (ECs) had potent angiogenic potential and were able to simultaneously differentiate into vascular smooth muscle cells (SMCs) and ECs in 2D culture. Under hypoxic conditions, iVPCs also secreted angiogenic cytokines such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as measured by enzyme-linked immunosorbent assay (ELISA). A longitudinal micro-scaffold made from poly(lactic-co-glycolic acid) was sufficient for the growth and delivery of iVPCs. Co-cultured ECs and SMCs aligned well on the micro-bundle scaffold similarly as in the vessels. 3D cell/polymer micro-bundles formed by iVPCs and micro-scaffolds were transplanted into the ischemic myocardium in a rat model of myocardial infarction (MI) with ligation of the left anterior descending artery. Our in vivo data showed that iVPCs on the micro-bundle scaffold had higher survival, and better retention and engraftment in the myocardium than free iVPCs. iVPCs on the micro-bundles promoted better cardiomyocyte survival than free iVPCs. Moreover, iVPCs and iVPC/polymer micro-bundles treatment improved cardiac function (ejection fraction and fractional shortening, endocardial systolic volume) measured by echocardiography, increased vessel density, and decreased infarction size [endocardial and epicardial infarct (scar) length] better than untreated controls at 8 weeks after MI. We conclude that iVPCs grown on a polymer micro-bundle scaffold are new promising approach for cell-based therapy designed for cardiovascular regeneration in ischemic heart disease.
Jamaiyar Anurag; Wan Weiguo; Ohanyan Vahagn; Enrick Molly; Janota Danielle; Cumpston Devan; Song Hokyung; Stevanov Kelly; Kolz Christopher L; Hakobyan Tatev; Dong Feng; Newby Bi-Min Zhang; Chilian William M; Yin Liya
Basic research in cardiology
2017
2017-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1007/s00395-017-0631-4" target="_blank" rel="noreferrer noopener">10.1007/s00395-017-0631-4</a>
Cell cycle arrest and autoschizis in a human bladder carcinoma cell line following Vitamin C and Vitamin K3 treatment.
Antioxidants/*pharmacology; Ascorbic Acid/*pharmacology; Calcium/metabolism; Cell Cycle/*drug effects; Cultured; DNA; Flow Cytometry; Humans; Hydrogen Peroxide/metabolism; Male; Neoplasm/drug effects; Sulfhydryl Compounds/metabolism; Tumor Cells; Urinary Bladder Neoplasms/pathology; Vitamin K 3/*pharmacology
Exponentially growing cultures of human bladder tumor cells (T24) were treated with Vitamin C (VC) alone, Vitamin K(3) (VK(3)) alone, or with a VC:VK(3) combination for 1, 2, or 4hr. Flow cytometry of T24 cells exposed to the vitamins for 1h revealed a growth arrested population and a population undergoing cell death. Cells in G(1) during vitamin treatment arrested in G(1) while those in S phase progressed through S phase and arrested in G(2)/M. DNA synthesis decreased to 14 to 21% of control levels which agreed with the percent of cells in S phase during treatment. Annexin V labeling demonstrated the majority of the cells died by autoschizis, but necrosis and apoptosis also were observed. Catalase treatment abrogated both cell cycle arrest and cell death which implicated hydrogen peroxide (H(2)O(2)) in these processes. Redox cycling of VC and VK(3) increased H(2)O(2) production and decreased cellular thiol levels and DNA content, while increasing intracellular Ca(2+) levels and lipid peroxidation. Feulgen staining of treated cells revealed a time-dependent decrease in tumor cell DNA, while electrophoresis revealed a spread pattern. These results suggest that Ca(2+) disregulation activates at least one DNase which degrades tumor cell DNA and induces tumor cell death.
