Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report the construction of three new vectors which can be used for the 'double-tagging' assay previously reported [Germino et al., Proc. Natl. Acad. Sci. USA 90 (1993) 933-937]. The vectors include two plasmids (pTrc.BCCP and pTrc.EZZ::BCCP) which encode different 'tags' for the capture of a target protein of interest on a filter coated with either avidin or IgG, respectively. The first plasmid (pTrc.BCCP) encodes the C terminus of the biotin carboxylase carrier protein (BCCP) under the control of the Ptac promoter, while the second produces fusions to an IgG-binding domain (EZZ). The gene encoding a protein of interest can be inserted into these plasmids and thereby direct the production of a fusion protein which is biotinylated in vivo and can bind to avidin, or a fusion protein which can bind to IgG. The third is a positive-selection, phase lambda expression vector (lambdaFJG2) which permits the construction of lacZ::cDNA fusion proteins which retain beta-galactosidase activity. The insertion of an active ecoRVR gene between the cloning sites (EcoRI and HindII or NotI) permits the positive selection of inserts. The C-terminal two-thirds of the mouse retinoblastoma-encoding gene (containing the E1A-binding pocket) was cloned into pTrc.BCCP and pTrc.EZZ::BCCP, while the 13S E1a gene was cloned into lambdaFJG2. We show that the interaction between these two proteins can be detected using the 'double-tagging' filter assay, and that this assay has high sensitivity and specificity for detecting this interaction. Finally, we have used these vectors to localize the CDK2-binding domain of the cyclin-dependent kinase inhibitor, p21. These results closely correspond to those obtained using the yeast two-hybrid assay, as well as in vitro binding assays.
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PMID:Vectors for a 'double-tagging' assay for protein-protein interactions: localization of the CDK2-binding domain of human p21. 896 91

In this work, we address the question of whether replicative senescence can be induced in immortal nontumorigenic human fibroblasts. The immortal fibroblasts used in this study were derived from two Li-Fraumeni (LF) patients who carry in their germ line one wild-type and one mutant p53 allele. Both immortal lines have lost the wtp53 allele and express no detectable p16INK4a protein, although they carry the p16INK4a gene. In contrast to immortal human fibroblasts, senescent human fibroblasts have a low content of 5-methyl-cytosine in their DNA. This observation suggested the possibility that a demethylating agent could revert the immortal phenotype and induce replicative senescence in the immortal cell lines. Cells of the two LF lines were exposed to the demethylating agent 5-aza-2'-deoxycytidine. Within 6 days, all cells were growth arrested and showed the enlarged and flat morphology characteristic of senescent cells, an accumulation of lipofuscin granules and senescence-associated beta-galactosidase activity at pH6, both biomarkers for senescence. Immunoblots of 5-aza-2'-deoxycytidine-treated cells showed a greatly increased expression of p16INK4a protein but no detectable change in the expression of p21CIP1, a gene known to be strongly expressed in senescent normal human fibroblasts. In two other experimental series, cells of the two LF lines were infected with retroviral constructs encoding either p16INK4a or p21CIP1. Each of the transduced genes induced senescence without affecting the expression of the other endogenous gene. The results show that induction of senescence in immortal LF fibroblasts can occur by different pathways: (a) by demethylation-dependent pathways that induce the expression of p16INK4a; and (b) by demethylation-independent pathways involving the expression of p21CIP1. The induction of senescence by p16INK4a and p21CIP1 occurred equally in the two human immortal fibroblast lines, which differed in the length of their telomeres and the activity of their telomerase.
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PMID:Independent induction of senescence by p16INK4a and p21CIP1 in spontaneously immortalized human fibroblasts. 948 50

After a limited number of population doublings (PDs), cultures of normal mammalian diploid cells undergo an irreversible growth arrest known as replicative senescence [1]. As well as contributing to cellular ageing, senescence is viewed as an important mechanism of tumour suppression by preventing the emergence of immortal cell clones [2-4]. Senescent cells have a number of characteristics that distinguish them from cycling or quiescent cells including elevated levels of two cyclin-dependent kinase (Cdk) inhibitors, p16INK4a and p21CIP1 [5-11]. Here, we demonstrate that both of these Cdk inhibitors, as well as other members of their protein families (the INK4 and CIP/KIP families, respectively [12]), induce several facets of the senescent phenotype when ectopically expressed in young human diploid fibroblasts. These include a reduced proliferative capacity, an altered size and shape, the presence of underphosphorylated retinoblastoma protein (pRb), increased expression of plasminogen activator inhibitor (PAI-1) and the appearance of senescence-associated beta-galactosidase (SA-beta-gal) activity [2,3,13-15]. A 20 amino acid peptide from p16INK4a that inhibits Cdks active in the G1 phase of the cell cycle [16] produces similar effects in a dose-dependent manner suggesting that, in primary fibroblasts, inhibition of G1-specific Cdk activity is sufficient to induce phenotypic changes that normally occur at the end of their finite lifespan.
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PMID:Inhibitors of cyclin-dependent kinases induce features of replicative senescence in early passage human diploid fibroblasts. 951 19

