scholarly journals Vault Poly(ADP-Ribose) Polymerase Is Associated with Mammalian Telomerase and Is Dispensable for Telomerase Function and Vault Structure In Vivo

2004 ◽  
Vol 24 (12) ◽  
pp. 5314-5323 ◽  
Author(s):  
Yie Liu ◽  
Bryan E. Snow ◽  
Valerie A. Kickhoefer ◽  
Natalie Erdmann ◽  
Wen Zhou ◽  
...  
Keyword(s):  
Telomere Length ◽  
Telomerase Activity ◽  
Molecular Assembly ◽  
Cellular Functions ◽  
Telomere Function ◽  
Minor Protein ◽  
The Stability ◽  
A Minor

ABSTRACT Vault poly(ADP-ribose) polymerase (VPARP) was originally identified as a minor protein component of the vault ribonucleoprotein particle, which may be involved in molecular assembly or subcellular transport. In addition to the association of VPARP with the cytoplasmic vault particle, subpopulations of VPARP localize to the nucleus and the mitotic spindle, indicating that VPARP may have other cellular functions. We found that VPARP was associated with telomerase activity and interacted with exogenously expressed telomerase-associated protein 1 (TEP1) in human cells. To study the possible role of VPARP in telomerase and vault complexes in vivo, mVparp-deficient mice were generated. Mice deficient in mVparp were viable and fertile for up to five generations, with no apparent changes in telomerase activity or telomere length. Vaults purified from mVparp-deficient mouse liver appeared intact, and no defect in association with other vault components was observed. Mice deficient in mTep1, whose disruption alone does not affect telomere function but does affect the stability of vault RNA, showed no additional telomerase or telomere-related phenotypes when the mTep1 deficiency was combined with an mVparp deficiency. These data suggest that murine mTep1 and mVparp, alone or in combination, are dispensable for normal development, telomerase catalysis, telomere length maintenance, and vault structure in vivo.

scholarly journals Swc4 positively regulates telomere length independently of its roles in NuA4 and SWR1 complexes

2020 ◽  
Vol 48 (22) ◽  
pp. 12792-12803
Author(s):  
Jia-Cheng Liu ◽  
Qian-Jin Li ◽  
Ming-Hong He ◽  
Can Hu ◽  
Pengfei Dai ◽  
...  
Keyword(s):  
Telomere Length ◽  
Essential Gene ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Direct Role ◽  
Telomere Shortening ◽  
Length Regulation ◽  
Telomere Length Regulation

Abstract Telomeres at the ends of eukaryotic chromosomes are essential for genome integrality and stability. In order to identify genes that sustain telomere maintenance independently of telomerase recruitment, we have exploited the phenotype of over-long telomeres in the cells that express Cdc13-Est2 fusion protein, and examined 195 strains, in which individual non-essential gene deletion causes telomere shortening. We have identified 24 genes whose deletion results in dramatic failure of Cdc13-Est2 function, including those encoding components of telomerase, Yku, KEOPS and NMD complexes, as well as quite a few whose functions are not obvious in telomerase activity regulation. We have characterized Swc4, a shared subunit of histone acetyltransferase NuA4 and chromatin remodeling SWR1 (SWR1-C) complexes, in telomere length regulation. Deletion of SWC4, but not other non-essential subunits of either NuA4 or SWR1-C, causes significant telomere shortening. Consistently, simultaneous disassembly of NuA4 and SWR1-C does not affect telomere length. Interestingly, inactivation of Swc4 in telomerase null cells accelerates both telomere shortening and senescence rates. Swc4 associates with telomeric DNA in vivo, suggesting a direct role of Swc4 at telomeres. Taken together, our work reveals a distinct role of Swc4 in telomere length regulation, separable from its canonical roles in both NuA4 and SWR1-C.

scholarly journals Metabolism of rat bone proteoglycans in vivo

1983 ◽  
Vol 216 (3) ◽  
pp. 589-596 ◽  
Author(s):  
C W Prince ◽  
F Rahemtulla ◽  
W T Butler
Keyword(s):  
Extraction Procedure ◽  
Total Radioactivity ◽  
Mineralized Tissue ◽  
Guanidinium Chloride ◽  
Disaccharide Composition ◽  
A Minor ◽  
Papain Digestion ◽  
Rat Bone

