161 MODULATION OF TELOMERASE ACTIVITY IN BOVINE EMBRYOS USING CYTOPLASMATIC PLASMID INJECTION

2010 ◽  
Vol 22 (1) ◽  
pp. 239
Author(s):  
W. Garrels ◽  
W. Kues ◽  
U. Baulain ◽  
H. Niemann
Keyword(s):  
Telomere Length ◽  
Telomerase Activity ◽  
Control Group ◽  
Activity Measurement ◽  
Bovine Embryos ◽  
Gfp Fluorescence ◽  
Plasmid Injection ◽  
Significant Extension ◽  
Human Telomerase ◽  
The Impact

Telomeres are repetitive, noncoding sequences at the ends of linear chromosomes that shorten with each cell division. They play an important role in aging and affect the regenerative capacity of cells. The holoenzyme telomerase rebuilds telomeres and is composed of 2 components, i.e. the catalytic protein component telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC). In mammals, telomerase is active during embryogenesis, in germ cells and a subset of stem and progenitor cells. In the present study, we set out to express the TERC component alone and then in combination with TERT, the human telomerase complex, in bovine embryos. The human telomerase components are highly homologous to bovine telomerase genes. Here, 3 different expression constructs encoding hTERC, hTERT, and a green fluorescent protein (GFP) reporter were co-injected into bovine zygotes cytoplasm, and three groups of 528, 1865, and 110 zygotes were constituted; hTERC/GFP (Group 1), hTERT/hTERC/GFP (Group 2), and GFP alone (Group 3), respectively. GFP fluorescence was used to identify successfully injected embryos. This method has recently been established in our laboratory. Injected and control embryos were cultured for 7 days to the blastocyst stage in vitro and the impact on early embryonic development and the physiological consequences of an ectopic overexpression of telomerase in early bovine embryos were assayed. We obtained 45 blastocysts with green fluorescence in the first, 192 in the second, and 28 in the third group. Embryos with GFP fluorescence were frozen for subsequent PCR analysis and telomerase activity measurement. Some blastocyts were analyzed using quantitative fluoresence in situ hybridization to monitor telomere length. Control groups were analyzed for the endogenous levels of TERC and TERT. Results indicate that endogenous TERC and TERT are up-regulated in morulae and blastocyts. In this study, we show that human TERC and TERT can be expressed in blastocysts by cytoplasmic plasmid injection in bovine zygotes. Statistical analyses were performed using the JMP 7.0.1 for Windows software (SAS Institute Inc., Cary, NC, USA). The Tukey-Kramer test was applied to compare the group means. The expression of hTERC alone resulted in a significant extension of telomere length of 280 telomere fluorescence units. Expression of both components also resulted in a significant extension of telomere length. In conclusion, TERC component alone is sufficient to elongate telomere length. The activity measurement showed that telomerase activity in the hTERT and hTERC injected group is 1.77 times higher than in the control group. Findings from this study will allow a comprehensive analysis of the functions of TERT and TERC in early embryogenesis. The ectopic expression of telomerase components in bovine embryos could pave new avenues for generating stem cells and for the development of novel regenerative therapies. Funded by DFG.

scholarly journals Impact of Yoga and Meditation on Cellular Aging in Apparently Healthy Individuals: A Prospective, Open-Label Single-Arm Exploratory Study

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Madhuri Tolahunase ◽  
Rajesh Sagar ◽  
Rima Dada
Keyword(s):  
Telomere Length ◽  
Exploratory Study ◽  
Telomerase Activity ◽  
Cellular Aging ◽  
Sirtuin 1 ◽  
Healthy Individuals ◽  
Open Label ◽  
The Mean ◽  
The Impact ◽  
Apparently Healthy