Jamison James M; Gilloteaux Jacques; Nassiri M Reza; Venugopal Meenakshi; Neal Deborah R; Summers Jack L
Biochemical pharmacology
2004
2004-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.bcp.2003.08.040" target="_blank" rel="noreferrer noopener">10.1016/j.bcp.2003.08.040</a>
Glycation Reduces the Stability of ApoAI and Increases HDL Dysfunction in Diet-Controlled Type 2 Diabetes.
Adult; Aged; Animal Studies; Animals; Apolipoprotein A-I/blood/*metabolism; Apolipoproteins – Blood; Apolipoproteins – Metabolism; Biochemical Phenomena; Case Control Studies; Case-Control Studies; Cells; Comparative Studies; Cultured; Diabetes Mellitus; Diet; Dyslipidemias/complications/diet therapy/*metabolism; Evaluation Research; Female; Funding Source; Glycosylation; HDL – Metabolism; HDL/*metabolism; Human; Humans; Hyperglycemia – Complications; Hyperglycemia – Diet Therapy; Hyperglycemia – Metabolism; Hyperglycemia/complications/diet therapy/*metabolism; Hyperlipidemia – Complications; Hyperlipidemia – Diet Therapy; Hyperlipidemia – Metabolism; Lipoproteins; Male; Mice; Middle Age; Middle Aged; Multicenter Studies; Protein Stability; Type 2 – Complications; Type 2 – Diet Therapy; Type 2 – Metabolism; Type 2/complications/*diet therapy/metabolism; Validation Studies
Context: Hyperglycemia plays a key role in the pathogenesis of cardiovascular complications of diabetes. Type 2 diabetes mellitus (T2DM) is associated with high-density lipoprotein (HDL) dysfunction and increased degradation of apolipoprotein I (ApoAI). The mechanism(s) of these changes is largely unknown. Objective: To study the role of hyperglycemia-induced glycation on ApoAI kinetics and stability in patients with diet-controlled T2DM. Design: 2H2O-metabolic labeling approach was used to study ApoAI turnover in patients with diet-controlled T2DM [n = 9 (5 F); 59.3 +/- 8.5 years] and matched healthy controls [n = 8 (4 F); 50.7 +/- 11.6 years]. The effect of Amadori glycation on in vivo ApoAI stability and the antioxidant and cholesterol efflux properties of HDL were assessed using a proteomics approach and in vitro assays. Results: Patients with T2DM had increased turnover of ApoAI and impaired cholesterol efflux and antioxidant properties of HDL. Glycated hemoglobin was negatively correlated with the half-life of ApoAI and cholesterol efflux function of HDL. Proteomics analysis identified several nonenzymatic early (Amadori) glycations of ApoAI at lysine sites. The kinetics analysis of glycated and native ApoAI peptides in patients with T2DM revealed that glycation resulted in a threefold shorter ApoAI half-life. Conclusions: The 2H2O method allowed the detection of early in vivo impairments in HDL metabolism and function that were related to hyperglycemia-induced glycation of ApoAI in T2DM.
Kashyap Sangeeta R; Osme Abdullah; Ilchenko Serguei; Golizeh Makan; Lee Kwangwon; Wang Shuhui; Bena James; Previs Stephen F; Smith Jonathan D; Kasumov Takhar
The Journal of clinical endocrinology and metabolism
2018
2018-02
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1210/jc.2017-01551" target="_blank" rel="noreferrer noopener">10.1210/jc.2017-01551</a>
Wogonin, a natural flavonoid, intercalates with genomic DNA and exhibits protective effects in IL-1beta stimulated osteoarthritis chondrocytes.