Former evaluations of the role of proteoglycans in mineralization have neglected to address the possibility that the metabolism of proteoglycans may be of significance in this regard. This problem was studied by using radiolabeling in vivo of rat calvaria with [35Sulphate for 2-72 h and a sequential extraction procedure to yield two pools of newly synthesized proteoglycans: one obtained from non-mineralized tissue by extraction with guanidinium chloride (GdmCl) and another obtained only after demineralization with EDTA. Total radioactivity in calvaria was maximal after 12 h of incorporation, but by 36 h had declined to a level that was about 55-65% of maximum. Radioactivity in the GdmCl extract declined steadily after 12 h, whereas that in the EDTA extract remained constant until 36 h, when it began to increase. Each extract contained a minor proteoglycan that eluted at the void volume (Vo) of a Sepharose CL-6B column. Unlike in the EDTA extract, this proteoglycan gradually disappeared from the GdmCl extract. Each extract also contained a major, smaller proteoglycan, with a Kav. of 0.24 and 0.36 in the GdmCl and EDTA extracts respectively. Papain digestion of each extract yielded glycosaminoglycan chains with Kav. values of 0.32 and 0.50 on CL-6B in the GdmCl and EDTA extracts respectively. Digestion of each extract with chondroitinase ABC and chondroitinase AC showed that the glycosaminoglycans were of similar disaccharide composition, with about 85% being 4-sulphated and the remainder 6-sulphated and/or iduronic acid-containing. These data suggest that about 45% of the newly synthesized proteoglycans are removed from the tissue during the course of mineralization.

Functional specificity of jejunal brush-border pteroylpolyglutamate hydrolase in pig

1991 ◽  
Vol 260 (6) ◽  
pp. G865-G872 ◽  
Author(s):  
C. J. Chandler ◽  
D. A. Harrison ◽  
C. A. Buffington ◽  
N. A. Santiago ◽  
C. H. Halsted
Keyword(s):  
Brush Border ◽  
Protein Band ◽  
Major Protein ◽  
Functional Specificity ◽  
Major Protein Band ◽  
Regional Location ◽  
Minor Protein ◽  
A Minor ◽  
Hydrolysis Of

To determine the functional specificity of intestinal brush-border pteroylpolyglutamate hydrolase (PPH), we compared the regional location of in vivo hydrolysis of pteroyltriglutamate (PteGlu3) with the location of activity and immunoreactivity of the enzyme in the pig. After in vivo incubations, PteGlu3 hydrolytic products were recovered from intestinal segments in the jejunum but not from the ileum. Brush-border PPH activity in fractionated mucosa was 10-fold greater in the jejunum than in the ileum, whereas the activity of intracellular PPH was increased in the distal ileum. Antibodies to purified brush-border PPH identified a major protein band at 120 kDa and a minor protein band at 195 kDa in solubilized jejunal brush border. Immunohistochemistry identified the enzyme only on the brush-border surface of the jejunum, whereas an immunoblot of solubilized brush-border membranes identified brush-border PPH in the jejunum but not in the ileum. The parallel of the regional location of in vivo hydrolysis of PteGlu3 with the location of brush-border PPH activity and immunoreactivity demonstrates the functional specificity of this enzyme in folate digestion.

scholarly journals HNRNPA2B1 promotes multiple myeloma progression by increasing AKT3 expression via m6A-dependent stabilization of ILF3 mRNA

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Fengjie Jiang ◽  
Xiaozhu Tang ◽  
Chao Tang ◽  
Zhen Hua ◽  
Mengying Ke ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cellular Proliferation ◽  
Aberrant Expression ◽  
The Stability ◽  
Induced Apoptosis ◽  
Proliferation In Vitro

AbstractN6-methyladenosine (m6A) modification is the most prevalent modification in eukaryotic RNAs while accumulating studies suggest that m6A aberrant expression plays an important role in cancer. HNRNPA2B1 is a m6A reader which binds to nascent RNA and thus affects a perplexing array of RNA metabolism exquisitely. Despite unveiled facets that HNRNPA2B1 is deregulated in several tumors and facilitates tumor growth, a clear role of HNRNPA2B1 in multiple myeloma (MM) remains elusive. Herein, we analyzed the function and the regulatory mechanism of HNRNPA2B1 in MM. We found that HNRNPA2B1 was elevated in MM patients and negatively correlated with favorable prognosis. The depletion of HNRNPA2B1 in MM cells inhibited cell proliferation and induced apoptosis. On the contrary, the overexpression of HNRNPA2B1 promoted cell proliferation in vitro and in vivo. Mechanistic studies revealed that HNRNPA2B1 recognized the m6A sites of ILF3 and enhanced the stability of ILF3 mRNA transcripts, while AKT3 downregulation by siRNA abrogated the cellular proliferation induced by HNRNPA2B1 overexpression. Additionally, the expression of HNRNPA2B1, ILF3 and AKT3 was positively associated with each other in MM tissues tested by immunohistochemistry. In summary, our study highlights that HNRNPA2B1 potentially acts as a therapeutic target of MM through regulating AKT3 expression mediated by ILF3-dependent pattern.