This study was designed to explore the impact of Yoga and Meditation based lifestyle intervention (YMLI) on cellular aging in apparently healthy individuals. During this 12-week prospective, open-label, single arm exploratory study, 96 apparently healthy individuals were enrolled to receive YMLI. The primary endpoints were assessment of the change in levels of cardinal biomarkers of cellular aging in blood from baseline to week 12, which included DNA damage marker 8-hydroxy-2′-deoxyguanosine (8-OH2dG), oxidative stress markers reactive oxygen species (ROS), and total antioxidant capacity (TAC), and telomere attrition markers telomere length and telomerase activity. The secondary endpoints were assessment of metabotrophic blood biomarkers associated with cellular aging, which included cortisol, β-endorphin, IL-6, BDNF, and sirtuin-1. After 12 weeks of YMLI, there were significant improvements in both the cardinal biomarkers of cellular aging and the metabotrophic biomarkers influencing cellular aging compared to baseline values. The mean levels of 8-OH2dG, ROS, cortisol, and IL-6 were significantly lower and mean levels of TAC, telomerase activity, β-endorphin, BDNF, and sirtuin-1 were significantly increased (all values p<0.05) post-YMLI. The mean level of telomere length was increased but the finding was not significant (p=0.069). YMLI significantly reduced the rate of cellular aging in apparently healthy population.

Expression Profile and up-Regulation of Telomere-Associated Proteins In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4050-4050
Author(s):  
Rafael Díaz de la Guardia ◽  
Carolina Elosua ◽  
Purificación Catalina ◽  
Brian A Walker ◽  
David C Johnson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Telomere Length ◽  
Conflicts Of Interest ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Post Translational Modification ◽  
Human Telomerase Reverse Transcriptase ◽  
Immortal Cells ◽  
Human Telomerase

Abstract Abstract 4050 The role of the telomeres in the mechanisms of ageing and carcinogenesis has generated a considerable interest as a novel approach to the treatment of many cancers. Telomeres are nucleoproteins structures that protect the ends of eukaryotic chromosomes, which are particularly vulnerable due to progressive shortening in almost all dividing cells. The telomere length was observed as a critical factor in the initiation and progression of human cancers, and it is associated to chromosomal instability. Most immortal cells possess enzymatic activity of telomerase. This suggests that telomerase activity and telomere length maintenance may be required for unlimited cell proliferation, tumorigenesis, and protection, allowing the evasion of apoptosis in cancer development. The telomerase activity could also be regulated positively or negatively by post-trancriptional and/or post-translational modification of the enzyme without transcriptional up-regulation of human telomerase reverse transcriptase (hTERT) mRNA. In this work, we analyze the expression data of all genes involved in telomerase activity. Patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), multiple myeloma (MM) and plasma cell leukemia (PLC) were studied through gene expression profiling analysis (Human Genome U133 Plus 2.0 arrays, Affymetrix). We identify 21 deregulated genes, implicated directly in telomere length maintenance activity in clonal plasma cells compared with normal cells (20 up-regulated and 1 down-regulated). These genes are MYC, KRAS, HSPA9, RB1 and members of the families: Small nucleolar ribonucleoproteins (H/ACA snoRNPs), A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs), and 14-3 -3 family. In conclusion, the myeloma cells acquire the telomere maintenance capability without deregulation of the human telomerase RNA gene (hTERC) and hTERT gene expression. It is an alternative lengthening of telomeres mechanism that has effect in the regulation of the BAD activity in apoptosis. The mechanism is based on preventing the partially-denatured proteins from aggregating, telomere maintenance through the correct processing and intranuclear trafficking of hTERC, telomerase reactivation and telomere stabilization, and efficient accumulation of hTERT in the nucleus. Thus, the findings of this study may help to improve telomerase-based therapy for multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

Telomere length, telomerase activity and mechanisms change in patients with type 2 diabetes mellitus

2016 ◽  
Vol 62 (1) ◽  
pp. 16-24 ◽  
Author(s):  
Natalia Vasil'evna Brailova ◽  
Ekaterina Nail'evna Dudinskaya ◽  
Olga Nikolaevna Tkacheva ◽  
Marina Vladimirovna Shestakova ◽  
Irina Dmitrievna Strazhesko ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Type 2 Diabetes Mellitus ◽  
Telomere Length ◽  
Chronic Inflammation ◽  
Telomerase Activity ◽  
Control Group ◽  
Diabetic Patients

