Human Telomerase Reverse Transcriptase (hTERT) Q169 Is Essential for Telomerase Function In Vitro and In Vivo

BACKGROUND:Telomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS:To understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT) Q169 to create neutral (Q169A), conservative (Q169N), and non-conservative (Q169D) mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function. CONCLUSIONS/SIGNIFICANCE:We provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme-primer interactions and nucleotide incorporation. We show that Q169 is critical for the structure and function of human telomerase, thereby identifying a novel residue in hTERT that may be amenable to therapeutic intervention

The associations between body and knee height measurements and knee joint structure in an asymptomatic cohort

<p>Abstract</p> <p>Background</p> <p>It has been suggested that knee height is a determinant of knee joint load. Nonetheless, no study has directly examined the relationship between anthropometric measures of height and knee joint structures, such as cartilage.</p> <p>Methods</p> <p>89 asymptomatic community-based adults aged 25-62 with no diagnosed history of knee arthropathy were recruited. Anthropometric data (knee height and body height) were obtained by standard protocol, while tibial cartilage volume and defects, as well as bone area were determined from magnetic resonance imaging. Static knee alignment was measured from the joint radiograph.</p> <p>Results</p> <p>All anthropometric height measures were associated with increasing compartmental tibial bone area (<it>p </it>≤ 0.05). Although knee height was associated with tibial cartilage volume (e.g. β = 27 mm³95% CI 7- 48; <it>p </it>= 0.009 for the medial compartment), these relationship no longer remained significant when knee height as a percentage of body height was analysed. Knee height as a percentage of body height was associated with a reduced risk of medial tibial cartilage defects (odds ratio 0.6; 95% confidence interval 0.4 - 1.0; <it>p </it>= 0.05).</p> <p>Conclusion</p> <p>The association between increased anthropometric height measures and increased tibial bone area may reflect inherently larger bony structures. However the beneficial associations demonstrated with cartilage morphology suggest that an increased knee height may confer a beneficial biomechanical environment to the chondrocyte of asymptomatic adults.</p

Primitive layered gabbros from fast-spreading lower oceanic crust

Three-quarters of the oceanic crust formed at fast-spreading ridges is composed of plutonic rocks whose mineral assemblages, textures and compositions record the history of melt transport and crystallization between the mantle and the sea floor. Despite the importance of these rocks, sampling them in situ is extremely challenging owing to the overlying dykes and lavas. This means that models for understanding the formation of the lower crust are based largely on geophysical studies and ancient analogues (ophiolites) that did not form at typical mid-ocean ridges. Here we describe cored intervals of primitive, modally layered gabbroic rocks from the lower plutonic crust formed at a fast-spreading ridge, sampled by the Integrated Ocean Drilling Program at the Hess Deep rift. Centimetre-scale, modally layered rocks, some of which have a strong layering-parallel foliation, confirm a long-held belief that such rocks are a key constituent of the lower oceanic crust formed at fast-spreading ridges. Geochemical analysis of these primitive lower plutonic rocks-in combination with previous geochemical data for shallow-level plutonic rocks, sheeted dykes and lavas-provides the most completely constrained estimate of the bulk composition of fast-spreading oceanic crust so far. Simple crystallization models using this bulk crustal composition as the parental melt accurately predict the bulk composition of both the lavas and the plutonic rocks. However, the recovered plutonic rocks show early crystallization of orthopyroxene, which is not predicted by current models of melt extraction from the mantle and mid-ocean-ridge basalt differentiation. The simplest explanation of this observation is that compositionally diverse melts are extracted from the mantle and partly crystallize before mixing to produce the more homogeneous magmas that erupt

The evolution and storage of primitive melts in the Eastern Volcanic Zone of Iceland: the 10 ka Grímsvötn tephra series (i.e. the Saksunarvatn ash)

Major, trace and volatile elements were measured in a suite of primitive macrocrysts and melt inclusions from the thickest layer of the 10 ka Grímsvötn tephra series (i.e. Saksunarvatn ash) at Lake Hvítárvatn in central Iceland. In the absence of primitive tholeiitic eruptions (MgO > 7 wt.%) within the Eastern Volcanic Zone (EVZ) of Iceland, these crystal and inclusion compositions provide an important insight into magmatic processes in this volcanically productive region. Matrix glass compositions show strong similarities with glass compositions from the AD 1783–84 Laki eruption, confirming the affinity of the tephra series with the Grímsvötn volcanic system. Macrocrysts can be divided into a primitive assemblage of zoned macrocryst cores (An_78–An_92, Mg#_cpx = 82–87, Fo_79.5–Fo_87) and an evolved assemblage consisting of unzoned macrocrysts and the rims of zoned macrocrysts (An_60–An_68, Mg#_cpx = 71–78, Fo_70–Fo_76). Although the evolved assemblage is close to being in equilibrium with the matrix glass, trace element disequilibrium between primitive and evolved assemblages indicates that they were derived from different distributions of mantle melt compositions. Juxtaposition of disequilibrium assemblages probably occurred during disaggregation of incompatible trace element-depleted mushes (mean La/Yb_melt = 2.1) into aphyric and incompatible trace element-enriched liquids (La/Yb_melt = 3.6) shortly before the growth of the evolved macrocryst assemblage. Post-entrapment modification of plagioclase-hosted melt inclusions has been minimal and high-Mg# inclusions record differentiation and mixing of compositionally variable mantle melts that are amongst the most primitive liquids known from the EVZ. Coupled high field strength element (HFSE) depletion and incompatible trace element enrichment in a subset of primitive plagioclase-hosted melt inclusions can be accounted for by inclusion formation following plagioclase dissolution driven by interaction with plagioclase-undersaturated melts. Thermobarometric calculations indicate that final crystal-melt equilibration within the evolved assemblage occurred at ~1140°C and 0.0–1.5 kbar. Considering the large volume of the erupted tephra and textural evidence for rapid crystallisation of the evolved assemblage, 0.0–1.5 kbar is considered unlikely to represent a pressure of long-term magma accumulation and storage. Multiple thermometers indicate that the primitive assemblage crystallised at high temperatures of 1240–1300°C. Different barometers, however, return markedly different crystallisation depth estimates. Raw clinopyroxene-melt pressures of 5.5–7.5 kbar conflict with apparent melt inclusion entrapment pressures of 1.4 kbar. After applying a correction derived from published experimental data, clinopyroxene-melt equilibria return mid-crustal pressures of 4±1.5 kbar, which are consistent with pressures estimated from the major element content of primitive melt inclusions. Long-term storage of primitive magmas in the mid-crust implies that low CO_2 concentrations measured in primitive plagioclase-hosted inclusions (262–800 ppm) result from post-entrapment CO_2 loss during transport through the shallow crust. In order to reconstruct basaltic plumbing system geometries from petrological data with greater confidence, mineral-melt equilibrium models require refinement at pressures of magma storage in Iceland. Further basalt phase equilibria experiments are thus needed within the crucial 1–7 kbar range.D.A.N. was supported by a Natural Environment Research Council studentship (NE/1528277/1) at the start of this project. SIMS analyses were supported by Natural Environment Research Council Ion Microprobe Facility award (IMF508/1013).This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s00410-015-1170-