Apoptosis/drug effects; Binding Sites; Cells; Chondrocytes/cytology/*drug effects/metabolism; Chondroprotective effects; Cultured; Denaturation; DNA binding; DNA/chemistry/*metabolism; Flavanones/chemistry/metabolism/*pharmacology; Flavonoids/chemistry/pharmacology; Fluorescence Resonance Energy Transfer; Humans; Intercalating Agents/chemistry/metabolism/*pharmacology; Interleukin-1beta/*pharmacology; Molecular Docking Simulation; Nucleic Acid Conformation; Osteoarthritis; Osteoarthritis/metabolism/pathology; Protective Agents/chemistry/metabolism/*pharmacology; Reactive Oxygen Species/metabolism; Up-Regulation/drug effects; Wogonin
Wogonin has recently been shown to possess anti-inflammatory and chondroprotective properties and is of considerable interest due to its broad pharmacological activities. The present study highlights that Wogonin binds DNA and exerts chondroprotective effects in vitro. Wogonin showed strong binding with chondrocytes genomic DNA in vitro. The mode of binding of Wogonin to genomic-DNA was assessed by competing Wogonin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. EtBr fluorescence reduced significantly with increase in Wogonin concentration suggesting possible DNA intercalation of Wogonin. Further, in silico molecular docking of Wogonin on mammalian DNA also indicated possible intercalation of Wogonin with DNA. The denaturation and FRET studies revealed that Wogonin prevents denaturation of DNA strands and provide stability to genomic DNA against a variety of chemical denaturants. The cellular uptake study showed that Wogonin enters osteoarthritis chondrocytes and was mainly localized in the nucleus. Wogonin treatment to OA chondrocytes protects the fragmentation of genomic DNA in response to IL-1beta as evaluated by DNA ladder and TUNEL assay. Treatment of chondrocytes with Wogonin resulted in significant suppression of IL-1beta-mediated induction of ROS. Further, Wogonin exhibited protective potential through potent suppression of extrinsic and intrinsic apoptotic pathways and induction of anti-apoptotic proteins in
Khan Nazir M; Ahmad Imran; Ansari Mohammad Y; Haqqi Tariq M
Chemico-biological interactions
2017
2017-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.cbi.2017.06.025" target="_blank" rel="noreferrer noopener">10.1016/j.cbi.2017.06.025</a>
Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis.
Humans; *Apoptosis; *Apoptosis; *ERK1/2; *Nrf2; *Osteoarthritis; *Redox; Oxidative Stress; Osteoarthritis/*metabolism; NF-E2-Related Factor 2/genetics/*metabolism; Up-Regulation; MAP Kinase Signaling System; Chondrocytes/*physiology; Caspases/metabolism; Cells; Cultured; 70-kDa/metabolism; Ribosomal Protein S6 Kinases; Carboxylic Ester Hydrolases/metabolism; ets-Domain Protein Elk-1/metabolism; Interleukin-1beta/immunology; 90-kDa/metabolism
Nrf2, a redox regulated transcription factor, has recently been shown to play a role in cartilage integrity but the mechanism remains largely unknown. Osteoarthritis (OA) is a multifactorial disease in which focal degradation of cartilage occurs. Here, we studied whether Nrf2 exerts chondroprotective effects by suppressing the oxidative stress and apoptosis in IL-1beta stimulated human OA chondrocytes. Expression of Nrf2 and its target genes HO-1, NQO1 and SOD2 was significantly high in OA cartilage compared to normal cartilage and was also higher in damaged area compared to smooth area of OA cartilage of the same patient. Human chondrocytes treated with IL-1beta resulted in robust Nrf2/ARE reporter activity, which was inhibited by pretreatment with antioxidants indicating that Nrf2 activity was due to IL-1beta-induced ROS generation. Ectopic expression of Nrf2 significantly suppressed the IL-1beta-induced generation of ROS while Nrf2 knockdown significantly increased the basal as well as
Khan Nazir M; Ahmad Imran; Haqqi Tariq M
Free radical biology & medicine
2018
2018-02
<a href="http://doi.org/10.1016/j.freeradbiomed.2018.01.013" target="_blank" rel="noreferrer noopener">10.1016/j.freeradbiomed.2018.01.013</a>
A wogonin-rich-fraction of Scutellaria baicalensis root extract exerts chondroprotective effects by suppressing IL-1beta-induced activation of AP-1 in human OA chondrocytes.