33 TELOMERASE ACTIVITIES IN CLONED BEAGLE DOGS

2015 ◽  
Vol 27 (1) ◽  
pp. 109
Author(s):  
G. A. Kim ◽  
H. J. Oh ◽  
M. J. Kim ◽  
Y. K. Jo ◽  
E. M. N. Setyawan ◽  
...  
Keyword(s):  
Telomere Length ◽  
Donor Cell ◽  
Telomerase Activity ◽  
Physiological Age ◽  
Biological Aging ◽  
Amplification Protocol ◽  
Donor Cells ◽  
Activity Measurements ◽  
Natural Breeding

Telomerase is important ribonucleoprotein for restoring telomere length from its own RNA template. Regarding cloned animals derived from somatic cell nuclear transfer (SCNT), interesting questions have been raised about whether the cloning process restores cellular telomerase activity undergone by their donor cells. The present study was conducted to determine the effects of cloning on telomerase activity in the dog and normality of telomerase activity in cloned dogs. Focusing our attention on differences in telomerase activity depending on the age, we analysed telomerase activity in dogs produced by natural breeding of various ages. Comparison of the telomerase activities of cloned dogs and those of dogs produced by natural breeding was also performed. For SCNT, 2 cell donors, 7- and 9-year-old beagles, were used and donor cells were isolated from ear skin. After establishing donor cell lines, the enucleated canine in vivo-matured oocytes and the cells were injected and fused by electrofusion. After 30 days from embryo transfer, pregnancy diagnosis was performed and 7 cloned dogs were produced on the due date. For standardization of telomerase activity in beagles produced by natural breeding, blood of total 14 dogs at each age (10 months, 20 months, 5, 7, and 8 years old) were collected and telomerase activity was measured by the telomeric repeat amplification protocol (TRAP) assay. Telomerase activity measurements of at least 6 replications in each dog were performed. For statistical analysis, one-way ANOVA with Dunn's Multiple Comparison Test was used. Significant differences in telomerase activity were observed between the blood of cloned and donor dogs. It was shown that mean telomerase activities were decreased according to biological aging with significances. Mean telomerase activities in 10 cloned dogs were higher than those of a donor dog. Cloned dogs also showed similar levels of telomerase activities as their age-matched natural bred dogs, suggesting that they are within the variation in normal dogs. These observations indicate that the cloning process restores the telomerase activity in the dog. Thus, complex regulation of telomerase activity during nuclear reprogramming may regulate and be involved in telomerase activity in cloned dogs. It remains to be determined whether telomere length is correlated with telomerase activity and if it accurately reflects the physiological age of cloned dogs.This study was supported by IPET (#311062–04–2-SB010), RDA (PJ008975022013), Research Institute for Veterinary Science, the BK21 program, Nestle Purina Korea, and TS Corporation.

scholarly journals TIN2 Functions with TPP1/POT1 To Stimulate Telomerase Processivity

2019 ◽  
Vol 39 (21) ◽  
Author(s):  
Alexandra M. Pike ◽  
Margaret A. Strong ◽  
John Paul T. Ouyang ◽  
Carol W. Greider
Keyword(s):  
Telomere Length ◽  
Telomerase Activity ◽  
Human Cells ◽  
Short Telomere ◽  
Gene Encoding ◽  
Important Regulator

ABSTRACT TIN2 is an important regulator of telomere length, and mutations in TINF2, the gene encoding TIN2, cause short-telomere syndromes. While the genetics underscore the importance of TIN2, the mechanism through which TIN2 regulates telomere length remains unclear. Here, we tested the effects of human TIN2 on telomerase activity. We identified a new isoform in human cells, TIN2M, that is expressed at levels similar to those of previously studied TIN2 isoforms. All three TIN2 isoforms localized to and maintained telomere integrity in vivo, and localization was not disrupted by telomere syndrome mutations. Using direct telomerase activity assays, we discovered that TIN2 stimulated telomerase processivity in vitro. All of the TIN2 isoforms stimulated telomerase to similar extents. Mutations in the TPP1 TEL patch abrogated this stimulation, suggesting that TIN2 functions with TPP1/POT1 to stimulate telomerase processivity. We conclude from our data and previously published work that TIN2/TPP1/POT1 is a functional shelterin subcomplex.

scholarly journals Expression of bovine superoxide dismutase in Drosophila melanogaster augments resistance of oxidative stress.