Aim.To study the association of chronic inflammation, oxidative stress with telomere biology in people with type 2 diabetes mellitus (T2DM).Material and Methods.A total 50 patients with T2D and without cardiovascular disease (CVD) and 139 people from control group were included in the study. All subjects were measured for carbohydrate metabolism; oxidative stress (malondialdehyde (MDA)); inflammation (C-reactive protein — CRP, fibrinogen, interleukin-6); lymphocyte telomere length, telomerase activity.Results.In diabetic patients telomeres were shorter than in controls (9.59±0.54 and 9.76±0.47; p=0.031), telomerase activity was lower (0.47±0.40 and 0.62±0.36; p=0.039), inflammation (CRP, elevated fibrinogen) was higher. All patients were divided by telomere length. In T2DM group CRP was higher in patients with «short» telomeres (7.39±1.47 and 3.59±0.58 mg/L; p=0.02). There were no significant differences in the level of chronic inflammation and oxidative stress in ‘long’ telomeres group: CRP 3.59±0.58 and 3.66±0.50 mg/L (p=0.93), MDA 2.81±0.78 and 3.24±0.78 mmol/l (p=0.08). Diabetic patients in «short» telomeres group had greater chronic inflammation: CRP 7.39±1.47 and 4.03±0.62 mg/L (p=0.046), increased fibrinogen, 0.371 and 0.159 (p=0.022). All patients were divided by telomerase activity. Severity of chronic inflammation was greatest in T2DM and the «low» activity of telomerase. There were relationship between telomere length and CRP in T2DM patients (r=–0.40; p=0.004).Conclusions. Chronic inflammation and cell aging were more pronounced in patients with T2DM. However, despite diabetes, signs of chronic inflammation were minimal in patients with «long» telomeres compared to healthy people. Perhaps long telomeres protect diabetic patients from the damaging effect of chronic inflammation.

scholarly journals Effect of Physical Exercise in Remodeling Telomere Length and Cancer Prevention in an Epigenetic Prospect – A Systematic Review

2021 ◽  
Vol 14 (02) ◽  
pp. 891-901
Author(s):  
Lavanya Prathap ◽  
Prathap Suganthirababu S ◽  
Praveen Kumar K ◽  
Preetha S
Keyword(s):  
Systematic Review ◽  
Physical Exercise ◽  
Cancer Prevention ◽  
Telomere Length ◽  
Telomerase Activity ◽  
Risk Of Bias ◽  
Epigenetic Mechanism ◽  
The Impact ◽  
Expression Status ◽  
P16 Expression

Background: Physical exercise has its impact at the molecular level and aids in healthy well-being of an individual. The current systematic review emphasis on the impact of physical exercise on the telomere length in cancer prevention through epigenetic mechanism. Evidences support the impact of physical exercise in alteration of telomere length through its influence in telomerase activity. The aim of the systematic review is to analyze the effect of physical exercise in remodeling the telomere length in cancer prevention in an epigenetic prospect. Material and Methods: We conducted a qualitative systematic review using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The systematic literature search covers articles ranging from the year 2010 to 2020. The Database used for literature searches are PubMed, Cochrane, Science Direct and Google scholar. The Medical Subject Headings (MeSH) used for search include ‘Cancer’ ‘exercise’ ‘Telomere length’ ‘telomerase expression’. The outcome variables include the telomere length, telomerase activity, telomere protein stabilizing gene expression status, Micro RNA expression status. Results: After exclusion of irrelevant articles 05 records are selected for final inclusion of the study and are analyzed using a Cochrane risk of bias assessment tool and SANRA tool found to be at low risk of bias and moderate quality respectively. The findings suggest chronic exercise is found to modulate the genetic and epigenetic equilibrium by either up regulation of p53 and p16 expression and stabilizing the telomerase activity within the limits or by increasing the telomerase activity and stabilizing the p53 and p16 expression within limits and impact telomere length, thus maintaining the genetic and epigenetic equilibrium. Conclusion: Based on the evidences collected it can be suggested that chronic moderate intensity aerobic exercise in a lifelong practice shows beneficial effects in a dose-response manner in cancer prevention in a novel way by modulating telomeres through epigenetic mechanism.