Cells; Chemical Fractionation/methods; Chondrocytes/*drug effects/metabolism; Cultured; Cyclooxygenase 2/genetics/metabolism; Flavanones/*pharmacology; Gene Expression Regulation/drug effects; Humans; Interleukin-1beta/pharmacology; Interleukin-6/genetics/metabolism; Osteoarthritis/pathology; Plant Extracts/*pharmacology; Plant Roots/*chemistry; Protective Agents/pharmacology; Reactive Oxygen Species/metabolism; Scutellaria baicalensis/*chemistry; Transcription Factor AP-1/genetics/*metabolism
Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation and sustained oxidative stress. The root extract of Scutellaria baicalensis (SBE) has been used for the treatment of inflammatory and other diseases. Here, we performed activity-guided HPLC-fractionation of SBE, identified the active ingredient(s) and investigated its chondroprotective potential. We found that the Wogonin containing fraction-4 (F4) was the most potent fraction based on its ability to inhibit ROS production and the suppression of catabolic markers including IL-6, COX-2, iNOS, MMP-3, MMP-9,
Khan Nazir M; Haseeb Abdul; Ansari Mohammad Y; Haqqi Tariq M
Scientific reports
2017
2017-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1038/srep43789" target="_blank" rel="noreferrer noopener">10.1038/srep43789</a>
Age-related expression patterns of Bag-1 and Bcl-2 in growth plate and articular chondrocytes.
Aging/*metabolism; Animals; Articular/cytology/*metabolism; Carrier Proteins/genetics/*metabolism; Cartilage; Cell Division/physiology; Cells; Chondrocytes/cytology/*metabolism; Cultured; DNA-Binding Proteins; Gene Expression Regulation; Growth Plate/*metabolism; Inbred C57BL; Mice; Proto-Oncogene Proteins c-bcl-2/*metabolism; Tissue Distribution; Transcription Factors
Aging cartilage displays increased chondrocyte apoptosis and decreased responsiveness of chondrocytes to growth factors. The molecular mechanisms responsible for these changes have not been identified. Bag-1 is a Bcl-2-binding protein that promotes cell survival, interacts with a diverse group of cellular proteins, and may integrate multiple pathways involved in controlling cell survival, growth, and phenotype. Bcl-2 is important for maintaining chondrocyte phenotype and delaying terminal differentiation and apoptosis of chondrocytes. Comparatively little is known about the role of Bag-1 in cartilage. Here we show that both growth plate and articular chondrocytes in the mouse express the Bag-1 protein. In the growth plate, Bag-1 expression is prominent in the late proliferative and prehypertrophic chondrocytes, displaying a pattern similar to what has been reported for Bcl-2. Further, the expression of both Bcl-2 and Bag-1 declines with age in the articular cartilage. Growth assays demonstrate that knocking down Bag-1 expression causes a decrease in growth rate. These results suggest that Bag-1 is involved in the regulation of chondrocyte phenotype and cartilage aging.
Kinkel Mary D; Yagi Rieko; McBurney Denise; Nugent Ashleigh; Horton Walter E Jr
The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology
2004
2004-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/ar.a.20063" target="_blank" rel="noreferrer noopener">10.1002/ar.a.20063</a>
Inhibition of the G2/M transition of the cell cycle by methyl-2,5-dihydroxycinnamate in human lymphoid cells.
Cell Line; Cinnamates/*pharmacology; Cultured; G2 Phase/*drug effects; Humans; Lymphocytes/cytology/*drug effects; Mitosis/*drug effects; Monocytes/cytology/drug effects; Transformed; Tumor Cells
Immortalized human lymphoid cells treated with Methyl-2,5-dihydroxycinnamate (MDHC), a stable analog of erbstatin, inhibited the G2/M transition of the cell cycle. The MDHC inhibition of the cell cycle was observed at concentrations well below the IC50 for the inhibition of the EGF receptor and sufficiently below that reported to induce protein cross-linking. The effect of MDHC upon the cell cycle is relatively stable, since unlike erbstatin, inhibition of the G2/M transition was observed 32 hours following removal of the drug. PHA stimulated human peripheral blood mononuclear cells (PBMC) were much less sensitive to MDHC. This study shows that MDHC acts on cells lacking an EGF receptor and the target of MDHC is involved in promoting progression of the cell cycle.