Normal human fibroblasts undergo only a limited number of divisions in culture and eventually enter a nonreplicative state designated senescence or mortality stage 1 (M1). Expression of certain viral oncogenes, such as the SV40 large T antigen (SV40 T-Ag), can elicit a significant extension of replicative life span, but these cultures eventually also cease dividing. This proliferative decline has been designated crisis or mortality stage 2 (M2). BrdU incorporation assays are commonly used to distinguish between senescence (<5% labeling index) and crisis (>30% labeling index). It has not been possible, however, to ascertain whether the high labeling index, indicative of ongoing DNA replication, was caused by the presence of T-Ag. We used gene targeting to knock out both copies of the p21(CIP1/WAF1) gene in presenescent human fibroblasts. p21 -/- cells displayed an extended life span but eventually entered a nonproliferative state. In their terminally nonproliferative state both p21 +/+ and p21 -/- cultures were positive for the senescence-associated beta-galactosidase (SA-beta-gal) activity; in contrast, the labeling index of p21 +/+ cells was low (<5%) whereas the labeling index of p21 -/- cells was high (>30%). The observation that p21 -/- and SV40 T-Ag-expressing cells behave identically with respect to life span extension as well as the high labeling index in the terminally nonproliferative state indicates that crisis is not a phenomenon induced solely by viral oncogenes, but a physiological state resulting from the bypass of normal senescence mechanisms. The widely used biomarker for senescence, SA-beta-gal, cannot distinguish between senescence and crisis. We propose that all SA-beta-gal-positive cultures should be further examined for their BrdU labeling index.
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PMID:Differentiation between senescence (M1) and crisis (M2) in human fibroblast cultures. 1058 75

The fluoroquinolone antibiotic, lomefloxacin, is phototoxic in human skin exposed to UVA radiation, photosensitises DNA strand breaks and pyrimidine dimers in human keratinocytes in vitro, and is phototumorigenic in mouse skin. The p53 tumour suppressor protein is activated by a variety of cellular insults including UV radiation, to become a transcription factor for downstream markers such as the cyclin-kinase inhibitor p21CIP1/WAF1 or cause caspase transactivation which cleaves poly ADP ribose polymerase (PARP) as an early step in apoptosis. We have investigated these molecular defence responses in human skin cells treated with lomefloxacin and UVA radiation in vitro. Western blots revealed that lomefloxacin photosensitised the stabilisation of p53 protein in human fibroblasts. Lomefloxacin also photosensitised p53 transcriptional activity in amelanotic melanoma cells expressing wild-type p53 and stably transfected with a construct containing a beta-galactosidase reporter gene downstream from a p53 consensus binding sequence. Neither photosensitised production of H2O2 nor the resultant DNA strand breaks, appeared to be involved in this effect. Interestingly, p21CIP1/WAFI protein was upregulated by lomefloxacin in the dark by a p53-independent mechanism. Lomefloxacin also photosensitised the degradation of nuclear PARP, suggestive of caspase mediated, early apoptotic events.
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PMID:The phototumorigenic fluoroquinolone, lomefloxacin, photosensitises p53 accumulation and transcriptional activity in human skin cells. 1119 49

Treatment of confluent monolayers of bovine aortic endothelial cells (BAEC) with gamma rays resulted in the delayed appearance of cells with an enlarged surface area that were morphologically similar to senescent cells. The majority of these cells stained positively for senescence-associated beta-galactosidase (SA-beta-gal), indicating that these cells are biochemically similar to senescent cells. The incidence of the senescence-like phenotype increased with dose (5-15 Gy) and time after irradiation. Cells with a senescence-like phenotype began to appear in the monolayer several days after irradiation. The onset of the appearance of this phenotype was accelerated by subculturing 24 h after irradiation. This acceleration was not entirely due to stimulation of progression through the cell cycle, since a high percentage of the senescent-like cells that appeared after subculture were not labeled with BrdUrd during the period after subculture. Prolonged up-regulation of expression of CDKN1A (also known as p21(CIP1/WAF1)) after irradiation was noted by Western blot analysis, again suggesting a similarity to natural senescence. Phenotypically altered endothelial cells were present in the irradiated monolayers as long as 20 weeks after irradiation, suggesting that a subpopulation of altered endothelial cells that might be functionally deficient could persist in the vasculature of irradiated tissue for a prolonged period after irradiation.
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PMID:Induction of a senescence-like phenotype in bovine aortic endothelial cells by ionizing radiation. 1150 Jan 32