A Non-Canonical Function of Zebrafish Telomerase Reverse Transcriptase Is Required for Developmental Hematopoiesis

Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from “authentic” telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words

Organic pollutants in sea-surface microlayer and aerosol in thecoastal environment of Leghorn—(Tyrrhenian Sea)

The levels of dissolved and particle-associated n-alkanes, alkylbenzenes, phthalates, PAHs, anionic surfactants and surfactant fluorescent organic matter ŽSFOM. were measured in sea-surface microlayer ŽSML. and sub-surface water ŽSSL. samples collected in the Leghorn marine environment in September and October 1999. Nine stations, located in the Leghorn harbour and at increasing distances from the Port, were sampled three times on the same day. At all the stations, SML concentrations of the selected organic compounds were significantly higher than SSL values and the enrichment factors ŽEFsSML concentrationrSSL concentration. were greater in the particulate phase than in the dissolved phase. SML concentrations varied greatly among the sampling sites, the highest levels Žn-alkanes 3674 mgrl, phthalates 177 mgrl, total PAHs 226 mgrl. being found in the particulate phase in the Leghorn harbour. To improve the knowledge on pollutant exchanges between sea-surface waters and atmosphere, the validity of spray drop adsorption model ŽSDAM. was verified for SFOM, surface-active agents, such as phthalates, and compounds which can interact with SFOM, such as n-alkanes and PAHs. q2001 Elsevier Science B.V. All rights reserved

How What We See and What We Know Influence Iconic Gesture Production

In face-to-face communication, speakers typically integrate information acquired through different sources, including what they see and what they know, into their communicative messages. In this study, we asked how these different input sources influence the frequency and type of iconic gestures produced by speakers during a communication task, under two degrees of task complexity. Specifically, we investigated whether speakers gestured differently when they had to describe an object presented to them as an image or as a written word (input modality) and, additionally, when they were allowed to explicitly name the object or not (task complexity). Our results show that speakers produced more gestures when they attended to a picture. Further, speakers more often gesturally depicted shape information when attended to an image, and they demonstrated the function of an object more often when they attended to a word. However, when we increased the complexity of the task by forbidding speakers to name the target objects, these patterns disappeared, suggesting that speakers may have strategically adapted their use of iconic strategies to better meet the task’s goals. Our study also revealed (independent) effects of object manipulability on the type of gestures produced by speakers and, in general, it highlighted a predominance of molding and handling gestures. These gestures may reflect stronger motoric and haptic simulations, lending support to activation-based gesture production accounts

Mechanisms of human telomerase reverse transcriptase (hTERT) regulation: clinical impacts in cancer

Background Limitless self-renewal is one of the hallmarks of cancer and is attained by telomere maintenance, essentially through telomerase (hTERT) activation. Transcriptional regulation of hTERT is believed to play a major role in telomerase activation in human cancers. Main body The dominant interest in telomerase results from its role in cancer. The role of telomeres and telomere maintenance mechanisms is well established as a major driving force in generating chromosomal and genomic instability. Cancer cells have acquired the ability to overcome their fate of senescence via telomere length maintenance mechanisms, mainly by telomerase activation. hTERT expression is up-regulated in tumors via multiple genetic and epigenetic mechanisms including hTERT amplifications, hTERT structural variants, hTERT promoter mutations and epigenetic modifications through hTERT promoter methylation. Genetic (hTERT promoter mutations) and epigenetic (hTERT promoter methylation and miRNAs) events were shown to have clinical implications in cancers that depend on hTERT activation. Knowing that telomeres are crucial for cellular self-renewal, the mechanisms responsible for telomere maintenance have a crucial role in cancer diseases and might be important oncological biomarkers. Thus, rather than quantifying TERT expression and its correlation with telomerase activation, the discovery and the assessment of the mechanisms responsible for TERT upregulation offers important information that may be used for diagnosis, prognosis, and treatment monitoring in oncology. Furthermore, a better understanding of these mechanisms may promote their translation into effective targeted cancer therapies. Conclusion Herein, we reviewed the underlying mechanisms of hTERT regulation, their role in oncogenesis, and the potential clinical applications in telomerase-dependent cancers.info:eu-repo/semantics/publishedVersio