Koch J A; Rosenthal K S; Jamison J M; Summers J L
Biochemical and biophysical research communications
1996
1996-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1006/bbrc.1996.1207" target="_blank" rel="noreferrer noopener">10.1006/bbrc.1996.1207</a>
Inhibition of nerve growth factor-stimulated neurite outgrowth by methylamine-modified alpha 2-macroglobulin.
alpha-Macroglobulins/metabolism/*pharmacology; Animals; Cells; Chick Embryo; Cultured; Drug Interactions; Female; Ganglia; Humans; Male; Methylamines/*pharmacology; Mice; Nerve Growth Factors/antagonists & inhibitors/metabolism/*pharmacology; Neurites/drug effects/*physiology/ultrastructure; Neurons/cytology/physiology; Placenta; Pregnancy; Spinal/physiology; Submandibular Gland
alpha 2-Macroglobulin (alpha 2M) is a rather ubiquitous protein in extracellular spaces of mammals. It is an inhibitor of endopeptidases, can be modified by aliphatic amines, and combines with a number of hormones/cytokines such as beta-nerve growth factor (NGF) [Koo PH, Stach RW (1989): J Neurosci Res 22:247]. The objective of this study is to compare the NGF-binding properties of methylamine-modified human alpha 2M (MA-alpha 2M) versus normal alpha 2M and their effects on the biological activity of NGF and neurite extension by embryonic chicken dorsal root ganglia. As determined by gel filtration, polyacrylamide gel electrophoresis, and equilibrium binding studies, these two forms of alpha 2M are similar in their binding affinities, with MA-alpha 2M binding about twice as much NGF as normal alpha 2M. Both normal alpha 2M and
Koo P H; Liebl D J
Journal of neuroscience research
1992
1992-04
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/jnr.490310412" target="_blank" rel="noreferrer noopener">10.1002/jnr.490310412</a>
Interaction of nerve growth factor with murine alpha-macroglobulin.
alpha-Macroglobulins/*metabolism; Animals; Cells; Chick Embryo; Cultured; Ganglia; Methylamines/pharmacology; Mice; Nerve Growth Factors/*metabolism; Spinal/*cytology/drug effects; Trypsin/pharmacology; Zinc/pharmacology
The murine nerve growth factor, when injected i.v. or, combined in vitro with plasma, was found largely associated with the mouse alpha-macroglobulin (a homologue of human alpha 2-macroglobulin). The nerve growth factor-alpha-macroglobulin complex produced is sufficiently stable to resist separation by gel filtration in 1.0 M sodium chloride, polyacrylamide gel electrophoresis, and immunoprecipitation by antibodies against alpha-macroglobulin. As determined by equilibrium binding studies and computer generated Scatchard analysis, alpha-macroglobulin apparently possesses two types of binding sites with the apparent dissociation constants of 1.2 x 10(-6) and 2.9 x 10(-9) M, respectively, saturable by 3.7 and 0.03 moles of nerve growth factor. Hence, about one mole of nerve growth factor is bound to each of the four subunits of alpha-macroglobulin. Nerve growth factor can be readily dissociated from alpha-macroglobulin in sodium dodecyl sulfate gel electrophoresis in the absence of a reductant. Procedures that affect the proteinase-binding or methylamine- activities of alpha-macroglobulin do not affect the binding of nerve growth factor, and the binding is unaffected by the presence of zinc ions or EDTA. Hence, nerve growth factor is noncovalently associated with alpha-macroglobulin at a site separate from that of the proteinase-, methylamine-, and zinc-binding sites of alpha-macroglobulin. Mouse alpha-macroglobulin can protect the nerve growth factor from inactivation by trypsin. Even in the presence of trypsin, alpha-macroglobulin-nerve growth factor complexes still can stimulate the neurite outgrowth by dorsal root ganglia of
Koo P H; Stach R W
Journal of neuroscience research
1989
1989-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/jnr.490220304" target="_blank" rel="noreferrer noopener">10.1002/jnr.490220304</a>
Subcellular localization and antiviral activity of carminic acid/poly r(A-U) combinations.