1991 ◽  
Vol 11 (2) ◽  
pp. 632-640 ◽  
Author(s):  
I Reveillaud ◽  
A Niedzwiecki ◽  
K G Bensch ◽  
J E Fleming
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Promoter ◽  
Adult Mortality ◽  
P Elements ◽  
Transgenic Lines ◽  
A Minor ◽  
Radical Damage ◽  
Positive Effect

Superoxide dismutases (SOD) play a major role in the intracellular defense against oxygen radical damage to aerobic cells. In eucaryotes, the cytoplasmic form of the enzyme is a 32-kDa dimer containing two copper and two zinc atoms (CuZn SOD) that catalyzes the dismutation of the superoxide anion (O2-) to H2O2 and O2. Superoxide-mediated damage has been implicated in a number of biological processes, including aging and cancer; however, it is not certain whether endogenously elevated levels of SOD will reduce the pathological events resulting from such damage. To understand the in vivo relationship between an efficient dismutation of O2- and oxidative injury to biological structures, we generated transgenic strains of Drosophila melanogaster overproducing CuZn SOD. This was achieved by microinjecting Drosophila embryos with P-elements containing bovine CuZn SOD cDNA under the control of the Drosophila actin 5c gene promoter. Adult flies of the resulting transformed lines which expressed both mammalian and Drosophila CuZn SOD were then used as a novel model for evaluating the role of oxygen radicals in aging. Our data show that expression of enzymatically active bovine SOD in Drosophila flies confers resistance to paraquat, an O2(-)-generating compound. This is consistent with data on adult mortality, because there was a slight but significant increase in the mean lifespan of several of the transgenic lines. The highest level of expression of the active enzyme in adults was 1.60 times the normal value. Higher levels may have led to the formation of toxic levels of H2O2 during development, since flies that died during the process of eclosion showed an unusual accumulation of lipofuscin (age pigment) in some of their cells. In conclusion, our data show that free-radical detoxification has a minor by positive effect on mean longevity for several strains.

scholarly journals Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

2008 ◽  
Vol 28 (7) ◽  
pp. 2380-2390 ◽  
Author(s):  
Hong Ji ◽  
Christopher J. Adkins ◽  
Bethany R. Cartwright ◽  
Katherine L. Friedman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Specific Binding ◽  
Telomerase Activity ◽  
Negative Regulator ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomere Length Homeostasis

ABSTRACT In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.

Telomerase Activity and Telomere Length of CD4+CD25+ Regulatory T-Cells under Conditions of In Vitro and In Vivo Expansion.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3857-3857
Author(s):  
Dominik G.F. Wolf ◽  
Anna M. Wolf ◽  
Christian Koppelstaetter ◽  
Holger F. Rumpold ◽  
Gert Mayer ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Telomere Length ◽  
Cancer Patients ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Healthy Human ◽  
Telomere Shortening

Abstract The expandability of CD4+CD25+ regulatory T-cells (Treg) has been shown in vitro and in vivo. Activation of telomerase activity is a prerequisite for clonal expansion and telomere maintenance in T-cells. There is currently no data available on the expression and function of telomerase in proliferating Treg. Analyses of telomere length by flow-FISH, real-time PCR and Southern blotting revealed that Treg isolated from healthy human volunteers have significantly shortened telomeres when compared to CD4+CD25− T-cells. However, telomere length is not further shortened in Treg isolated from the peripheral blood of cancer patients, despite the observation that the regulatory T-cell pool of these patients was significantly enlarged. To gain further insight into maintenance of telomere length of Treg, we induced in vitro proliferation of Treg by stimulation with anti-CD3 and IL-2. This led to a rapid increase of telomerase activity, as determined by PCR-ELISA. However, when we focused on the proliferating fraction of Treg using a sorting strategy based on the dilution of CFSE, we could show a significant telomere shortening in Treg with high proliferative and immmuno-suppressive capacity. Of note, proliferating CFSElow Treg are characterized by high telomerase activity, which however seems to be insufficient to avoid further telomere shortening under conditions of strong in vitro stimulation. In contrast, under conditions of in vivo expansion of Treg in cancer patients, the induction of telomerase activity is likely to compensate for further telomere erosion. These data might be of importance when considering the application of in vitro expanded Treg for the treatment of GvHD or autoimmune diseases, as telomere shortening might be associated with genomic instability.

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