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

Reliability of Synaptic Transmission at the Synapses of Held In Vivo under Acoustic Stimulation

BACKGROUND:The giant synapses of Held play an important role in high-fidelity auditory processing and provide a model system for synaptic transmission at central synapses. Whether transmission of action potentials can fail at these synapses has been investigated in recent studies. At the endbulbs of Held in the anteroventral cochlear nucleus (AVCN) a consistent picture emerged, whereas at the calyx of Held in the medial nucleus of the trapezoid body (MNTB) results on the reliability of transmission remain inconsistent. In vivo this discrepancy could be due to the difficulty in identifying failures of transmission. METHODS/FINDINGS:We introduce a novel method for detecting unreliable transmission in vivo. Based on the temporal relationship between a cells' waveform and other potentials in the recordings, a statistical test is developed that provides a balanced decision between the presence and the absence of failures. Its performance is quantified using simulated voltage recordings and found to exhibit a high level of accuracy. The method was applied to extracellular recordings from the synapses of Held in vivo. At the calyces of Held failures of transmission were found only rarely. By contrast, at the endbulbs of Held in the AVCN failures were found under spontaneous, excited, and suppressed conditions. In accordance with previous studies, failures occurred most abundantly in the suppressed condition, suggesting a role for inhibition. CONCLUSIONS/SIGNIFICANCE:Under the investigated activity conditions/anesthesia, transmission seems to remain largely unimpeded in the MNTB, whereas in the AVCN the occurrence of failures is related to inhibition and could be the basis/result of computational mechanisms for temporal processing. More generally, our approach provides a formal tool for studying the reliability of transmission with high statistical accuracy under typical in vivo recording conditions

Presynaptic NMDA Receptors Mediate IPSC Potentiation at GABAergic Synapses in Developing Rat Neocortex

NMDA receptors are traditionally viewed as being located postsynaptically, at both synaptic and extrasynaptic locations. However, both anatomical and physiological studies have indicated the presence of NMDA receptors located presynaptically. Physiological studies of presynaptic NMDA receptors on neocortical GABAergic terminals and their possible role in synaptic plasticity are lacking.We report here that presynaptic NMDA receptors are present on GABAergic terminals in developing (postnatal day (PND) 12-15) but not older (PND21-25) rat frontal cortex. Using MK-801 in the recording pipette to block postsynaptic NMDA receptors, evoked and miniature IPSCs were recorded in layer II/III pyramidal cells in the presence of AMPA/KA receptor antagonists. Bath application of NMDA or NMDA receptor antagonists produced increases and decreases in mIPSC frequency, respectively. Physiologically patterned stimulation (10 bursts of 10 stimuli at 25 Hz delivered at 1.25 Hz) induced potentiation at inhibitory synapses in PND12-15 animals. This consisted of an initial rapid, large increase in IPSC amplitude followed by a significant but smaller persistent increase. Similar changes were not observed in PND21-25 animals. When 20 mM BAPTA was included in the recording pipette, potentiation was still observed in the PND12-15 group indicating that postsynaptic increases in calcium were not required. Potentiation was not observed when patterned stimulation was given in the presence of D-APV or the NR2B subunit antagonist Ro25-6981.The present results indicate that presynaptic NMDA receptors modulate GABA release onto neocortical pyramidal cells. Presynaptic NR2B subunit containing NMDA receptors are also involved in potentiation at developing GABAergic synapses in rat frontal cortex. Modulation of inhibitory GABAergic synapses by presynaptic NMDA receptors may be important for proper functioning of local cortical networks during development

Influence of Psychological Factors on Pain and Disability in Anterior Knee Pain Patients

AKP patients express chronic pain but also disability. However, the correlation between pain and disability is not complete and linear. Some patients with a lot of pain show mild disability while others with much less pain also show great disability. The disability is profoundly influenced by other emotional and cognitive factors that are associated with the perception of pain. Therefore, the clinical efforts do not have to be focused only on treating the pain as a feeling but on identifying and modifying these factor

Cancer risk in 680,000 people exposed to computed tomography scans in childhood or adolescence: data linkage study of 11 million Australians.

OBJECTIVE: To assess the cancer risk in children and adolescents following exposure to low dose ionising radiation from diagnostic computed tomography (CT) scans. DESIGN: Population based, cohort, data linkage study in Australia. COHORT MEMBERS: 10.9 million people identified from Australian Medicare records, aged 0-19 years on 1 January 1985 or born between 1 January 1985 and 31 December 2005; all exposures to CT scans funded by Medicare during 1985-2005 were identified for this cohort. Cancers diagnosed in cohort members up to 31 December 2007 were obtained through linkage to national cancer records. MAIN OUTCOME: Cancer incidence rates in individuals exposed to a CT scan more than one year before any cancer diagnosis, compared with cancer incidence rates in unexposed individuals. RESULTS: 60,674 cancers were recorded, including 3150 in 680,211 people exposed to a CT scan at least one year before any cancer diagnosis. The mean duration of follow-up after exposure was 9.5 years. Overall cancer incidence was 24% greater for exposed than for unexposed people, after accounting for age, sex, and year of birth (incidence rate ratio (IRR) 1.24 (95% confidence interval 1.20 to 1.29); P<0.001). We saw a dose-response relation, and the IRR increased by 0.16 (0.13 to 0.19) for each additional CT scan. The IRR was greater after exposure at younger ages (P<0.001 for trend). At 1-4, 5-9, 10-14, and 15 or more years since first exposure, IRRs were 1.35 (1.25 to 1.45), 1.25 (1.17 to 1.34), 1.14 (1.06 to 1.22), and 1.24 (1.14 to 1.34), respectively. The IRR increased significantly for many types of solid cancer (digestive organs, melanoma, soft tissue, female genital, urinary tract, brain, and thyroid); leukaemia, myelodysplasia, and some other lymphoid cancers. There was an excess of 608 cancers in people exposed to CT scans (147 brain, 356 other solid, 48 leukaemia or myelodysplasia, and 57 other lymphoid). The absolute excess incidence rate for all cancers combined was 9.38 per 100,000 person years at risk, as of 31 December 2007. The average effective radiation dose per scan was estimated as 4.5 mSv. CONCLUSIONS: The increased incidence of cancer after CT scan exposure in this cohort was mostly due to irradiation. Because the cancer excess was still continuing at the end of follow-up, the eventual lifetime risk from CT scans cannot yet be determined. Radiation doses from contemporary CT scans are likely to be lower than those in 1985-2005, but some increase in cancer risk is still likely from current scans. Future CT scans should be limited to situations where there is a definite clinical indication, with every scan optimised to provide a diagnostic CT image at the lowest possible radiation dose