Antiviral Agents/*pharmacology; Carmine/*analogs & derivatives/pharmacokinetics/pharmacology; Cell Nucleolus/metabolism; Cells; Chromatin/metabolism; Cultured; Doxorubicin/pharmacology; Drug Combinations; Drug Synergism; Fluorescence; Humans; Interferon-beta/biosynthesis/physiology; Microscopy; Phase-Contrast; Poly A-U/*pharmacokinetics/*pharmacology; Vesicular stomatitis Indiana virus/drug effects/growth & development; Viral Plaque Assay
Carminic acid (CAR) enhances the antiviral activity of poly r(A-U) twelve-fold without increasing interferon induction, inactivating the vesicular stomatitis virus or inducing host cell cytotoxicity. Phase contrast photomicrographs of human foreskin fibroblasts (HSF) incubated with CAR alone, poly r(A-U) alone or with a CAR/poly r(A-U) combination illustrate that the CAR/poly r(A-U) combinations display altered subcellular distribution with the CAR being localized in the nucleoli and chromatin. Phase contrast and fluorescence photomicrographs of adriamycin (ADR)-treated and ADR/poly r(A-U)-treated HSF cells corroborate these findings. These results suggest that modulation of one or more nucleolar processes may be responsible for the enhanced antiviral activity.
Krabill K; Jamison J M; Gilloteaux J; Summers J L
Cell biology international
1993
1993-10
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1006/cbir.1993.1014" target="_blank" rel="noreferrer noopener">10.1006/cbir.1993.1014</a>
Enhanced antiviral activity and altered subcellular distribution of magnesium/poly r(A-U) combinations.
Antiviral Agents/*pharmacology; Cells; Chromatin/drug effects; Cultured; Ethidium/*pharmacology; Humans; Magnesium/metabolism/*pharmacology; Nucleolus Organizer Region/*drug effects; Poly A-U/*pharmacology; Vesicular stomatitis Indiana virus/drug effects
When Mg2+ or ethidium bromide (EB) were combined with poly r(A-U) at a ligand/ribonucleotide ratio of 1/4, the antiviral activity of the Mg2+ and EB increased 136-fold and 154-fold. Eriochrome Blue SE was employed to visualize the subcellular distribution of Mg2+ following co-incubation of Human Foreskin Fibroblasts (HSF) with Mg2+ alone or with the Mg2+/poly r(A-U) combination. Phase contrast micrographs of these Mg(2+)-treated HSF cells as well as phase contrast and fluorescence micrographs of EB-treated or EB/poly r(A-U)-treated HSF cells illustrated that the Mg2+ (or EB)/poly r(A-U) combinations display altered subcellular distribution with the Mg2+ and EB being localized in the nucleoli and chromatin of the HSF cells. These results suggest that modulation of nuclear processes may be responsible for the enhanced antiviral activity.
Krabill K; Jamison J M; Gilloteaux J; Summers J L
Cell biology international reports
1992
1992-10
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/s0309-1651(06)80050-0" target="_blank" rel="noreferrer noopener">10.1016/s0309-1651(06)80050-0</a>
RNA interference for CFTR attenuates lung fluid absorption at birth in rats.