Cellular senescence is a state of irreversible growth arrest induced either by telomere shortening (replicative senescence) or by telomere-independent signals (stress-induced senescence). The alveolar epithelium is often injured by a variety of inhaled toxins, including cigarette smoke (CS). In the present study, we investigated whether exposure to CS induces senescence of alveolar epithelial cells. In vitro experiments showed that exposure of A549 cells or normal human alveolar epithelial cells to sublethal concentrations of aqueous CS extracts induced cellular senescence. The senescence was characterized by a dose- and time-dependent increase in senescence-associated beta-galactosidase activity, senescence-associated changes in cell morphology, an increase in cell size and lysosomal mass, accumulation of lipofuscin, overexpression of p21(CIP1/WAF1/Sdi1) protein, and irreversible growth arrest. In vivo experiments in Institute for Cancer Research mice showed that inhalation of CS for 2 wk induced increases in senescence-associated beta-galactosidase activity, lipofuscin accumulation, and p21(CIP1/WAF1/Sdi1) protein expression in alveolar epithelial cells. These results suggest that CS induces a phenotype that is indistinguishable from that of senescence in alveolar epithelial cells. The induction of cellular senescence by CS may contribute to impaired re-epithelialization, leading to CS-related chronic lung diseases.
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PMID:Cigarette smoke induces senescence in alveolar epithelial cells. 1533 26

MBP-1 acts as a general transcriptional repressor. Overexpression of MBP-1 induces cell death in a number of cancer cells and regresses tumor growth. However, the function of endogenous MBP-1 in normal cell growth regulation remains unknown. To unravel the role of endogenous MBP-1, we knocked down MBP-1 expression in primary human foreskin fibroblasts (HFF) by RNA interference. Knockdown of MBP-1 in HFF (HFF-MBPsi-4) resulted in an induction of premature senescence, displayed flattened cell morphology, and increased senescence-associated beta-galactosidase activity. FACS analysis of HFF-MBPsi-4 revealed accumulation of a high number of cells in the G1-phase. A significant upregulation of cyclin D1 and reduction of cyclin A was detected in HFF-MBPsi-4 as compared to control HFF. Senescent fibroblasts exhibited enhanced expression of phosphorylated and acetylated p53, and cyclin-dependent kinase inhibitor, p21. Further analysis suggested that promyolocytic leukemia protein (PML) bodies are dramatically increased in HFF-MBPsi-4. Together, these results demonstrated that knockdown of endogenous MBP-1 is involved in cellular senescence of HFF through p53-p21 pathway.
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PMID:Knockdown of MBP-1 in human foreskin fibroblasts induces p53-p21 dependent senescence. 1885 84

The aim of this study was to explore the changes in cellular senescence related indexes of bone marrow mesenchymal stem cells (BMMSCs) after total body irradiation (TBI). At different time points after 4 Gy irradiation, BMMSCs were isolated from male C57BL/6 mice and cultured. Morphology, senescence-associated beta-galactosidase (SA-beta-gal) staining and cell cycle analysis were used to evaluate the changes in BMMSCs at cellular level while real-time RT-PCR was used to detect the alterations in senescence related gene expression including p16INK4a, p21Cip1/Waf1, p53 and TGF-beta1. The results showed that within 4 weeks after exposure to 4 Gy TBI, the morphology of BMMSCs and the expression level of SA-beta-gal were not significantly changed, the cellular senescence-related cell cycle arrest was not occurred and the senescence related gene expression level was not increased. It is concluded that at the early stage after 4 Gy TBI, the related molecular level of cellular senescence in BMMSCs is not changed.
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PMID:[Effect of total body irradiation on cellular senescence related indexes of bone marrow mesenchymal stem cells]. 1909 50

This study was purposed to investigate the relation of the Notch signaling pathway to senescence of murine bone marrow stromal cells in vitro. Intracellular domain of Notch 1 (ICN) was transfected into cultured murine bone marrow stromal cells by lipofectamine transfection. After transfection for three days the proliferation of transfected cells was measured by MTT, cell cycle distribution was analyzed by flow cytometry. The percentage of senescence associated beta-galactosidase (SA-beta-Gal) positive cells were measured by cytochemical method, and the expression rates of P53 and p21Cip1/Waf1 at gene and protein levels were analyzed by RT-PCR and Western blot respectively. The results showed that after transfection for 3 days the proliferation of murine bone marrow stromal cells was inhibited with induction of G1 arrest, the percentage of SA-beta-gal positive cells increased and the p53 and p21Cip1/Waf1 mRNA and protein expression levels were upregulated. It is concluded that the activated Notch signaling can induce premature senescence of bone marrow stromal cells through the p53-p21Cip1/Waf1 pathway.
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PMID:[Relation of notch pathway to senescence of murine bone marrow stromal cells]. 2041 78


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