103 ECTOPIC EXPRESSION OF TELOMERASE IN BOVINE PREIMPLANTATION EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 132
Author(s):  
K. Iqbal ◽  
W. A. Kues ◽  
H. Niemann
Keyword(s):  
Telomere Length ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Fluid Medium ◽  
Gfp Fluorescence ◽  
Morula Stage

Recently, we have demonstrated a stage-specific increase of telomere length at morula-blastocyst transition in bovine and murine embryos (Schaetzlein et al. 2004 PNAS 101, 8034–8038). Telomeres are composed of repetitive hexanucleotide sequences (TTAGGG) encompassing several kilobasepairs and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage up to the morula stage, telomerase is not active or only found at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) was selected as the target molecule due to its 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from local abattoir followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. In preliminary tests, it was shown that co-injection of green fluorescent protein (GFP) and red fluorescent protein (dsRed) constructs resulted in the simultaneous expression of the 2 proteins. The onset of fluorescent protein expression was recorded 30 to 40 hours after injection by fluorescence microscopy. Because all cDNA were driven by the cytomegalovirus (CMV) promoter, it was assumed that hTERT is expressed in parallel. In the main experiment, 2 constructs encoding human TERT and GFP were co-injected to allow live separation of embryos. A total of 400 bovine embryos were injected, 209 (53%) of the treated embryos showed specific GFP fluorescence; out of a total of 104 blastocysts (26%), 55 showed GFP fluorescence (53%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic human TERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. Surprisingly, expression pattern of the endogenous TERT revealed a transient increase at the 2–4 cell stage and a major increase at the blastocyst stage. The mRNA level of the ectopic hTERT started to rise from 4 to 8 cell stage and remained high up to the morula stage. Currently, qFISH and TRAP techniques are being employed to assess enzymatic activity of hTERT and to quantitatively determine telomere length. In conclusion, this study demonstrates the ectopic expression of TERT and fluorescent proteins in early embryos; overexpression of TERT may facilitate the derivation of bovine ES cells. Supported by DFG and Goyaike S.A.A.C.I. Y.F.

277 PLASMID MEDIATED EXPERIMENTAL TELOMERE EXTENSION IN BOVINE EMBRYOS BY ECTOPIC EXPRESSION OF HUMAN TERT

2009 ◽  
Vol 21 (1) ◽  
pp. 235 ◽  
Author(s):  
K. Iqbal ◽  
W. A. Kues ◽  
H. Niemann
Keyword(s):  
Telomere Length ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Mrna Levels ◽  
Bovine Embryos ◽  
Fluid Medium ◽  
Gfp Fluorescence ◽  
Morula Stage

Telomeres are composed of repetitive hexanucleotide sequences, (TTAGGG)n, encompassing several kilobase pairs, and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage and up to the morula stage, telomerase is not active or is found only at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) has 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from a local abattoir, followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. Two plasmid encoding CMV promoter-driven sequences of hTERT and green fluorescent protein (GFP) were coinjected in bovine zygotes, and GFP driven by a muscle specific promoter was injected for mock experiments. The hTERT and GFP were co-injected to allow live separation of embryos. A total of 768 bovine embryos were injected; 468 (61%) of the treated embryos showed specific GFP-fluorescence. Of a total of 132 blastocysts (17%), 45 showed GFP fluorescence (34%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic hTERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. The mRNA level of the ectopic hTERT began to increase from the 4- to the 8-cell stage and remained high up to the morula stage. Embryos at the morula and blastocyst stages were spread on slides and analyzed by quantitative fluorescence in situ hybridization (qFISH). A Cy3-labeled 18-mer peptide nucleic acid (PNA) probe was used to hybridize the telomeres. The resulting spot intensities were quantified by using TFL-Telo software and were statistically analyzed. A modest increase in telomere length was observed in hTERT injected [775 ± 69 fluorescence unit (fu)] group compared to uninjected control (679 ± 75 fu) group at blastocyst stage. In conclusion, this study demonstrates that the ectopic expression of hTERT in embryos results in telomere elongation; overexpression of TERT may facilitate the derivation of bovine embryonic stem cells. Supported by DFG and Goyaike SAACIYF.

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