*RNA Interference; Animals; Biological Transport/drug effects; Cells; Cultured; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics/*metabolism; Epithelial Sodium Channels/genetics/metabolism; Female; Gene Expression Regulation/drug effects; Lung/cytology/drug effects/*metabolism; Messenger/metabolism; Newborn; Pregnancy; Rats; Respiratory Mucosa/cytology/drug effects/metabolism; RNA; Small Interfering/*pharmacology; Sprague-Dawley; Water/*metabolism
BACKGROUND: Small interfering RNA (siRNA) against alphaENaC (alpha-subunit of the epithelial Na channel) and CFTR (cystic fibrosis transmembrane conductance regulator) was used to explore ENaC and CTFR function in newborn rat lungs. METHODS: Twenty-four hours after trans-thoracic intrapulmonary (ttip) injection of siRNA-generating plasmid DNA (pSi-0, pSi-4, or pSi-C2), we measured CFTR and ENaC expression, extravascular lung water, and mortality. RESULTS: alphaENaC and CFTR mRNA and protein decreased by approximately 80% and approximately 85%, respectively, following alphaENaC and CFTR silencing. Extravascular lung water and mortality increased after alphaENaC and CFTR-silencing. In pSi-C2-transfected isolated DLE cells there were attenuated CFTR mRNA and protein. In pSi-4-transfected DLE cells alphaENaC mRNA and protein were both reduced. Interestingly, CFTR-silencing also reduced alphaENaC mRNA and protein. alphaENaC silencing, on the other hand, only slightly reduced CFTR mRNA and protein. CONCLUSION: Thus, ENaC and CFTR are both involved in the fluid secretion to absorption conversion around at birth.
Li Tianbo; Koshy Shyny; Folkesson Hans G
Respiratory research
2008
2008-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1186/1465-9921-9-55" target="_blank" rel="noreferrer noopener">10.1186/1465-9921-9-55</a>
Glucose stimulates cholesterol 7alpha-hydroxylase gene transcription in human hepatocytes.
*Gene Expression Regulation; Acetylation; AMP-Activated Protein Kinases/metabolism; ATP Citrate (pro-S)-Lyase/genetics/metabolism; Bile Acids and Salts/metabolism; Cells; Cholesterol 7-alpha-Hydroxylase/genetics/*metabolism; Cultured; DNA-Binding Proteins/metabolism; Enzymologic; Epigenesis; Genes; Genetic; Glucose/*administration & dosage; Hep G2 Cells; Hepatocyte Nuclear Factor 4/metabolism; Hepatocytes/*enzymology/metabolism; Histones/metabolism; Humans; Hyperglycemia/enzymology/*metabolism; Messenger/metabolism; Methylation; Reporter; RNA; RNA Interference
Bile acids play important roles in the regulation of lipid, glucose, and energy homeostasis. Recent studies suggest that glucose regulates gene transcription in the liver. The aim of this study was to investigate the potential role of glucose in regulation of bile acid synthesis in human hepatocytes. High glucose stimulated bile acid synthesis and induced mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory gene in bile acid synthesis. Activation of an AMP-activated protein kinase (AMPK) decreased CYP7A1 mRNA, hepatocyte nuclear factor 4alpha (HNF4alpha) protein, and binding to CYP7A1 chromatin. Glucose increased ATP levels to inhibit AMPK and induce HNF4alpha to stimulate CYP7A1 gene transcription. Furthermore, glucose increased histone acetylation and decreased H3K9 di- and tri-methylation in the CYP7A1 chromatin. Knockdown of ATP-citrate lyase, which converts citrate to acetyl-CoA, decreased histone acetylation and attenuated glucose induction of CYP7A1 mRNA expression. These results suggest that glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. This study uncovers a novel link between hepatic glucose metabolism and bile acid synthesis. Glucose induction of bile acid synthesis may have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions.
Li Tiangang; Chanda Dipanjan; Zhang Yanqiao; Choi Hueng-Sik; Chiang John Y L
Journal of lipid research
2010
2010-04
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1194/jlr.M002782" target="_blank" rel="noreferrer noopener">10.1194/jlr.M002782</a>