Organization and In Vitro Expression of Human tRNAGlu Genes

Prior to the start of this work several recombinants containing tRNA genes were selected from a bank of 15-20 kb fragments of a human foetal DNA, cloned in l Charon 4A,by screening with a mixed placentae tRNA[32P]pCp probe (Goddard et al. , 1983). Two of these recombinants, lht137 and lht190, generated restriction fragment profiles which were very similar but not identical. Two common 2.4 kb Hindlll fragments which hybridized the probe used, were subcloned from lht137 and lht190 into the vector pAT153 to generate recombinants pLB4 and pTC51 respectively. The characterized recombinant pLB4 was found to contain a single tRNA Glu gene, notable for its high transcriptional activity and the potential of its 5'-flanking sequence for forming a tRNA-like structure (Goddard et al., 1983)

Human tRNAGlu genes: their copy number and organisation

AbstractThe tRNAGlu gene copy number, determined by genomic blot analysis of human plaeental DNA, is approximately thirteen. These studies, using several probes and DNA digested with several restriction enzymes singly or in combination, show that most of these tRNAGlu genes are flanked by DNA of very similar sequence for at least 5 kb. This conclusion is supported by the close similarity of the restriction maps of two λ Charon-4A recombinants of human genomic DNA containing two different tRNAglu genes

Protein modification and maintenance systems as biomarkers of ageing

Changes in the abundance and post-translational modification of proteins and accumulation of some covalently modified proteins have been proposed to represent hallmarks of biological ageing. Within the frame of the Mark-Age project, the workpackage dedicated to "markers based on proteins and their modifications" has been firstly focused on enzymatic and non-enzymatic post-translational modifications of serum proteins by carbohydrates. The second focus of the workpackage has been directed towards protein maintenance systems that are involved either in protein quality control (ApoJ/Clusterin) or in the removal of oxidatively damaged proteins through degradation and repair (proteasome and methionine sulfoxide reductase systems). This review describes the most relevant features of these protein modifications and maintenance systems, their fate during ageing and/or their implication in ageing and longevity

Biological Monitoring of Hexavalent Chromium and Serum Levels of the Senescence Biomarker Apolipoprotein J/Clusterin in Welders

Welding fumes contain metals and other toxic substances known or strongly suspected to be related with oxidative stress and premature cellular senescence. Apolipoprotein J/Clusterin (ApoJ/CLU) is a glycoprotein that is differentially regulated in various physiological and disease states including ageing and age-related diseases. In vitro data showed that exposure of human diploid fibroblasts to hexavalent chromium (Cr(VI)) resulted in premature senescence and significant upregulation of the ApoJ/CLU protein. In this study we analyzed blood and urine samples from shipyard industry welders being exposed to different levels of Cr(VI) over a period of five months in order to assay in vivo the relation of ApoJ/CLU serum levels with Cr(VI). Our findings confirmed the previously reported in vitro data since reduction of Cr levels, after a worksite intervention, associated with lower levels of ApoJ/CLU serum levels. We concluded that the human ApoJ/CLU gene is responsive to the acute in vivo oxidative stress induced by heavy metals such as hexavalent chromium

18 alpha-Glycyrrhetinic Acid Proteasome Activator Decelerates Aging and Alzheimer's Disease Progression in Caenorhabditis elegans and Neuronal Cultures

Aims: Proteasomes are constituents of the cellular proteolytic networks that maintain protein homeostasis through regulated proteolysis of normal and abnormal (in any way) proteins. Genetically mediated proteasome activation in multicellular organisms has been shown to promote longevity and to exert protein antiaggregation activity. In this study, we investigate whether compound-mediated proteasome activation is feasible in a multicellular organism and we dissect the effects of such approach in aging and Alzheimer's disease (AD) progression. Results: Feeding of wild-type Caenorhabditis elegans with 18 alpha-glycyrrhetinic acid (18 alpha-GA; a previously shown proteasome activator in cell culture) results in enhanced levels of proteasome activities that lead to a skinhead-1- and proteasomeactivation-dependent life span extension. The elevated proteasome function confers lower paralysis rates in various AD nematode models accompanied by decreased A beta deposits, thus ultimately decelerating the progression of AD phenotype. More importantly, similar positive results are also delivered when human and murine cells of nervous origin are subjected to 18 alpha-GA treatment. Innovation: This is the first report of the use of 18 alpha-GA, a diet-derived compound as prolongevity and antiaggregation factor in the context of a multicellular organism. Conclusion: Our results suggest that proteasome activation with downstream positive outcomes on aging and AD, an aggregation-related disease, is feasible in a nongenetic manipulation manner in a multicellular organism. Moreover, they unveil the need for identification of antiaging and antiamyloidogenic compounds among the nutrients found in our normal diet.Peer reviewe

Postmitotic neurons develop a p21-dependent senescence-like phenotype driven by a DNA damage response

In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro-inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence like state in mature postmitotic neurons in vivo. About 40 80% of Purkinje neurons and 20–40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl ⁄ 6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL-6 production, heterochromatinization and senescence-associated b-galactosidase activity. Frequencies of these senescence-like neurons increased with age. Short term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late-generation TERC) ⁄ ) mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late- generation TERC) ⁄ )CDKN1A) ⁄ ) mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence-like phenotype in neurons, as in senescing fibroblasts and other proliferation competent cells. We conclude that a senescence-like phenotype is possibly not restricted to proliferation-competent cells. Rather, dysfunctional telomeres and ⁄ or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence-like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging

Involvement of proteasome activation

Alzheimer's disease (AD) is the most common form of dementia worldwide, characterized by a progressive decline in a variety of cognitive and non-cognitive functions. The amyloid beta protein cascade hypothesis places the formation of amyloid beta protein aggregates on the first position in the complex pathological cascade leading to neurodegeneration, and therefore AD might be considered to be a protein-misfolding disease. The Ubiquitin Proteasome System (UPS), being the primary protein degradation mechanism with a fundamental role in the maintenance of proteostasis, has been identified as a putative therapeutic target to delay and/or to decelerate the progression of neurodegenerative disorders that are characterized by accumulated/aggregated proteins. The purpose of this study was to test if the activation of proteasome in vivo can alleviate AD pathology. Specifically by using two compounds with complementary modes of proteasome activation and documented antioxidant and redox regulating properties in the 5xFAD transgenic mice model of AD, we ameliorated a number of AD related deficits. Shortly after proteasome activation we detected significantly reduced amyloid-beta load correlated with improved motor functions, reduced anxiety and frailty level. Essentially, to our knowledge this is the first report to demonstrate a dual activation of the proteasome and its downstream effects. In conclusion, these findings open up new directions for future therapeutic potential of proteasome-mediated proteolysis enhancement.publishersversionpublishe

Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARKAGE Study

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project ‘MARK-AGE’. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly

Age-dependent expression of DNMT1 and DNMT3B in PBMCs from a large European population enrolled in the MARK-AGE study

Aging is associated with alterations in the content and patterns of DNA methylation virtually throughout the entire human lifespan. Reasons for these variations are not well understood. However, several lines of evidence suggest that the epigenetic instability in aging may be traced back to the alteration of the expression of DNA methyltransferases. Here, the association of the expression of DNA methyltransferases DNMT1 and DNMT3B with age has been analysed in the context of the MARK-AGE study, a large-scale cross-sectional study of the European general population. Using peripheral blood mononuclear cells, we assessed the variation of DNMT1 and DNMT3B gene expression in more than two thousand age-stratified women and men (35-75 years) recruited across eight European countries. Significant age-related changes were detected for both transcripts. The level of DNMT1 gradually dropped with aging but this was only observed up to the age of 64 years. By contrast, the expression of DNMT3B decreased linearly with increasing age and this association was particularly evident in females. We next attempted to trace the age-related changes of both transcripts to the influence of different variables that have an impact on changes of their expression in the population, including demographics, dietary and health habits, and clinical parameters. Our results indicate that age affects the expression of DNMT1 and DNMT3B as an almost independent variable in respect of all other variables evaluated

Ресурсоэффективные системы в управлении и контроле: взгляд в будущее (т. 2): сборник научных трудов VII Международной конференции школьников, студентов, аспирантов, молодых ученых, 8 -13 октября 2018 г., г. Томск

В сборнике представлены материалы VII Международной конференции школьников, студентов, аспирантов, молодых ученых "Ресурсоэффективные системы в управлении и контроле: взгляд в будущее". Более 500 авторов из 35 вузов, предприятий и научных исследовательских университетов России, ближнего и дальнего зарубежья представили тезисы своих докладов, в которых рассматриваются актуальные проблемы неразрушающего контроля и технической диагностики, внедрения систем менеджмента, качества образования, управления в современной экономике. Материалы предназначены для специалистов, преподавателей, аспирантов и студентов вузов, а также для всех интересующихся проблемами ресурсоэффективных технологий